an injectable gelatin-based conjugate incorporating egf
TRANSCRIPT
An Injectable Gelatin-Based Conjugate Incorporating EGF Promotes Tissue
Repair and Functional Recovery After Spinal Cord Injury in a Rat Model
By
Adhvait M. Shah
BS, Biomedical Engineering
Tufts University, 2012
SUBMITTED TO THE HARVARD-MIT PROGRAM IN HEALTH SCIENCES AND TECHNOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF
DOCTOR OF PHILOSOPHY IN MEDICAL ENGINEERING AND MEDICAL PHYSICS AT
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
FEBRUARY 2019
© 2019 Adhvait M. Shah. All rights reserved.
The author hereby grants to MIT permission to reproduce and distribute publicly paper and electronic copies of this thesis document in whole or in part in any medium now
known or hereafter created.
Signature of Author:
Harvard MIT Program in Health Sciences & Technology December 21, 2018
Certified by:
Myron Spector, PhD
Professor of Orthopedic Surgery (Biomaterials), HMS
Department of Orthopedic Surgery, BWH
Director, Tissue Engineering/Regenerative Medicine Laboratory, VABHS
Mechanical engineering and HST, MIT
Thesis Supervisor
Accepted by:
Emery N. Brown, MD, PhD
Director, Harvard-MIT Program in Health Sciences and Technology
Professor of Computational Neuroscience and Health Sciences and Technology
2
An Injectable Gelatin-Based Conjugate Incorporating EGF Promotes Tissue
Repair and Functional Recovery After Spinal Cord Injury in a Rat Model
By
Adhvait M. Shah
Submitted to the Harvard-MIT program in Health Sciences and Technology on December 21st, 2018 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in
Medical Engineering and Medical Physics
ABSTRACT
Spinal cord injury (SCI) is a devastating condition drastically reducing the quality of life that affects
about 300,000 patients in the USA. As a result of the injury, sensory perception and motor
functions are lost. Current treatments do not address the root cause - degeneration and loss of
neural tissue. The overall goal of this pre-clinical work was to evaluate a novel gelatin-based
conjugate (gelatin-hydroxyphenyl propionic acid; Gtn-HPA) capable of undergoing covalent
cross-linking in vivo after being injected as a liquid. Gtn-HPA incorporating epidermal growth
factor (EGF) and/or stromal cell-derived factor - 1α (SDF-1α) was evaluated for promoting tissue
healing and functional recovery using a standardized 2-mm hemi-resection SCI rat model, four
weeks after injection.
Injection of Gtn-HPA/EGF immediately after the surgical excision injury significantly improved
motor functional recovery, compared to gel alone and non-treated controls. Bladder function was
also improved in Gtn-HPA/EGF-treated animals. Functional improvement correlated with the
amount of spared tissue. The volume of gel in the defects was quantified by a newly developed
MRI-based method employing T1-weighted inversion recovery to unambiguously image Gtn-HPA
in the injury site in a non-destructive manner. Histological analysis showed the presence of
multiple islands of Gtn-HPA in the injury site after four weeks. There was a significantly greater
number of cells migrating into the Gtn-HPA/EGF, compared to the gel alone, and these cells
displayed neural progenitor cell markers: nestin, vimentin, and Musashi. The cells infiltrating Gtn-
HPA were negative for glial fibrillary acidic protein (GFAP), a marker for astrocytes. Injection of
the gel reduced the reactive astrocytic presence at the border outlining the injury site indicating
the reduction of glial scar. There was no notable inflammatory response to the Gtn-HPA gel,
reflected in the number of CD68-positive cells, including macrophages. Of note was the
demonstration by immunohistochemistry that the Gtn-HPA remaining at 4 weeks post-injection
contained EGF. MMP2 was found to be playing a role in in vivo degradation of the Gtn-HPA gel.
Additional behavioral and histological results were acquired injecting Gtn-HPA/EGF in 2-mm
complete resection SCI rat model. Collectively, the findings signaled that injury sites injected with
Gtn-HPA/EGF had greater potential for regeneration.
In summary, this work commends an injectable, covalently cross-linkable formulation of Gtn-HPA
incorporating EGF for further investigation in promoting functional recovery and potential
regeneration for treatment of SCI and thereby improve the quality of life of SCI patients.
3
Committee Members:
Thesis Supervisor:
Myron Spector, PhD
Professor of Orthopedic Surgery (Biomaterials), HMS
Department of Orthopedic Surgery, BWH
Director, Tissue Engineering/Regenerative Medicine Laboratory, VABHS
Mechanical Engineering and HST, MIT
Thesis committee Chair:
Michael J. Cima, PhD David H. Koch Professor of Engineering
Professor of Materials Science and Engineering, MIT
Thesis Readers:
Zhigang He, PhD, BM
Professor of Neurology and Ophthalmology, HMS
F. M. Kirby Neurobiology Center, BCH
Srinivasan Mukundan, Jr., MD, PhD
Department of Radiology, BWH
Associate Professor of Radiology, HMS
4
Acknowledgements
I am enormously grateful to my thesis advisor Dr. Myron Spector. I was inspired
during my first course with him at MIT. He has always supported me for my professional
and personal growth throughout my doctoral work. He has always given me ‘in-person’
time whenever I needed it. I was able to accomplish the work done in this project as a
result of various discussions with him. His suggestions have brought us closer to our goal.
He has been very accommodating of my other commitments as well. He has been my
guiding light in difficult days. Overall, he has groomed me into an able research scientist
and critical thinker. I could not have asked for a better mentor, teacher and motivator. He
is a legend and role model in my view.
I would like to thank my colleagues in the Tissue engineering/regenerative
medicine laboratory at Veterans Affairs Medical Center at Jamaica Plains. I thank Dr. Hu-
ping Hsu for his time and assistance with the animal surgeries. His excellent and
experienced hands created consistent and standardized defect in our rat model. He has
been an excellent mentor and teacher in the surgical suite. I would like to thank my current
lab members Dr. Wanting Niu, Christopher Love, and Dr. Yunfei Ma for their help in
various aspects of the project. Specifically, I wish to thank Wanting for her help with
animal care and in vitro experiments, Christopher Love for discussions and help with
mechanical testing, and Yunfei for his help with imaging of slides and ELISA. I also wish
to express my gratitude to previous lab members Dr. Daniel Macaya, Dr. Ravi Rajappa
and Dr. Teck Chuan Lim for laying the foundation of my work and teaching me various
skills that were crucial in successful completion of the doctoral work. I truly felt that our
lab operated as a family with celebrations and support in each other’s work. I thank Dr.
Spector for giving me an opportunity to work in stimulating and supportive environment.
I would like to express my gratitude to my thesis committee: Dr. Michael Cima, Dr.
Zhigang He and Dr. Srinivasan Mukundan. Their time, invaluable feedback and genuine
interest in my work is truly appreciated. Their feedback has led to form more
collaborations and pursue interdisciplinary projects that included MRI. Discussions with
all three members have given me broader view of my project and its impact on the patients
suffering from spinal cord injury. It was my honor to have them on my committee.
I would like to thank my collaborators for their assistance in various aspects of the
thesis project. Dr. Motoichi Kurisawa at A*STAR in Singapore provided the Gtn-HPA
conjugate to fabricate various formulations of the Gtn-HPA. I am grateful to Dr. Yaotang
Wu and Michael Marcotrigiano for their technical assistance with MRI at SAIL facility in
Children’s Hospital. I would like to acknowledge Dr. Srinivasan Mukundan, Dr. Samuel
Patz, Dr. Sharon Peled and Tehya Johnson for their MRI expertise at SAIL facility at
Brigham and Women’s hospital. I am thankful to the staff, Diane Ghera and Sylvia Smith
at animal research facility (ARF) at Veterans Affairs Medical center, Jamaica Plains for
their assistance in the animal surgeries and animal care. I am thankful to Dr. Bertrand
Huber for his help with automated slide scanner at VA Jamaica Plains campus. This work
5
was possible due to the support of these collaborators. I would also like to thank anybody
who has directly and indirectly assisted me professionally with my research work.
The funding for this work was provided by the following sources: Veterans Affairs
administration, Harvard-MIT Division of Health, Science and Technology (HST) fellowship
and Neuroimaging Training Program (NTP) fellowship from NIBIB at NIH.
Personal acknowledgements:
I would like to dedicate this thesis work to my grandmother, Madhurika Shah. She
would have been the happiest person to see me finish my doctoral degree. As a teacher
herself, the seeds of curiosity and education were sown in me by her very early in my
childhood. Her smile and accommodating nature to all situations is very inspiring. Words
are not enough to express my gratitude to her – I miss you Ba!
I would like to thank my parents – Mitesh Shah and Dipti Shah – from the bottom
of my heart. They have supported all my endeavors from my childhood to today. My father
has worked very hard to ensure that we have all the comforts of the world. There have
been times when he had to stay away from the family as well. Moreover, his
inquisitiveness and dedication are an inspiration for me. He has left no stone unturned to
ensure we have the best of education. Thank you pappa! My mother is a very strong
woman – she has endured all the hardships with a smile! She has instilled discipline in
me which has been very helpful in my life as a doctoral student. Thank you mammy! Both
of you have always encouraged me to pursue my dreams and supported me in difficult
times. Your upbringing has made me who I am today! Thank you both for being my
parents.
A special thank you to my wife, Rinkal. Rinkal, my life has found a new meaning
due to your cheerfulness, support and loving attitude. You are the most selfless person
that I have known. We have been with each other and supported each other during the
various highs and lows of our life. Your unwavering support and words ‘everything will be
good’ have given me hope during the tough times during my last two years of PhD. I
cherish our discussions and all the fun times that we have spent together. I look forward
to our journey together. I am extremely proud of your achievements in such a short time
and look forward to celebrating our graduations together. I love you!
I would like to thank my brother, Namank. Namank, you truly are an inspiration to
many. You have set an example of how an ideal life should be. You have excelled in all
aspects of your life. You have been extremely supportive during my years in the PhD.
You have taken on more responsibility so that I can focus on my studies. You have
provided continued technical guidance as necessary, especially when my laptop crashed.
You have saved me from panic attacks. Moreover, I truly cherish our car rides together. I
will always remember your support and feedback during my qualifying exam preparation.
Now that I will be more available, I look forward to hanging out with you and Priya.
6
I would like to also acknowledge continued support of my extended family
members here in the USA and in India. Your company and conversations have made my
time as graduate student more enjoyable. I would like to thank all my friends at MIT,
Cityview and in India. I sincerely appreciate your love and support. I will leave MIT with
fond memories, which shall be cherished throughout my life.
In the end, I would like to express my immense gratitude to revered Pandurang
Shastri Athavale, inspirer of the Swadhyay movement all across the globe. Pujniya
Dadaji, your thoughts have guided me and molded my life for the better. You have given
me the tools to develop myself in all the aspects of my life. Your path has taken me closer
to God. You are truly responsible for giving me a comprehensive view of the world –
external and internal. I can’t imagine the state of my life had you not come into my life. I
will give my best to live according to your teachings with the guidance of Aadarniya Didiji.
Jay Yogeshwar!
7
Table of Contents
Acknowledgments ________________________________________________________ 4
Table of Contents _________________________________________________________ 7
Chapter 1
1. Introduction ___________________________________________________________ 11
1.1. Spinal cord injury – clinical motivation
1.2. Hypothesis and specific aims
1.3. Summary of the chapters of the thesis
1.4. References
Chapter 2
2. Clinical motivation and background
2.1. Introduction to spinal cord injury _________________________________________ 17
2.1.1. Prevalence of spinal cord injury
2.1.2. Clinical presentation of SCI and functional impairments
2.1.3. Clinical management and current treatment
2.1.4. Anatomy and pathophysiology of SCI
2.1.5. Cavitary lesions – potential for regeneration and recovery
2.1.6. Pre-clinical SCI models – an overview
2.2. Tissue engineering approaches in SCI ____________________________________ 34
2.2.1. Three pillars of tissue regeneration – an overview
2.2.2. Scaffolding materials for SCI
2.2.2.1. Natural vs. synthetic materials
2.2.2.2. Pre-formed vs. injectable biomaterials
2.2.2.3. Injectable biomaterials for SCI
2.2.2.4. Criteria for biomaterials for cavitary lesions
2.2.3. Biomolecules for SCI – an overview
2.2.3.1. Neurotrophins and growth factors
2.2.3.2. ECM and scar modifying molecules
2.2.4. Cell-based treatment for SCI – an overview
2.3. References _________________________________________________________ 46
Chapter 3
3. Rationale and development of the formulation with Gtn-HPA matrix with EGF and
SDF-1α for spinal cord injury rat model evaluation
3.1. Biomaterial: Gelatin-hydroxyphenyl propionic acid (Gtn-HPA) __________________ 55
3.1.1. Introduction
3.1.2. Synthesis of Gtn-HPA
3.1.3. Mechanical properties of Gtn-HPA
3.1.4. Prior in vitro studies with Gtn-HPA
3.1.5. Gtn-HPA: promising biomaterial for spinal cord injury repair
3.2. Growth factor: Epidermal growth factor (EGF) ______________________________ 63
3.2.1. Introduction
3.2.2. Prior relevant in vitro studies with EGF
8
3.2.3. Prior in vivo studies incorporating EGF in SCI models
3.3. Chemokine: Stromal-cell derived factor-1α (SDF-1α) _________________________ 67
3.3.1. Introduction
3.3.2. Prior relevant in vitro studies with SDF-1α
3.3.3. Prior in vivo studies incorporating SDF-1α in SCI models
3.4. Two formulations of Gtn-HPA for injection in the SCI models ___________________ 71
3.5. Release profile of EGF from Gtn-HPA formulations for four weeks ______________ 72
3.5.1. Methods
3.5.2. Results and discussion
3.6. References _________________________________________________________ 74
Chapter 4
4. Investigating the effects of Gtn-HPA matrix with EGF and SDF-1α in a T8 2 mm complete resection rat model of spinal cord injury after four weeks 4.1. Introduction and clinical motivation _______________________________________ 79 4.2. Overall goal and hypotheses of the chapter ________________________________ 80
4.3. Methods ____________________________________________________________ 81
4.3.1. Gtn-HPA gel fabrication
4.3.2. Animal surgical procedure and Gtn-HPA injection
4.3.3. Experimental design
4.3.4. Animal care post-surgery
4.3.5. Behavioral assessment – BBB scale
4.3.6. Animal sacrifice and harvesting of the spinal cord
4.3.7. Tissue embedding procedure and cryosection
4.3.8. Histology and histomorphometric analysis
4.3.9. Immunofluorescent staining and analysis
4.3.10. Statistical Analysis
4.4. Results _____________________________________________________________ 90
4.4.1. Qualitative observations of the animals and Gtn-HPA injection
4.4.2. Functional evaluation using BBB scale
4.4.3. Gross histological outcomes
4.4.4. Histomorphometric evaluation of the injury site
4.4.5. Immunohistochemistry evaluation of the injury site
4.4.5.1. GFAP – astroytes
4.4.5.2. Iba1 – microglia/macrophages
4.4.5.3. NeuN – mature neurons
4.4.5.4. MMP2 – Gtn-HPA degradation
4.4.5.5. EGF – Gtn-HPA incorporated EGF
4.4.5.6. Nestin – neural stem cells
4.5. Discussion _________________________________________________________ 107
4.5.1. Functional recovery assessment
4.5.2. Histological evaluation of the injury site
4.5.3. Immunohistochemical evaluation of the injury site
4.6. References _________________________________________________________117
9
Chapter 5
5. Developing an MRI protocol to visualize the Gtn-HPA matrix in the spinal cord
5.1. Introduction and motivation ___________________________________________ 121
5.2. Overall goal and hypotheses of the chapter ______________________________ 123
5.3. Approach 1: T2-weighted MRI _________________________________________ 124
5.3.1. Introduction
5.3.2. Methods
5.3.2.1. Surgery procedure and experimental design
5.3.2.2. Sample preparation and acquisition of MRI
5.3.2.3. Image analysis
5.3.3. Results
5.4. Approach 2: Magnetic resonance elastography (MRE) ______________________ 128
5.4.1. Introduction
5.4.2. Methods
5.4.3. Results
5.5. Approach 3: T1-weighted Inversion Recovery (T1IR) ________________________ 130
5.5.1. Introduction
5.5.2. Preliminary phantom work - Relaxometry
5.5.2.1. Methods
5.5.2.2. Results
5.5.3. Preliminary phantom work – T1IR
5.5.3.1. Methods
5.5.3.2. Results
5.5.4. Ex vivo spinal cord T1IR – Protocol 1.0
5.5.4.1. Methods – surgical procedure and MRI acquisition
5.5.4.2. Results
5.5.5. Ex vivo spinal cord T1IR – Protocol 2.0
5.5.5.1. Methods - surgical procedure and MRI acquisition
5.5.5.2. Results
5.6. Summary and discussion _____________________________________________ 140
5.7. References ________________________________________________________ 144
Chapter 6
6. Investigating the effects of Gtn-HPA matrix with EGF and SDF-1α in a T8 2-mm hemi-
resection rat model of spinal cord injury after four weeks
6.1. Introduction and clinical motivation ______________________________________ 147
6.2. Overall goal and hypotheses of the chapter _______________________________ 149
6.3. Methods ___________________________________________________________ 150
6.3.1. Gtn-HPA gel fabrication
6.3.2. Animal surgical procedure and Gtn-HPA injection
6.3.2.1. Improvement in the dural replacement technique
6.3.3. Experimental design
6.3.4. Animal care post-surgery and bladder function evaluation
6.3.5. Behavioral assessment – BBB scale
6.3.6. Animal sacrifice and harvesting of the spinal column
6.3.7. MRI ex vivo scan of the spinal column
10
6.3.8. Gtn-HPA volume determination using MRI images
6.3.9. Cryoprotection, tissue embedding and cryosection
6.3.10. Histology and histomorphometric analysis
6.3.11. Spared tissue area and injury area quantification
6.3.12. Immunofluorescent staining and analysis
6.3.13. Statistical analysis
6.4. Results ____________________________________________________________ 163
6.4.1. Surgical observations and Gtn-HPA injection
6.4.2. Functional evaluation – bladder function
6.4.3. Functional evaluation – motor recovery (BBB scoring)
6.4.4. Spared tissue and injury area quantification
6.4.5. Spared tissue correlation with functional evaluation
6.4.6. Gtn-HPA gel volume measurement using MRI images
6.4.7. Gross histological outcomes
6.4.7.1. Control group – no injection
6.4.7.2. 8 wt% Gtn-HPA gel groups
6.4.7.3. EGF in suspension group
6.4.7.4. 12 wt% Gtn-HPA groups
6.4.8. Histomorphometric evaluation of the injury site
6.4.9. Immunohistochemistry evaluation of the injury site
6.4.9.1. GFAP – astrocytes
6.4.9.2. Iba1 - resident and/or activated microglia
6.4.9.3. CD68 – macrophages/monocytes
6.4.9.4. NeuN – mature neurons
6.4.9.5. MMP2 – Gtn-HPA gel degradation
6.4.9.6. EGF – Gtn-HPA incorporated EGF
6.4.9.7. Nestin – neural stem cells
6.4.9.8. vWF – endothelial cells
6.5. Discussion ________________________________________________________ 195
6.5.1. Functional evaluation
6.5.1.1. Bladder function – urine retention volume
6.5.1.2. Motor function – BBB scale
6.5.2. Visualizing Gtn-HPA using MRI
6.5.3. Histological evaluation of the injury site
6.5.4. Immunohistochemical evaluation of the injury site
6.6. References ________________________________________________________ 210
Chapter 7
7. Conclusions __________________________________________________________ 215
Chapter 8
8. Limitations and Future directions
8.1. Limitations of the thesis ______________________________________________ 218
8.2. Future directions ___________________________________________________ 221
Appendix ______________________________________________________________ 223
11
Chapter 1:
Introduction
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1.1. Spinal cord injury – clinical motivation
Spinal cord injury (SCI) is a devastating disease with significant loss of neuronal function.
Any damage – mostly mechanical - to the spinal cord tissue resulting in temporary or permanent
changes in sensory, motor or autonomic function is defined to be SCI [1]. According to WHO,
roughly 250,000 new SCI cases are registered every year throughout the globe [2]. In the USA,
SCI currently affects 288,000 patients with around 17,700 new cases every year. 78% patients
are male with average age for the onset of injury 43 years. The leading causes of traumatic SCI
are automobile acccidents and falls that constitute roughly 70% of SCI patients [3]. In terms of
anatomy, injury in upper thoracic region could results in complete loss of function in all four limbs
(tetraplegia) while SCI at lower thoracic and lumbar level could lead to paraplegia depending upon
the severity of the injury. It is shocking that 67% of patients suffer from tetaplegia leading to
complete bed-rest [4]. As a result, they suffer secondary complications such as pressure ulcers
(52%) and depression (22.2%) [5, 6]. The lifetime costs could be as high as $5 million for
tetraplegic patients. It is reported that less than 1% of the patients experience complete gain in
the function after hospital discharge [3]. Clinically, three major approaches that mitigate the
secondary damage caused by the primary injury are taken – the cord is decompressed as a result
of pinching of bone fragments, acute anti-inflammatory agents are administered and rehabilitation
efforts are made for functional recovery. Although clinical ‘treatment’ of the SCI include
management of the symptoms, the root cause – degeneration of the neural tissue – is not
addressed.
SCI initiates a complex cascade of pathophysiological responses that hinder possibility of
any intrinsic regeneration of the severed axons. Various molecules that are released as a result
of myelin degradation. These molecules that are recruited to the injury site are inhibitors of
regeneration [7]. Moreover, the parenchyma and stroma of the tissue are lost post injury, often
resulting in a fluid-filled cavitary defect. Our thesis is that, an intrinsic regenerative process will be
facilitated by filling the fluid-filled cavity with a biomaterial matrix (also referred to as a scaffold)
capable of serving as a stroma to enable population of the defect with endogenous neural
reparative cells. Since the surgical intervention necessary of placing pre-formed scaffolds in the
injury site would cause further damage, we propose to develop a minimally invasive injectable
matrix that is permissive of endogenous cell migration, proliferation, and differentiation. Moreover,
injectable materials can also conform to the heterogeneous cavities post SCI, which are of
variable size and shape. In addition, these injectable biomaterials can incorporate signaling
molecules and factors that could promote a reparative/regenerative tissue response, eventually
functional recovery.
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1.2. Hypothesis and specific aims
The overall goal of this thesis was to improve functional outcome after SCI through a new
treatment employing the tools of tissue engineering and regenerative medicine: a biomaterial
scaffold and recombinant regulatory molecules (viz., a growth factor and chemokine). While it
may not be possible to fully regenerate a spinal cord after its injury, the goal was to improve short-
and long-term outcomes, respectively, by: interrupting degenerative processes in the acute phase
post-SCI; and populating the defect with cells capable of re-establishing neural circuitry. To this
end, this thesis investigated a novel gelatin-based conjugate (gelatin-hydroxyphenyl propionic
acid; Gtn-HPA) capable of undergoing covalent cross-linking in vivo after being injected as a
liquid. Gtn-HPA incorporating epidermal growth factor (EGF) and/or stromal cell-derived factor -
1α (SDF-1α) was evaluated for promoting tissue healing and functional recovery using a
standardized 2-mm surgical excision SCI rat model, four weeks after injection. Specifically, the
supposition was that EGF and SDF-1α would act to reduce degenerative processes acutely post-
SCI and recruit endogenous neural stem cells to repopulate the defect in order to promote a
wound healing response for potential regeneration of certain neural processes. Moreover, the
goal was also to evaluate the neuroprotective effect and short-term functional recovery outcomes
four weeks post injection of Gtn-HPA, alone and incorporating EGF and SDF-1α.
Our principal hypothesis is that, when injected immediately after injury, the gelatin-based
gel (Gtn-HPA) incorporating EGF and SDF-1α results in: short term functional improvement (at
four weeks) post injury; and recruitment of endogenous neural stem cells (NSCs) into the gel-
filled lesion.
The specific aims of the thesis were as follows:
1) To develop and study the release of EGF from 8% and 12% Gtn-HPA for injection into in vivo
SCI model evaluated in specific aims 2 and 3, and to develop a magnetic resonance imaging
(MRI) protocol to visualize the Gtn-HPA gel, and to distinguish it from the surrounding tissue.
2) To investigate the potential of EGF and SDF-1α delivered by 8% Gtn-HPA matrix in two
different rodent SCI models (2 mm complete resection and 2 mm hemi-resection) four weeks post
injection.
3) To investigate the effects of 12% Gtn-HPA/EGF with an improved dural replacement technique
in a 2 mm hemi-resection model four weeks post injection.
14
1.3. Summary of the chapters of the thesis
This thesis is divided into eight chapters – the contents of which is summarized below:
Chapter 2 provides clinical motivation and a detailed background related to SCI. It discusses the
prevalence of the SCI and the functional deficits that the patients suffer post SCI followed by the
current practices of the clinical management after SCI. The chapter describes the temporal and
anatomical changes at the molecular level after the injury leading the formation of spinal cord
cavities usually seen in the patients. After describing relevant pre-clinical models to study SCI, a
detailed background into the prior regenerative research work both in vitro and in vivo to promote
regeneration of the defect lesion after SCI is discussed.
Chapter 3 gives background and rationale for the specific tissue engineering tools that were
employed in this thesis work – Gtn-HPA biomaterial and the factors EGF and SDF-1α. By way of
background, several prior in vitro studies characterizing the Gtn-HPA are presented, and the
results from the current ELISA assessment for EGF release from Gtn-HPA are presented over
four weeks are described as evaluated in the first specific aim. The chapter also presents the
history of the prior biomaterials studied in our laboratory for SCI regeneration. Importantly, the
rationale and the formulations of Gtn-HPA that were evaluated in Chapter 4 and Chapter 6 are
discussed.
Chapter 4 describes the evaluation of Gtn-HPA incorporating EGF and SDF-1α in a standardized
2-mm complete transection model of SCI for evaluation of second specific aim. These
experiments were guided by the optimizing the formulation of the Gtn-HPA that was described in
Chapter 3. The animals were assessed each week for their motor function followed by their
histological and immunohistochemical assessment of the injury site. The learnings from this
Chapter were applied in Chapter 6. In addition, a challenge regarding visualizing the Gtn-HPA in
a non-destructive manner was overcome in the work described in Chapter 5.
Chapter 5 describes various approaches that were taken to develop a noninvasive and non-
destructive imaging protocol to visualize the Gtn-HPA gel as per the first specific aim. The results
from three MRI-based approaches – T2-weighted imaging, Magnetic resonance elastography and
T1-weighed inversion recovery – are presented. The final protocol that was able to unambiguously
distinguish the Gtn-HPA based on the T1-weighted inversion recovery method is presented. All
the animals evaluated in Chapter 6 in hemi-resection model were imaged using this protocol.
Chapter 6 describes a comprehensive in vivo evaluation of two different – 8% and 12% - Gtn-
HPA formulations incorporating EGF and SDF-1α in a standardized 2-mm hemi-resection SCI
15
model as evaluated for specific aim 2 and 3. The chapter also described various surgical
improvements that were made based on the outcomes of Chapter 4. The formulation of the Gtn-
HPA was chosen based on the findings from Chapter 4. Histologic, immunohistochemical and
functional evaluation was performed on the rats post SCI. Specifically, motor function and bladder
function was evaluated to gain greater insights into the effects of Gtn-HPA/EGF matrix.
Furthermore, all animals were scanned using MRI-based T1-weighted inversion recovery protocol
developed in Chapter 5.
Chapter 7 details the conclusions that are supported based on the findings of the thesis work.
Chapter 8 discusses the limitations of this work and provides thoughts on the future investigation
in regard to the work presented in this thesis.
At the end, Appendix includes protocols and relevant information for meticulous planning of the
future experiments.
1.4. References
1. Staff, M.C. Spinal cord injury. 2014 10/8/2014 [cited 2014 12/20/2014]; Available from: http://www.mayoclinic.org/diseases-conditions/spinal-cord-injury/basics/definition/con-20023837.
2. Spinal cord injury. 2014 11/2013 [cited 2014 12/19]; Available from: http://www.who.int/mediacentre/factsheets/fs384/en/.
3. Facts and Figures at a Glance, in SCI Data Sheet. 2018, University of Alabama at Birmingham: National SCI Statistical Center. p. 2.
4. Ramer, L.M., M.S. Ramer, and E.J. Bradbury, Restoring function after SCI: towards clinical translation of experimental strategies. Lancet Neurol, 2014. 13(12): p. 1241-1256.
5. McKinley, W.O., et al., Long-term medical complications after traumatic SCI: A regional model systems analysis. Archives of Physical Medicine and Rehabilitation, 1999. 80(11): p. 1402-1410.
6. Williams, R. and A. Murray, Prevalence of Depression After SCI: A Meta-Analysis. Arch Phys Med Rehabil, 2014.
7. Schwab, M.E. and S.M. Strittmatter, Nogo limits neural plasticity and recovery from injury. Curr Opin Neurobiol, 2014. 27: p. 53-60.
16
Chapter 2:
Clinical motivation and background
17
2.1. Introduction to spinal cord injury
2.1.1. Prevalence of spinal cord injury: SCI is a debilitating condition that greatly affects
the quality of life for the patients. SCI is a clinically complex and life-changing condition. The
incidence of SCI is also high with roughly 17,700 new cases every year in the US - equivalent to
54 new cases per million in the USA [1]. As per the World Health Organization, SCI affects nearly
500,000 people worldwide and higher incidence of SCI of around 80 new cases per million
compared to the USA, which is a conservative estimate given the poor reporting of the cases in
various countries [2]. Leading causes of SCI are motor vehicle accidents (38%), falls (31%),
violence (13%), and sports (8%) [1]. These causes lead to differing level and extent of the SCI
depending upon the location and injury to the cord. Most severe form of the injury is injury in the
cervical region with severe mechanical insult, for example, resulting from a diving accident. Such
injury to the spinal cord would lead to complete paralysis of four limbs – complete tetraplegia.
Spinal cord injuries are typically categorized into four types – complete tetraplegia, incomplete
tetraplegia, complete paraplegia and incomplete paraplegia. Most SCI patients suffer from
incomplete tetraplegia (47%) followed by incomplete paraplegia (20%). Considering these two
most prevalent types of injury, lifetime costs for the patient could be as high as 4 million dollars if
they are injured at a young age [2]. It is reported that less than 1% patients experienced complete
recovery after the SCI. Furthermore, it is interesting to note that the average age of SCI has
increased from 29 in 1970s to 43 in 2018. There are two age groups where peaks are observed
in the incidence rates of the SCI – younger age group (20-29 years) due to motor accidents and
sports while the older age group (65-75 years) due to falls in the home or hospitals [3]. Therefore,
sincere efforts to prevent falls have led to reduced incidence of falls in the hospitals. As these
numbers suggest, there could be direct and indirect costs to an individual due to SCI. Direct costs
include health care costs, rehabilitation costs, more expensive transportation modes and special
needs. Indirect costs include reduced life expectancy, reduced productivity, stress, societal
challenges and disability. In addition to these costs, there are notable clinical functional
impairments that drastically reduce the quality of life for the patient.
2.1.2. Clinical presentation of the SCI and functional impairments: SCI is a debilitating
condition that leads to significant loss in the neuronal function resulting in autonomic, motor and
sensory impairments. Traumatic SCI usually starts with an abrupt mechanical insult resulting in
fracture or dislocation to the vertebrae intruding into the spinal canal. This process initiates a
cascade of pathophysiological changes at the site of the injury depending upon the severity of the
SCI. This leads to irreversible damage of the axons and break of the neuronal circuitry prompting
18
loss of function for the patient. As a result, SCI patients suffer from multiple adverse secondary
consequences that drastically reduce their quality of life [4].
In the acute phase of the SCI, the level and the severity of the injury determines the clinical
presentations of the SCI. Level of the SCI could be classified into two – tetraplegia and paraplegia.
Typically, cervical injuries would lead to tetraplegia affecting the functions of the neck, trunk, upper
and lower limbs. On the other hand, paraplegic injury refers to loss of function in the lower limbs
and the trunk without affecting the upper limbs. Severity of the injury is categorized in two types
– complete and incomplete. A complete SCI would lead to a complete loss of motor and sensory
below the level of the injury while an incomplete SCI retains some residual function below the
level of the injury [2]. Therefore, depending upon the level and the severity of the SCI, relevant
clinical management is necessary in the acute phase of the SCI, specifically cardiovascular and
pulmonary symptoms, which could be life-threatening [5]. The direct mechanical insult results in
secondary damages to the cord ensuing in functional loss. Most common secondary
complications include pressure ulcers, incontinence, pneumonia, autonomic dysreflexia, urinary
tract infections, loss of sexual function, fractures and depression-related disorders [6].
Acute clinical manifestations post SCI include pathologies in the cardiovascular, nervous
and respiratory systems. Cervical injuries cause loss of the sympathetic tone leading to imbalance
between the sympathetic and parasympathetic nervous system known as neurogenic shock.
Neurogenic shock results from the interruption of sympathetic tone due to disruption in
Figure 2.1 Prevalence of SCI with respect to age and gender. Reference - WHO Report on SCI, 2014
19
supraspinal control, and an intact parasympathetic influence via the vagus nerve, leading to an
imbalance in the autonomic control. Immediately, patient would suffer from bradycardia and
hypotension. Bradycardia is reported in between 64 to 77 percent of patients with cervical SCI.
Hypotension leads to further secondary damage in the cord due to reduced blood flow to the cord.
In addition, cervical injuries cause pathologies in the cardiovascular system – hypotension,
instability in the blood pressure and abnormalities in the heart rate [7, 8]. Autonomic dysreflexia
is another commonly reported clinical outcome post high cervical SCI. Autonomic dysreflexia is
characterized by extreme hypertension, in which the systolic blood pressure could reach up to
300 mm Hg accompanied by headache and slow heart rate. Untreated autonomic dysreflexia
could lead to intercranial hemorrhage, seizures, cardiac arrythmia and even death [9]. In addition
to the autonomic and cardiovascular system, respiratory system is also clinically affected due to
cervical injuries. In fact, respiratory complications are leading cause of death after SCI. Studies
have found that 67% of acute SCI patients experience severe respiratory complications within the
first days after the injury – namely atelectasis (36.4%), pneumonia (31.4%), and respiratory failure
(22.6%) [10]. People with an SCI above C3 may require constant mechanical ventilation or
implantation of a phrenic or diaphragm pacemaker to maintain adequate breathing [3, 5].
Chronic clinical presentations post SCI include pressure ulcers, loss of sexual function,
renal abnormalities, bladder and bowel incontinence and depression-related disorders (Table
2.1). Pressure ulcers are most common long term complication post SCI, especially in tetraplegic
patients followed by autonomic dysreflexia and renal abnormalities [11]. Moreover, loss of bladder
and bowel control affect the patients on a day-to-day basis. Due to SCI, patient loses voluntary
control on the sphincter resulting in recurrent and spontaneous voiding of the bladder. The onset
of symptoms is also dependent on the type of neuron that is affected to cause incontinence of the
bladder – upper or lower motor neuron because the management would depend upon the type of
the injury causing loss of bladder control. Similarly, bowel movements are also recurrent with loss
of control. It is shown that these symptoms could lead to social anxiety and isolation due to the
nature of the symptoms [12]. Another chronic clinical outcome of the SCI is spasticity. Around
70% of the patients develop varying degrees of spasticity that could cause significant disabilities.
Spasticity results because of the inability to control the muscles. Usually, muscles become very
stiff as a result of continuous contraction. This is due to imbalance in signals from the brain and
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the cord. For example, if an upper motor neuron is severed, electrical signal does not reach the
synapse between the upper and lower motor neuron near the spinal cord roots. However, lower
motor neuron continuously is supplied with signal without any inhibition resulting in ‘permanent’
contraction of the muscles. Spasticity restrict the range of motion and thereby hinder daily
functions of the patient. Therefore, spasticity is primarily focused on by the rehabilitation care
team to making the patients more independent with their daily works, e.g. picking up a spoon,
opening a lid of the bottle and similar actions [13, 14].
Figure 2.2 Common acute and chronic clinical presentation of SCI patients. Reference – Expert Chikitsa, http://wiki.expertchikitsa.com
Table 2.1 Prevalence of various chronic secondary complications after SCI. Reference – McKinley, et al. Arch of Phys Med and Rehab, 1999. 80(11): p. 1402-1410.
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All the above clinical outcomes were directly related to the primary and secondary
damages to the tissue (Figure 2.2). However, chronic clinical manifestations of the SCI also
include anxiety, societal isolation, post-traumatic stress disorder (PTSD) and depression. Given
how the quality of life is reduced and prognosis of their injury, it is not surprising that many patients
undergo psychological stress. Patients need to be given proper care and attention to deal with
this stress and anxiety that could come with the injury. It is reported that 20-30% of patients show
symptoms of clinically significant symptoms of depression [15]. Taking into consideration all the
clinical symptoms that patients experience, they have very little life satisfaction after the injury.
Although many patients do cope up well with their injury and new accommodations, better clinical
management and treatments are needed for the patients. Currently, SCI clinical management
involves management of the symptoms but not the root cause – degeneration of the spinal cord.
2.1.3. Clinical management and current treatment: SCI is managed by an integrated
team consisting for surgeons, physicians, rehabilitation therapist, occupational therapist,
recreational therapist, speech and hearing therapist, and many more depending upon the
symptoms of the patients. Therefore, professional societies have laid down management
guidelines. Initial care highlights the importance of addressing the airways, breathing and
circulation (ABC) first to ensure no life-threatening episode occurs [16]. In order to prevent further
spinal damage, patient’s spine is immobilized by the means of rigid cervical collar and backboard
for transfers. Once the patient is conscious and responding, a motor and sensory examination is
performed as per the American Spinal Injury Association (ASIA) impairment scale to assess the
level and extent of the injury (Table 2.2). The findings would be used for further care and
management [17]. Hypotension is corrected to systolic pressure of more than 90 mm Hg and
hypovolemia is treated on a case-by-case basis. Patients are kept in ICU setting for continuous
monitoring of cardiac, hemodynamic and respiratory indicators [16]. Once the patient is stable
and conscious, either CT and MRI imaging is performed to confirm the location and extent of the
SCI. X-rays are not recommended because they have been shown to miss nearly 6% of the
injuries [18].
After the appropriate imaging modality, efforts are focused on neuroprotection of the
healthy tissue to minimize further secondary damage. Neuroprotection includes efforts to
preserve the spared tissue after the injury from further secondary damage. Studies have shown
correlation between the amount of spared tissue and functional outcome. Given that
neuroprotective efforts are employed in the acute phase, we aim to evaluate the neuroprotective
effects of acute Gtn-HPA injection immediately after the creation of SCI cavity in our surgical
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excision models discussed later. The first clinical treatment considered is early surgical
decompression. After SCI, continuous mechanical compression of the cord due to pinching of a
broken bone or an intact bone could significantly impair blood flow to the healthy tissue, thereby
resulting in ischemia and in increased zone of neuronal injury. Early surgical decompression
relives the compression improving the blood supply to the healthy neural tissue and thereby
preventing further expansion of the neural injury. Early surgical decompression has shown to be
very effective for improving pathological and functional outcomes by 31% in the pre-clinical
studies [19]. Patients who underwent early surgical decompression within 24 hours of injury
showed improvement of at least 2 ASIA grades within 6 months of injury compared to patients
undergoing the decompression later in the acute care [20]. However, depending on the level of
trauma, the type of SCI and risk-benefits analysis, not all patients are considered for the early
surgical decompression. For example, patients with central cord injury are typically not considered
for the surgical decompression due to poor surgical outcomes. Moreover, surgical decompression
could involve breaching the dura that surrounds the spinal cord thereby making patients
vulnerable for more infection-related clinical problems [21].
ASIA grade Clinical state
ASIA Grade A
(complete)
Complete lack of motor and sensory function below the level of
injury and no sacral functions
ASIA Grade B
(incomplete)
Loss of motor function but preserved sensory function below the
level of the injury and sacral functions
ASIA Grade C
(incomplete)
Motor function is preserved below the level of injury with more
than half of key muscles below the injury level has muscle
strength of less than 3
ASIA Grade D
(incomplete)
Motor function is preserved below the level of injury with at least
half of key muscles below the injury level having muscle strength
of more than 3
ASIA Grade E Normal motor and sensory functions
Table 2.2 ASIA grade categories for classification of the SCI. Clinical management is dependent on the
ASIA scale assessment. Reference – American Spinal Injury Association
The second clinical treatment with the goal of neuroprotection involves the use of steroids
to minimize the neuroinflammation. Methylprednisolone is a synthetic glucocorticoid that
upregulates the release of anti-inflammatory cytokines and reduces the oxidative stress to the
injured tissue. Methylprednisolone have shown to neutralize acute inflammation and reduce
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swelling to reduce injury to remaining tissue in addition to decreasing lipid peroxidation. Therefore,
methylprednisolone was recommended for the acute administration within 48 hours of the injury
in the 1990s and 2000s [22, 23]. However, case reports and studies showed that
methylprednisolone increased the risk of infections thereby outweighing the neurological benefits
it provided if it was administered with high dose for 48 hours. Current guidelines do not
recommend the use of methylprednisolone after 8 hours of the injury either in 24-hour dose
protocol or 48-hour dose protocol. A 2012 review meta-analysis from six important randomized
clinical trials found that patients receiving methylprednisolone within 8 h of injury had a 4-point
greater ASIA motor score improvement [24]. Therefore, current guidelines recommend IV
administration of methylprednisolone only if it can be received within 8 hours of the traumatic
injury for the duration of 24 hours [25].
The third clinical treatment aimed at protecting the neural tissue from further damage is
blood pressure augmentation. Reduced blood flow and local edema due to neuroinflammation
contributes to ischemia in the area immediately surrounding the injury. This could further worsen
the functional sparing after the SCI. Therefore, blood pressure augmentation is a viable strategy
to protect the perilesional neural tissue from further degeneration by enhancing blood perfusion
in the area. The technique aims to maintaining the mean arterial pressure (MAP) to be greater
than 80-90 mm Hg for 7 days post injury. This method has shown to be beneficial for long-term
ASIA grade improvement in the patients [26]. However, some patients are not considered for this
treatment if they cannot be immobilized significantly. Additionally, systemic hypothermia was
considered as a therapy to reduce the rate of spread of the neuronal damage as a neuroprotective
therapy. However, it is not shown to be highly effective at improving functional outcomes and
therefore, it is not recommended by the current guidelines [27].
Another long-term clinical treatment that could assist patient improve their quality of life by
augmenting their functional capabilities is rehabilitation. Various forms of rehabilitative care has
shown to be beneficial in improving functional outcomes by improving on the ASIA grade scale of
up to 2 grades [28]. Rehabilitation therapy range from bodyweight supported treadmill training,
functional electrical stimulation and bicycle training. Functional electrical stimulation (FES) is an
interesting method to send signals to the preserved neural circuit or to the muscles directly.
Transplantation of the electrodes near the nerves of interest have shown electrical activity near
the nerves and functional stimulation of the corresponding muscles. Overall, the type of
rehabilitative measures are dependent on the type of the injury, level of the injury and the current
function that patients have preserved [29].
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Current clinical treatments are aimed at the clinical management of multiple symptoms in
the acute setting while rehabilitation being the clinical intervention in the chronic phase. Taking
all clinical treatments into consideration, these do not address the primary reason for the
functional impairment – degeneration of the neuronal tissue. Therefore, restorative and
regeneration approaches are the future in the context of developing effective clinical treatments
to improve the functional recovery in the patients following a SCI. Given the complexity of the SCI
affecting wide range of tissues and functions, better understanding of the pathophysiological
processes and the chronology of the injury progression is essential in designing restorative and
regenerative therapies for the SCI.
2.1.4. Anatomy and pathophysiology of the SCI: Spinal cord is an integral link
between the brain and the peripheral nervous system. It extends from the foramen magnum to
lumbar level vertebrae. Various ascending and descending tracts run along the length of spinal
cord. Ascending tracts carry sensory information such as pain, temperature, light touch,
proprioception of arms and legs to brainstem and cerebrum. Descending tracts carry motor and
autonomic information controlling the voluntary and involuntary movements of our body (Figure
2.3). Transverse section of the spinal cord shows that it is divided into two regions – gray matter
and white matter. H-shaped horns are gray matter while the surrounding area is the white matter.
Gray matter consists of the neuronal nuclei while the white matter consists of the axonal
ascending and descending tracts [10, 30]. At cellular level, spinal cord tissue consists of neuronal
parenchymal cells. Supportive cells, specifically known as glial cells, include astrocytes,
oligodendrocyte, and microglia. There is also a stem cell niche near the subependymal layer
surrounding the central canal and the meninges.
Typically, traumatic SCI is defined as neuronal damage to the cord that results in change
or loss of function. Traumatic SCI involves mechanical insult to the cord as a result of vehicular
accidents, falls, gun wounds or sports activities. The mechanical force initiates a cascade of
pathophysiological responses that causes critical neural degeneration – the process that leads to
break in the neuronal circuitry and thereby contributes to functional impairment. These functional
impairments manifest as clinical symptoms of the SCI. It is critical to review these
pathophysiological processes to develop various targets for the development of novel treatments
of SCI. Pathophysiological response of the SCI is considered to be biphasic – primary damage
and secondary damage [31].
Primary damage refers to the immediate mechanical damage to the cord. Most spinal cord
injuries are contusion injuries due to blunt force directly applied on the cord as a result of
25
dislocation of the spinal column and vertebrae. Laceration, shearing, sudden jerk due to varying
acceleration and deceleration and compression are other types of mechanical injuries to the cord.
Cause of the SCI determines the type of primary damage caused to the cord [32]. For example,
a knife or gun wound would cause shearing or laceration type of primary injury while an accident
would cause contusion injury. Sheer force from the primary injury severs the axons, breaches the
blood-brain barrier (BBB), causes spinal and neurogenic shock and disrupts the vasculature.
Primary injury triggers the wave of the secondary damage [33].
The secondary damage deals with the cascade of cellular and molecular mechanisms in
response to the primary injury after the traumatic event at the injury site. The secondary damage
could be categorized into four phases based on the chronology – immediate (0-2 hours), acute
Figure 2.3 Anatomy of the spinal cord (a) Spinal cord has multiple ascending and descending axonal tracts either originating or reaching the brain responsible for transmitting functional information (b) Spinal cord is well protected by the spinal column and perfused by the spinal arteries (c) Spinal cord classified into cervical, thoracic, lumbar and sacral segments.
Image modified from Ahuja, CS, et al, Nat Rev Dis Primers, 3: 1017. Apr 2017.
26
(2-48 hours), subacute (48 hours – 2 weeks), intermediate (2 weeks – 6 months), and chronic
(beyond 6 months) (Figure 2.4) [34]. Immediate phase (0-2 hours) starts at the time of the injury
and runs parallel with the primary injury phase after the traumatic event. This includes immediate
death of the neurons and glial cells due to the mechanical disturbance. Moreover, disruption of
the blood vessel membrane leads to the hemorrhage of the parenchyma of the cord, especially
of the gray matter of the spinal cord. As a result, cells immediately start undergoing necrotic cell
death due to local ischemia. Microglial activation due to the hemorrhage upregulates the
proinflammatory cytokines such as IL-1β and TNF-α. These cytokines further increased vascular
permeability through the acute and subacute phase up to about 2 weeks [35]. Extracellular
glutamate levels also increase – in certain cases to levels which are determinantal to the
functioning of neurons [36, 37]. Acute phase (2-48 hours) pathophysiology is very critical to the
clinical management and acute therapies because patients would be presented in the ER room
in this phase [34, 38]. Acute phase is characterized by sustained hemorrhage, progressive
edema, neuroinflammation and resulting ischemia. Increased cell permeabilization permits the
movement of vasoactive peptides, cytokines and inflammatory cells such as neutrophils and
microglia. Endogenous microglia are continually activated while other inflammatory cells such as
astrocytes, neutrophils, T cells and monocytes invade the injury site [39]. These mechanisms
initiate production of free radicals, ionic imbalance and immune-associated neurotoxicity. Ionic
imbalance, especially that of calcium, is the most common reason for the necrotic and apoptotic
cell death. Free radicals released in this phase initiate the process of lipid peroxidation – oxidative
degradation of the lipids - thereby marking the beginning of the demyelination and
oligodendrocyte cell death [23]. Furthermore, glutamate levels keep rising in this phase and are
not reabsorbed by the astrocytes increasing glutamate levels result in excitotoxicity for
surrounding neurons [40, 41]. Continuous hypotension and reduced blood flow cause severe
prolonged ischemia that contributes to cell swelling and overall, swelling of the cord. Swelling
applies more pressure on the peri-lesional healthy tissue and therefore spread the injury through
multiple spinal cord segment [42]. Given that most pathological processes initiate in the acute
phase, most restorative and pharmacological approaches target these pathological processes to
improve functional outcomes and thereby, protecting the further spread of the injury. Several
potential molecules are currently in clinical trials and have shown promise in the initial Phase I
and II clinical trials.
Subacute phase (48 hours – 2 weeks) is characterized by continuation of all the
pathological processes from the acute phase and their consequences. The blood brain barrier is
reestablished while the edema is being resolved at the injury site. The invaded inflammatory and
27
immune cells aid the ongoing the inflammatory response and contribute to the apoptosis of
oligodendrocytes and neurons. Phagocytic cells such as neutrophils and macrophages start to
Figure 2.4 Key pathological events after a contusive spinal cord injury. The pathophysiological process is divided into five phases - (a) - Immediate (0-2 hours) and acute phase (2-48 hours) (b) subacute phase (48 hours - 2 weeks) and (c) Intermediate (2 weeks – 6 months) and chronic phase (beyond 6 months). Reference - Ahuja, CS, et al, Nat Rev Dis Primers, 3: 1017. Apr 2017.
28
clear the debris from the injury site – phagocytic activity is highest in this phase. However, they
also release free radicals, ATP and neurotransmitters that propagate the injury to nearby tissues
[43]. Clearing the myelin debris is believed to be an important process in promoting some axonal
growth because the molecules from the myelin degradation such as myelin-associated proteins
(MAP) and Nogo are inhibitory to the axonal cone regeneration [44]. The clearing of the debris
leaves behind a cystic cavity that would be surrounded by the reactive astrocytes to form a glial
scar. Towards the end of the subacute phase, astrocytes around the cavity become proliferative
and reactive. Most cell-based therapies target their intervention in the subacute phase because
a very dense glial scar is formed in the intermediate phase making it difficult for the exogenous
and endogenous cells to migrate to the injury site [45].
Intermediate phase (2 weeks – 6 months) involves the maturation of the astrocyte-
mediated glial scar and cavity formation. This phase also sees the attempts made by the tissue
to regenerate the severed axons, remyelination and remodeling of the neuronal circuitry.
However, it is insufficient to cause any functional improvement in the patient. Astrocytic glial scar
surrounds the cystic cavity that houses anti-regenerative molecules barrier to the axonal growth.
In the subacute phase and intermediate phase, activated microglia, macrophages and astrocytes
release various ECM molecules such as chondroitin sulfate proteoglycan (CSPG) [46, 47]. These
CSPG molecules along with reactive astrocytes processes and macrophages create a dense
interwoven barrier around the cavity. This barrier is chemically and physically impenetrable by the
surrounding cells or molecules and therefore impedes any regenerative processes such as axonal
cone sprouting and neurite outgrowth [48]. Moreover, Wallerian degeneration continues through
the intermediate phase as well degenerating distal axons. Numerous ECM proteins are laid down
by the fibroblasts and astrocytes, specifically fibrous connective tissue and thick collagen deposits
[49]. Chronic phase (beyond 6 months) witnesses limited Schwann cell remyelination and minor
axonal outgrowth. Neural plasticity plays an important role in regaining of function through
rehabilitation and function-specific training. The perilesional astrogliosis continues to form a
mature glial scar that is inhibitory of any potential regeneration [50]. Wallerian degeneration and
demyelination of the axons continues even in this phase and could last up to one-two years post
injury. Phagocytic processes are still ongoing and it could take similar time for severed axons with
their debris to be cleared from the injury site [51, 52]. In most patients, the lesion has stabilized
but the duration could vary from patient to patient. However, unfortunately, in nearly 30% of the
SCI patients, these lesions do not remain static and further grow into syrinxes, which can cause
delayed neuronal dysfunction and functional impairment. These syrinxes, also known as
29
Syringomyelia, are described by extension of fluid-filled cysts through multiple spinal cord
segments in the chronic phase of the injury [53, 54].
2.1.5. Cavitary lesions – potential for regeneration and recovery: Interestingly,
despite the similar pathophysiological processes, spontaneous regeneration is observed in the
peripheral nervous system to a certain degree. Pre-formed guidance channels and suturing of the
nerves have shown the elongation of axons to appreciate distances to promote recovery [55].
After PNS injury, Wallerian degeneration follows resulting in the disintegration of the distal axons
while the proximal axons sprout, elongate and re-establish synaptic connection to the nerve or
the muscles [56]. Schwann cells remyelinate the axons, guide the axons, secrete growth factors
and synthesize the necessary ECM molecules to rebuild the tissue [57]. However, in CNS, the
equivalent oligodendrocytes are few in number compared to the axons and therefore are not able
to contribute significantly in the myelination of spared axons [58]. In addition, many of the
oligodendrocytes die after the injury reducing the support for axonal regeneration and
spontaneous recovery. In fact, the neural progenitor stem cells are reported to commit to
oligodendrocyte precursor cells after injury [59]. Moreover, the degraded myelin from the severed
axons and demyelinated axons as a result of spinal cord injury release potent growth inhibitors
such as Nogo, myelin-associated glycoproteins, and oligodendrocyte myelin glycoprotein [60].
SCI injury phases Key pathological processes
Immediate phase (0-2 hours)
• Mechanical insult to the cord
• Severing of the axons
• Microglial activation and release of proinflammatory cytokines
• Hemorrhagic necrosis
Acute phase (2-48 hours)
• Edema & ionic imbalance
• Glutamate-mediated excitotoxicity
• Hemorrhage, ischemia and free radical production
• Lipid peroxidation & early demyelination (oligodendrocyte death)
• Neutrophil invasion and activated microglia
• Increased permeability through the blood-brain barrier (BBB)
Subacute phase (48 hours – 2 wks)
• Macrophage infiltration and reactive astrocytes
• Edema resolution but continued cell death
• Start of glial scar formation and repair of the blood-brain barrier
Intermediate phase (2 wks - 6 months)
• Cyst and cavity formation
• Maturation of glial scar
Chronic phase (beyond 6 months)
• Continued demyelination due to Wallerian degeneration
• Functional gain due to spared axons and plasticity
• Spinal cord tissue repair
• Cavity extension into syrinxes and syringomyelia
Table 2.3 Key pathophysiological processes summarized with their chronological phases after a SCI
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These molecules break down the cytoskeleton of the regenerating axonal growth cones inhibiting
their regeneration [61]. Typically in PNS, these molecules are rapidly cleared by macrophages
but in CNS, it takes a long time for microglia to clear the debris [50]. In addition to the inhibitory
molecules, CSPGs and reactive astrocytes create a physical barrier for the which are barriers to
spontaneous regeneration. The pathophysiological response after CNS injury typically leads to
the formation of cyst-filled cavity surrounded by a very dense glial scar by these reactive
astrocytes [62]. There are beneficial and harmful effects of the cavity and the glial scar that is
formed [63]. Glial scar limits the spread of the secondary damage to the nearby healthy tissue
because the cystic fluid contains molecules that are anti-regeneration and would induce injury
processes to the nearby tissue. Therefore, the glial scar protects the neural tissue from ongoing
damage [64]. However, the contents of the cystic fluid and dense nature of the glial scar
constituting chondroitin sulphate proteoglycan (CSPGs) are inhibitory to the migration of any
reparative cells inside the cavity [65]. These are several intrinsic and extrinsic factors that lead to
the failure of regeneration in the CNS.
While there are significant barriers to spontaneous regeneration after SCI, research during
the last three decades have shown promise for repair. In fact, regeneration of numerous tracts
might not be necessary to promote functional recovery after SCI. There are various approaches
that are used to promote regeneration after SCI (Figure 2.5). These include inhibition of pathways
to lead to disintegration of the cytoskeleton of the axonal cones, remyelination of the spared
axons, recruitment of endogenous neural progenitor stem cells to the injury site using chemotactic
factors, implanting exogeneous cells that secrete growth-stimulating factors and re-establishing
neural connections lost as a result of the injury [17]. A number of pre-clinical studies have shown
functional recovery and injury site reorganization after an early intervention before the glial scar
is formed [66-69]. These studies indicate that redundancy in the nervous system and neural
plasticity can organize the re-wiring of the axons to promote recovery. Moreover, some of the
studies employing rat and monkey SCI models have shown axonal sprouting and axonal
regeneration after SCI [70-73]. Various potential therapeutics such as anti-NogoA and Cethrin
target the pathway of growth-inhibitory molecules to promote axonal growth [74, 75] . Therefore,
there is potential for regeneration in the injury site after SCI. If the local environment in the injury
site can be modulated using signaling molecules, if the tissue can be filled with regeneration-
31
promoting stroma and if the injury site is repopulated with the lost cells, there is sufficient evidence
that there is potential in regeneration of the spinal cord tissue following SCI. Since there are no
well-established and validated in vitro models that could be used to study the potential repair after
SCI, most investigations focus on the in vivo investigation of proposed treatment and therapy in
relevant animals models [76]. The choice of animal models is critical to evaluating the type of
therapy and the anticipated outcomes of the treatment.
2.1.6. Pre-clinical SCI models – an overview: SCI models have been pivotal in
investigating the efficacy of various proposed therapeutic interventions and in obtaining useful
information regarding the pathophysiological processes after SCI. The first animal SCI model was
weight-drop contusion model developed in 1911 [77]. Rats are the most commonly used species
for studying SCI because of several factors. Although there are some differences between the
pathophysiological progression of the injury post SCI, they very closely resemble the human SCI
[45]. Rats have demonstrated similar histological, behavioral and electrophysiological outcome
measures compared to humans following SCI [78]. Moreover, rats are easier to handle, relatively
inexpensive and readily available making them a preferred choice over other SCI models.
Although nonhuman primate SCI models better approximate human SCI, rats are preferred in
Figure 2.5 Various neuroregenerative strategies after spinal cord injury. Figure Reference - Ahuja, CS, et al, Nat Rev Dis Primers, 3: 1017. Apr 2017.
32
preliminary proof of principle investigations [79]. Positive findings then drive the investigation into
higher animal models such as primates, pigs and dogs.
Considering the incredibly multisystem complex nature of SCI, there are various SCI
models available for pre-clinical evaluation based on the mechanism of the injury. They are
classified as contusion, compression, dislocation, chemical and transection models. Contusion
models are the most clinically relevant SCI models as most injuries inflict a transient mechanical
force on the cord without breaching the dura [80]. In a typical contusion model, a transient force
is applied on the cord exposed by laminectomy. This mechanical force displaces and damages
the cord. The transient force is applied using several mechanisms – weight-drop method, air
pressure and electromagnetic impactor. Various devices such as NYU-MASCIS, IH impactor,
OSU impactor and air gun impactor have been developed to perpetrate a controlled and
consistent force on the spinal cord [81]. However, the lesion created is not reproducible and lacks
the volume to inject a biomaterial. Moreover, there is technical problems with the clamping
technique especially in the rodent model [82]. Compression models involve compressing the
spinal cord for a prolonged period. Fracture dislocations and burst fractures primarily result in the
compression type injuries. Clip compression model is the most widely used SCI compression
model. One of the foremost advantages of the clip compression model is that it can be adapted
to any region in the cord. Along with clips, balloon and forceps compression models have also
been used. Although compression models are less expensive to implement, the compression
parameters are typically not recorded making it difficult to compare across several studies using
the compression model. Moreover, it does not create a cavity into which a biomaterial can be
injected [83]. Chemically-induced SCI models are different because they were developed to study
the specific facet of the secondary injury mechanism post SCI. Chemically-induced SCI models
are primarily used to evaluate the molecular mechanisms involved post SCI and to evaluate the
effect of proposed therapeutic agents on these molecular mechanisms. Although these are very
specific to the nature of the mechanism, they do not approximate the overall effect of SCI.
Moreover, the location and the method of delivery of these chemicals to induce SCI varies in the
experimental procedures throughout the field. They do not create a reproducible injury for valid
comparison between the groups and with other models [84].
In the context of tissue engineering, transection models have been widely utilized to
investigate the effects of various biomaterials, neurotrophic factors and implanted cells to study
neuronal regeneration in the injury site [85]. Transection models ensures total loss of the neuronal
circuitry at the site of injury making them ideal setups to investigate axonal regeneration and
33
functional recovery. It is important to note that these transection models are not ideal to study the
pathophysiological processes because they are less common in clinical settings [81]. There are
two types of transection models – complete transection and partial transection. Complete
transaction involves complete loss of neuronal circuitry at the site of the injury with disassociation
between the rostral and caudal segments of the injury. Moreover, complete section models better
mimic the most severe clinical ‘complete SCI’ inducing complete break of the neuronal circuitry
as observed in most violence-related SCIs such as gun and knife wounds [86]. The most important
advantage of the complete resection model is the creation of a reproducible standardized defect
at a pre-defined location on the cord. In addition, complete resection is a model that has been
used to study the ‘bridging gap’ therapies such as scaffolds and similar devices [87]. Partial
resection is yet another type of transection model that has been applied in various ways. One
method involves severing of a particular spinal tract of interest. Specifically, transection of
corticospinal tract has been widely studied using the partial transection SCI model in mice, rats
and monkeys [88-90]. The second method involves severing of all the axons in one half of the
cord known as hemi-resection SCI model. This type of model allow for comparison with the
contralateral side for the effects of the proposed treatment [91]. Overall, these surgical excision
models are ideal for studying the outcome of injectable biomaterial therapies because it creates
a standardized unambiguous defect at the pre-defined location in the cord. Additionally, the cavity
provides a voluminous injection of injectable gels proposed to replace the stroma of the injured
site. In addition, these models offer advantage to visually confirm the injection of the injectable
therapeutics into the cavity, which is not achievable with any other pre-clinical SCI models. As
discussed, these models are widely used in all animal models from mice and rats to pigs and
monkeys. The typical outcomes measures for transection models include behavioral assessment,
imaging and histological evaluation of the injury site along with the immunohistochemical
investigation. For the advantages that transaction models provide to study axonal regeneration
and functional recovery as a result of biomaterial-based intervention, we will employ both 2-mm
complete and hemi-resection rat SCI model to investigate the effect of our proposed treatment –
injectable gelatin-based matrix incorporating epidermal growth factor (EGF) and stromal cell-
derived factor-1α (SDF-1α). However, prior promising work in the context of tissue engineering
approaches to treat SCI commends a review.
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2.2. Tissue engineering approaches in SCI
2.2.1. Three pillars of tissue regeneration – an overview: Tissue engineering has shown
great promise in providing a systematic approach to promote regeneration in the injured tissue. It
is an extremely interdisciplinary field that has applications cutting across various industries
including food industry. Tissue engineering utilizes biomaterials, biochemical signals, physical
signals and cells, alone or in combination, to generate tissues that mimic the native tissue [92].
Clinically, organ transplantation is the gold standard to replace the ailing organ. However, limited
availability and rejection has led to finding alternatives. Early applications of tissue engineering
aimed at maintaining, restoring and improving the function of damaged tissue has been
encouraging in various organ systems [93]. Considering these advances, tissue engineering
approaches have become popular clinically as well. Traditional medical tools are not capable of
providing a functional restoration after injuries, especially in case of CNS injuries. The first tissue
engineered skin products were introduced in 1970s and 1980s [94]. Tissue engineering typically
involves a triad – three pillars for regeneration of the tissue. Scaffolds, signals and cells form the
‘central dogma’ of the field of tissue engineering. Depending upon the goal of the specific
application and the complexity of the tissue in consideration, one or all three approaches in
combination are essential for success in tissue engineering [95].
In the context of SCI, all three pillars are essential because of the complex organization
of the spinal cord tissue. Post SCI, there is a significant cell death of different types of cells. As a
result, signaling molecules that promote regeneration and repair the tissue are ineffective.
Moreover, the stroma is completely lost due to pathological process post injury. Therefore, all
three aspects are critical to promote regeneration in the spinal cord after injury. In fact, all three,
either alone or in combination, have proved useful in promoting regeneration and functional
recovery after SCI [96]. Historically, however, regeneration was thought to be impossible in CNS.
In ‘Edwin Smith Papyrus’ book, an Egyptian medical textbook dating 1700 BC, SCI was thought
to be ‘ailment not to be treated’ [97]. As a result of Wallerian degeneration, distal nerves
degenerate and lose their connection to the target. Therefore, true regeneration involves three
major steps – 1) axons proximal to the injury has to grow and infiltrate through the cyst-like
environment at the injury site, 2) axons has to grow all the way to the correct target, and 3) axon
has to synapse correctly at the target. In addition, new glial cells have to establish contact with
nerve axons for true regeneration. Currently, research in SCI regeneration is at the initial stages.
In 1981, David, et al, showed that it was indeed possible to achieve significant axon elongation
using a peripheral nerve graft in SCI if a permissive environment is provided [98]. Researchers
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have come a long way in SCI regeneration by means of three pillars of Tissue engineering –
scaffold, soluble factors and cells.
2.2.2. Scaffolding materials for SCI: Various biomaterials, both individually or in
combination with factors and cells, have been tested for promoting regeneration and repair post
SCI. Biomaterials provide passive structural support, bioactive modulation of matrix, delivery
vehicle for factors and attachment surface for cells. In addition, cystic cavitation created as a
result of the pathophysiologic progression completely destroys the stroma in the injured tissue,
thereby inhibiting any support to potentially growing axons post SCI. Therefore, biomaterials have
emerged as a novel strategy to fill the cavitary lesions to provide a multifunctional substitute of
extracellular matrix molecules [99]. Generally, natural polymers such as collagen, laminin, PLGA,
and chitosan have been widely researched with cells and factors, which have shown decrease in
the cyst volume, increase in angiogenesis and axonal regrowth [99, 100]. For example, when
equine collagen alone was implanted in hemi-resection SCI model, it showed increase in the
number of axons [101]. Usually, pre-formed scaffolds are not suitable for SCI due to large variation
in injury size and volume. So, injectable gels offer a minimally invasive novel way to deliver
biomaterial matrix accounting for injury specifics. A detailed introduction to the various
biomaterials evaluated to promote SCI follows.
2.2.2.1. Natural vs. synthetic materials: Prior to evaluating a material for scaffold
fabrication for SCI, consideration need to be made whether to employ a natural or a synthetic
material. Natural materials offer a range of advantages over the use of synthetic polymers. First,
natural polymers consist of moieties such as RGD sequences that can aid in cell adhesion. In
addition, viability of cells grown on natural biomaterials is generally higher than that of synthetic
polymers [102]. Moreover, they can be designed to match the stiffness of the host tissue and are
degradable by the body [103]. Natural materials typically have high water content aiding in cellular
migration, diffusion of particles and molding into various forms [99]. In addition, they are readily
available from various sources such as plants and animals. However, one of the limitations of
using natural biomaterials is a potential transmission of diseases from the host source. Usually,
natural materials provide minimal control on the important properties such as degradation rate
and mechanical properties. Although synthetic biomaterials provide various advantages such as
easily controlled customized mechanical properties and easier sterilization techniques, they are
usually non-biocompatible eliciting a severe immune reaction. Moreover, they would need to be
functionalized extensively to gain ligands for cell adhesion. Considering the complex nature of the
injury site in SCI, synthetic polymers would induce further secondary damage would not be
36
beneficial. Therefore, most spinal cord applications utilize natural biomaterials to provide a
supportive and bioactive scaffold for cellular migration and proliferation post SCI [104-106].
2.2.2.2. Pre-formed vs. injectable biomaterials: Another consideration to be made is
whether a pre-formed scaffold or an injectable biomaterial would be beneficial and feasible for
SCI. Typically, contusion type injuries are most common in clinical setting. These contusions
create an irregularly shaped cavity heterogeneous in both shape and the volume. Pre-formed
scaffolds of specific size and dimensions would not conform to the type of cavitary lesions
observed in the spinal cord. Although pre-formed scaffold such as guidance channels have shown
great promise in pre-clinical models in several SCI studies using both natural and synthetic
polymers, it is becoming increasing evident that injectable biomaterials are more feasible to
implant the biomaterial in a minimally invasive manner to minimize additional damage to the
unaffected spinal cord tissue [107]. Moreover, injectable gels can conform to the irregularly
shaped defect and can completely fill the defect to build a better interface with the surrounding
host tissue – extremely useful in the context of CNS injuries. In addition, injectable gels can also
be functionalized to deliver encapsulated factors and transplant cells in the injury site. Although
there is reduced control on the porosity of the injectable gels, the materials can be chosen such
that it can be degraded by the migratory cells by releasing ECM enzymes such as matrix
metalloproteinases (MMPs) [108, 109].
2.2.2.3. Injectable biomaterials for SCI: Various injectable biomaterials have been
developed and designed to be evaluated in pre-clinical SCI models. Transaction and contusion
models are typically utilized to be able to inject into a cavity created by these models [110].
Collagen-based injectable biomaterials are probably the most widely studied in the context
of SCI. Collagen is a naturally occurring most abundant ECM molecule [111]. It has highly tunable
mechanical properties to be fabricated into an injectable gel. The gelation of collagen-based
scaffolds could depend upon the pH, temperature and the chemical cross-linker, if any [112]. In
the context of SCI repair, it has been utilized as hydrogel, aligned fibers, drug-delivery vehicle,
and cell-transplantation substrate [113]. Considering that collagen degrades relatively quickly
compared to the timeline for axonal regeneration in SCI, it is usually formulated with other
biomaterials or linkers to reduce the degradation rate. In a study by Marchand et al, self-
assembling glyoxal-linked collagen gel into a thoracic hemi-resection model. Collagen-glyoxal gel
persisted for 8 weeks in the injury site. However, glyoxal induced an inflammatory response.
Nevertheless, axonal regeneration was observed in the treatment groups three months are
injection into the hemi-resection cavity [114]. In a recent study, Li et al implanted a collagen
37
scaffold with cetuximab in the lower thoracic 4-mm hemi-resection model. They reported that
collagen-cetuximab scaffolds encouraged neuronal differentiation while decreasing the
differentiation of the NPCs into astrocytes. Overall, the scaffolds promoted axonal regeneration
and functional recovery 12 weeks after surgery [115].
Laminin is a glycoprotein that has capability to self-assemble by controlling the pH,
temperature and concentration [116]. Laminin signals through a wide variety of integrins and
therefore has been shown to induce neurite outgrowth in several studies [117, 118]. A study by
Menezes et al acutely injected polylaminin in the injury site. The results showed improved motor
function in three different SCI models - partial transaction, complete transection and compression.
Moreover, neurons retrogradely labeled were identified in the spina cord tissue near the injury
indicates axonal growth through the injury site. In addition, laminin also contributed to mitigate the
inflammatory response after the injury.
Fibronectin is another glycoprotein evaluated for SCI repair. Since fibronectin is not
capable of undergoing gelation on its own despite environmental modifications, it is usually
combined with other ECM molecules such as fibrin to fabricate a long-lasting scaffold. In a study
by King et al, combination of fibrin and fibronectin was used to fabricate an injectable hydrogel.
Fibrin is shown to degrade very quickly in vivo as early as one week [119]. However, the
combination gel persisted for four weeks after injection into a dorsal hemi-resection cavity. By one
week, the gel showed good integration with the surrounding tissue and promoted deposition of
laminin. After four weeks, the combination gel supported growth of axons and invasion of
Schwann cells and blood vessels in the injury site. Moreover, the combination gel showed greater
viability of neurons compared to the controls and gels alone [120].
Hyaluronan/hyaluronic acid is a naturally occurring non-sulfated glucosamine in the CNS
[121]. Given its greater degradation rate, it is typically combined with other factors and materials.
For example, hyaluronic acid was cross-linked with different concentration of thiols in a in vitro
and in vivo evaluation by Horn et al. Although there were promising findings in vitro of increased
neurite outgrowth in these gels compared to the fibrin, there were no differences among any
assessment parameters in vivo [122]. However, another study that cross-linked hyaluronic acid
with IKVAV peptide delivering BDNF showed neuronal regeneration after six weeks in a clip
compression model of SCI. These hydrogels were injected into the intrathecal space near the
injury. Compared to all other groups, animals injected with BDNF-containing hydrogels showed
the greatest improvement in the functional recovery assessed using the well-established BBB
scale [123].
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Few non-mammalian biomaterials such as agarose, chitosan, and alginate have been
studied in few studies for treatment of SCI. Few studies have shown neuronal survival in vitro and
promotion of potential axonal regeneration in vivo, however to a minor degree [124, 125]. For
example, BDNF-loaded microtubules were prepared to be delivered using an agarose gel in a
hemi-resection models of SCI in a study by Jain et al. The agarose gel was persistent even after
6 weeks of injection in the injury site. Although there were some encouraging findings of reduced
inflammatory response and presence of neurons at the border outlining the defect, it did not show
axonal regeneration even after eight weeks. The authors hypothesized that the persistence of
agarose after eight weeks was impinging on the new tissue formation [124]. There are two major
limitations of these polymers – they typically elicit a inflammatory response due to non-
biocompatibility and do not have the ligand-associated interactions with the host tissue [99]. Given
a wide range of injectable biomaterials available and evaluated for spinal cord repair, certain
criteria must be established for ideal injectable biomaterial for SCI.
2.2.2.4. Criteria for biomaterials for cavitary lesions: Several criteria in designing an
ideal biomaterial for cavitary lesions of the spinal cord are listed based on the desired
characteristics for SCI repair [103, 109]. This comprehensive list was based on review papers
and discussions from our laboratory. Figure 2.6 gives a good schematic view of the considerations
for biomaterial apt for SCI repair.
1) It should be biocompatible without inducing more than typical inflammatory response
2) It should be injectable so that it can be delivered to the injured cavity in minimally invasive
manner to abate surgical damage during implantation
3) It should be able to gelate in situ to conform to the heterogeneous spinal cord cavities
4) It should be non-swelling not to apply pressure to the surrounding tissue, which is known to
initiate further secondary damage after the injury
5) It should have similar mechanical properties as that of the spinal cord to minimize secondary
damage and non-shrinking so that it can integrate well with the surrounding tissue
6) The constituents of the biomaterial should not induce cytotoxicity in the tissue
7) It should serve as a viable replacement of the disappearance of stroma lost due to SCI and be
permissive of cellular migration, proliferation and differentiation into the defect site to promote
neural repair
8) It should have tunable physical properties to control gelation time, stiffness and degradation
characteristics. Specifically, for experimental models, it should be able to complete gelation in
two-four minutes to visually confirm the gelation in the surgical hemi-resection model
39
9) It should have chemical moieties such as RGD sequences as ligand for cell integrins
10) It should be able to persist in vivo for timeline of our experiment (i.e. four weeks) and longer
11) It should be able to incorporate and locally deliver signaling molecules, growth factors, and
cells in the injury site without the particular use of vehicles
In the context of this work, we believe a gelatin-based matrix – gelatin-hydroxyphenyl
propionic acid (Gtn-HPA) meets all the desired criteria. A detailed discussion of the properties
and benefits of Gtn-HPA is presented in Chapter 3.
Figure 2.6 Schematic of various properties of a desired scaffold for SCI repair
Reference – Straley et al J Neurotrauma. 2010 Jan; 27(1): 1–19.
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2.2.3. Biomolecules for SCI – an overview: In addition to replacing the cystic cavity and
degraded stroma in the injured spinal cord tissue with injectable biomaterials, biomolecules are
locally delivered in the injury site. Biomolecules play a variety of role after SCI. These factors can
enhance survival of the neurons, promote myelination of spared axons, alter the phenotype of
stem cells to differentiate into neuronal and glial fate, promote axonal regrowth and promote
plasticity [126]. Moreover, these factors can induce the migration, proliferation and differentiation
of endogenous and exogenously transplanted cells in the injury site. In additions, few
biomolecules aid functional recovery by means of modifying the ECM and the glial scar. These
factors, alone or in combination with scaffolds and cells, have been shown to improve
morphological and functional outcomes in pre-clinical models of SCI [127].
2.2.3.1. Neurotrophins and growth factors: A wide range of growth factors have been
evaluated in the spinal cord models to assess the therapeutic potential in promoting functional
recovery and regeneration in the SCI. Some of these have been or are currently being evaluated
in clinical trials [128].
Brain-derived neurotrophic factor (BDNF) is one of the most extensively studied factors in
pre-clinical models of SCI. BDNF has been reported to play a role in neuroprotection, axonal
sprouting, neurogenesis, remyelination and adaptive plasticity after SCI [129]. In a study by
Blesch et al, administering BDNF in a transient manner was shown to increase the number of
spared axon and induce regrowth of the axons in the injury site indicating that sustained delivery
of these factors is essential [130]. Moreover, when BDNF was delivered using collagen matrix,
improved functional recovery along with axonal regeneration of CST axons was observed by Han
et al in a complete transection model of SCI [131]. Several studies have shown that BDNF
increases the myelination of axons and confers neuroprotection to the healthy spinal cord tissue
in rat or cat models [132, 133].
Another neurotrophin to be widely studied in experimental SCI models is NT-3. Typically,
NT-3 is delivered near the injury site acutely. It has been delivered with a wide range of scaffolds
ranging from collagen, fibrin and chitosan to hydrogels [127]. Most studies show axonal sprouting
in the injured axons after injection of NT-3 at the injury site mostly in the thoracic models of SCI
[134]. In a study by Paiantino et al, an injectable hydrogel incorporating NT-3 was implanted in
the 1.5-mm hemisection model of SCI. The results showed increased axonal growth of CST axons
and better functional recovery in the treatment groups compared to the controls [135]. In addition,
a number of studies showed myelinating and neuroprotective effects of NT-3 suggesting NT-3 to
be a promising biomolecule to promote repair in the injured spinal cord tissue after SCI [136, 137].
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Various other notable neurotrophins and growth factors include nerve growth factor (NGF),
NT-4/5, fibroblast growth factor (FGF), Epidermal growth factor (EGF), Glial cell-derived
neurotrophic factor, IGF-1 and platelet-derived growth factor (PDGF) [127]. Martens et al pursued
an in vivo investigation in a mouse model where they injected a combination of EGF and FGF-2
in the fourth ventricle in the brain and central canal of adult male CD1 mice. Just six days post
injection of EGF and FGF, greater proliferating cells were observed in both the cervical spinal
cord and near fourth ventricle in the brain. Specifically, in the spinal cord, these cells were in the
ependymal and subependymal tissue closer to the central canal. Indeed, these cells were nestin
positive indicating the presence of neural progenitor stem cells. The results from this study
indicated that endogenous neural progenitor stem cells near the central canal in the spinal cord
proliferate in response to EGF and FGF-2 [138]. In a more comprehensive in vivo study by
Hamann et al investigating the same EGF and FGF-2 combination in a clip compression rat
model, EGF and FGF-2 were delivered in a highly concentrated collagen solution to localize the
growth factors in the injury site. Interestingly, they labeled the injected EGF and FGF-2 with
markers. The results indicated that EGF readily penetrated the injury site in the spinal cord while
FGF-2 was predominantly observed in the dura. Furthermore, significantly less cavitations were
seen in the growth factors group indicating that EGF promoted tissue sparing including greater
white matter sparing in the injury site. Moreover, as reported by Kojima et al, they also observed
greater BrdU+ cells in the EGF and FGF-2 group than the controls in the ependymal region [69].
A study by Johnson et al investigated the combined effect of PDGF and NT-3 delivered using a
fibrin scaffold in subacute model of SCI. These increased the migration of SCIs to the injury site
and promoted differentiation of SCIs into neurons [119]. Despite abundant studies with many
factors, ideal combination of the factors needs to be tailored to the anticipated outcome after SCI.
2.2.3.2. ECM and scar modifying molecules: Chondroitinase ABC (chABC) is an
enzyme that breaks down the chondroitin sulphate proteoglycans (CSPGs) that form the physical
barrier around fluid-filled cystic cavity. Therefore, to promote migration of factors and endogenous
cells into the cavity site, chABC, alone or in combination, has been evaluated in various spinal
cord models [139]. In a recent canine SCI model study by Hu et al, administration of chABC
significantly improved the forelimb and hindlimb coordination after 6 months compared to the
control group [140]. Moreover, Alluin et al investigated a combinatorial approach of delivering
chABC with cocktail of factors containing FGF-2, EGF and PDGF-AA in a clip spinal compression
model. This combination was injected into the injury site via subarachnoid catheter connected to
an osmotic pump. The combination showed reduced GFAP presence in the perilesional area
indicating reduction of astrogliosis near the injury site. Interestingly, even the macrophages
42
response was attenuated in the defect area due to the administration of the combination of chABC
and growth factors. One of the key findings was collateral sprouting of the corticospinal tract using
BDA tracing. There was greater spared tissue observed in the cord due to implantation of the
chABC with growth factors compared to the controls. Lastly, this intervention also shown improved
functional recovery suggesting augmentation of neuronal plasticity [141]. Overall, biomolecules
with different effects modulate the injury site environment and are critical to regeneration in SCI.
In addition to biomolecules, repopulating the injury site with exogeneous cells has also proven to
be beneficial as described next.
2.2.4. Cell-based treatment for SCI – an overview: Cell implantation could confer notable
activities in the injury site that could promote neural regeneration and functional recovery. First,
cells can secrete neurotrophic factors at the injury site to initiate signaling pathways that can lead
to neural recovery. Second, cells can synthesize extracellular matrix to replace the lost stroma
due to the pathophysiological cascade post injury and to provide a scaffold to regenerating axons.
Third, cells can repopulate the lost cell types such as neurons, oligodendrocytes and endothelial
cells. The cells to be implanted should meet general criteria as follows – cells should show
proliferative ability in vitro, should exhibit good viability and function retention, should be safe for
injection meaning they should not elicit any responses that can lead to cancer-like tissue formation
and should be readily available or isolated using established procedure [142]. Different types of
cells meet the general criteria and have been studied extensively for treatment of SCI.
Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are an attractive
choice for cell implantation because they have the ability to differentiate into various neural cell
types that include oligodendrocytes, astrocytes and functional neurons [143]. Both cell types have
shown tremendous therapeutic benefit in vivo after SCI. In an interesting experiment, Bottai et al
injected non-differentiated ESCs through the tail vein acutely in mouse SCI model. They observed
a significant improvement of functional recovery BBB scores in the treatment groups compared
to the controls. Moreover, they reported reduction in the number of invading macrophages and
neutrophils suggesting that the cells were able to modulate inflammatory response post SCI. The
authors also speculated that the injection of ESCs could potentially preserve greater spared tissue
through reducing the inflammatory response. No teratoma formation was observed in the injury
site in the treatment groups [144]. Besides injecting ESCs or iPSCs directly, another strategy is
to transplant cells of specific fate derived from them. For example, Niapour et al implanted ESC
derived neural stem cells (NSCs) along with Schwann cells (SCs) in a contusion model of SCI.
They hypothesized that ESC-derived NSCs would lead to greater neuronal differentiation. Their
43
results showed significant motor function recovery due in the experimental groups when
compared with the controls. Moreover, experimental groups showed significantly increased
expression of TUJ1 and MAP2 biomarkers, and reduced GFAP signal five weeks post
implantation. Another strategy is to transplant the cells in a multifunctional scaffold. Hatami et al
employed a rat hemi-resection model to implant ESC-derived NPCs encapsulated in collagen
scaffolds into the cavity created by hemi-resection. They showed that transplanted ESC-derived
NPCs differentiated into neurons and glial cells in vivo. Moreover, the intervention promoted
significant hindlimb functional recovery and sensory responses in the groups treated with cells
compared to the controls [145].
Mesenchymal stem cells (MSCs) are yet another preferred cell types for SCI studies
considering that they could be readily isolated from the patients and preserved with minimal loss
in the potency [146]. MSCs have been shown to differentiate into all the cell types of neural tissue
under preferred differentiating medium [147]. Moreover, transplanted MSCs are capable of
guiding axonal growth, promote axonal cone growth, mitigate the extent of demyelination and
reduce anti-inflammatory molecules in the injury site in SCI [148]. One goal for using MSCs for
SCI is to mitigate the inflammatory response thereby conferring greater neuroprotection to the
unaffected tissue. For example, Nakajima et al transplanted bone-marrow derived MSCs (bMSCs)
in a rat contusion model and evaluated their effect up to five weeks post transplantation. After
transplantation of bMSCs into the contusion epicenter, the bMSCs significantly upregulated the
levels of IL-4 and IL-13 while downregulated the levels of TNF-alpha and IL-6. These processes
led to phenotype change in macrophages from M1 to M2. The authors suggest that this
mechanism led to more preserved axons, greater myelin sparing and reduced scar formation.
Moreover, the treatment groups also exhibited better locomotion recovery compared to the
controls [149]. However, there was no indication of differentiation of these MSCs into neurons –
a limitation observed in several studies [150, 151]. MSCs have been shown to secrete various
growth factors such as GDNF and NGF, guide axonal elongation, decrease demyelination and
promote axonal regeneration [152]. Moreover, MSCs have been shown to test positive for
neuronal markers NeuN confirming neuronal differentiation [153]. Also, Cho et al showed that
MSCs were able to improve functional recovery and neural regeneration along with neural
differentiation [154]. No adverse effects were reported when bone-marrow derived MSCs
(bmMSCs) were injected into rats in various SCI models [152, 155].
Neural stem cells (NSCs) have been found to exist in the subependymal layer around the
central canal and meninges in spinal cord. Moreover, NSCs are capable of self-renewal and
44
generating various neural fate cells such as neurons, oligodendrocytes and astrocytes both in
vitro and in vivo. Therefore, NSCs are thought to be important for regeneration after SCI [156,
157]. NSCs from human fetal spinal cord were grafted into the lumbar cord of adult nude rats by
Yan et al. They reported differentiation of NSCs into neurons, axon regeneration, and formation
of synapses with host motor neurons. Interestingly, the differentiated neurons from NSCs were
able to integrate with host neuronal circuitry pointing to the potential axonal regeneration and
recovery of function at anatomical level [158]. In yet another interesting approach using NSCs by
Yasuda et al, NSCs were preconditioned with the ability for remyelination. The results were
significant and observed that at the end of experiment, the remyelination potential of NSCs
increased and was probably linked with the observed improved motor and electrophysiological
functional recovery in treatment groups [159]. In a very recent study, neural stem cells (NSCs)
enriched with interneurons were transplanted at T3-T4 level of spinal cord. They showed that the
transplanted cells survived, differentiated and integrated with the host spinal cord tissue at the
injury site. Electromyography results suggested improved recovery one month post the
implantation. Moreover, animals that received donor cells showed significantly greater motor
functional improvement than the controls and point to the important role of NSCs in re-establishing
neuronal circuits in the injured adult spinal cord [160]. In the context of our work, we propose to
deliver EGF and SDF-1α to recruit the endogenous NSCs at the injury site to promote functional
recovery and regeneration after SCI. It has been shown that EGF induces migration and
proliferation of NSCs both in vitro and in vivo [161, 162].
Olfactory ensheathing cells (OECs) and Schwann cells (SCs) have also been transplanted
in SCI models and have shown to promote neuroprotection, expression of the neurotrophic
factors, remyelination and axonal regeneration [152]. Richter et al demonstrated remyelination,
reduced scar formation, and better axonal regeneration after transplantation of lamina propria-
derived OECs (LP-OECs) in a T3 compression SCI model [163]. Moreover, Zhang et al. also
transplanted LP-OECs in a chronically contused SCI model. They observed greater axonal
regeneration, remyelination and reduced lesion volume after scar ablation. However, they did not
observe any improvement in the motor functional recovery [164]. Since Schwan cells play a critical
role in promoting regeneration in the peripheral nervous system, SCs have been transplanted in
the SCI models to promote remyelination of the spared axons and improvement in the functional
recovery [57]. An interesting approach was taken by Agudo et al by transplanting the Schwann
cell precursors (SCPs) immediately after inducing a crush SCI. The authors presented that they
observed proliferation of SCPs and SCPs were instrumental in promoting angiogenesis.
Remarkably, the transplanted groups with SCPs had reduced glial scar formation. Moreover,
45
anterograde tracing revealed regeneration of BDA-labeled CST axons beyond the injury site
[165]. Overall, cells transplanted in the lesion cavity especially in scaffolds offer an innovative
approach to repair and regeneration after SCI. Given the complexity of the spinal cord tissue after
an injury, a combinatorial approach with all three pillars of tissue engineering should be evaluated
for synergistic effects of the treatment.
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2.3. References
1. Facts and Figures at a Glance, in SCI Data Sheet. 2018, University of Alabama at Birmingham: National Spinal Cord Injury Statistical Center. p. 2.
2. Chan, M., International Perspectives on Spinal Cord Injury. 2013, World Health Organization. 3. Spinal Cord Injury. 2014 11/2013 [cited 2014 12/19]; Available from:
http://www.who.int/mediacentre/factsheets/fs384/en/. 4. McKinley, W.O., et al., Long-term medical complications after traumatic spinal cord injury: A
regional model systems analysis. Archives of Physical Medicine and Rehabilitation, 1999. 80(11): p. 1402-1410.
5. Hagen, E.M., Acute complications of spinal cord injury. World Journal of Orthopedics, 2015. 6(1): p. 17-23.
6. Munce, S.E., et al., Impact of quality improvement strategies on the quality of life and well-being of individuals with spinal cord injury: a systematic review protocol. Syst Rev, 2013. 2: p. 14.
7. Rouanet C, R.D., Rocha E, Gagliardi V, Silva GS, Traumatic spinal cord injury: current concepts and treatment update. Arquivos de Neuro-Psiquiatria, 2017. 75(6).
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165. Agudo, M., et al., Schwann cell precursors transplanted into the injured spinal cord multiply, integrate and are permissive for axon growth. Glia, 2008. 56(12): p. 1263-70.
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Chapter 3:
Rationale and development of the
formulation of Gtn-HPA matrix
formulation with EGF and SDF-1α for
spinal cord injury rat model evaluation
55
3.1. Biomaterial: Gelatin-hydroxyphenyl propionic acid (Gtn-HPA)
3.1.1. Introduction: Various biomaterials, both individually or in combination with factors
and cells, have been tested for promoting regeneration and repair post SCI. Biomaterials provide
passive structural support, bioactive modulation of matrix, delivery vehicle for factors and
attachment surface for cells. Endogenous cells such as neural stem cells (NSCs) and glial cells
need a supportive matrix to migrate into the defect. Therefore, natural polymers such as collagen,
laminin, PLGA, and chitosan have been widely researched with cells and factors, which have
shown decrease in the cyst volume, increase in angiogenesis and axonal regrowth [1, 2]. For
example, when equine collagen alone was implanted in hemi-resection SCI model, it showed
increase in the number of axons [3]. Generally, pre-formed scaffolds are not suitable for SCI due
to large variation in injury size and volume. So, injectable gels offer a minimally invasive novel
way to deliver biomaterial matrix accounting for injury specifics conferring greater preservation of
the spared tissue.
The general design criteria of the injectable matrix for SCI applications could be as follows:
1) can undergo gelation in vivo within 1-2 minutes; 2) modulus matches that of spinal cord; 3)
have tunable physical properties to control degradation rate; and 4) should be able to persist for
timeline of our experiment and longer [4]. There various hydrogels that are based on the HRP-
based crosslinking mechanism by decomposing hydrogen peroxide (H2O2) that offer independent
tuning of properties of the gel [5, 6]. Considering these criteria, we propose to use a conjugated
gelatin (Gtn)-hydroxyphenyl propionic acid (HPA) gel that is capable of being cross-linked by
peroxidase and hydrogen peroxide, which allow for independent tuning of rate and degree of
cross-linking, respectively. This independent tunability is essential in obtaining an optimal
degradation rate, and in matching the mechanical properties (shear modulus) of the gel and of
the spinal cord tissue [7]. Moreover, arginine-glycine-aspartic acid (RGD peptide sequences) are
important moiety for cell attachment, cell anchorage, cell spreading and cell signaling [8]. Since
gelatin-based hydrogels such as Gtn-HPA comprises of abundance of RGD sequences that aid
in establishing ECM-like material for the migrating cells, Gtn-HPA is a promising biomaterial for
tissue repair applications [9].
3.1.2. Synthesis of Gtn-HPA: Gtn-HPA hydrogels were developed by Wang et al with the
intent to be utilized as scaffolds for tissue engineering applications [10]. Fabricating Gtn-HPA gel
is two step procedure: First, a lyophilized conjugate of gelatin (Gtn) and 3-(4-hydroxyphenyl)
propionic acid (HPA) is prepared from raw materials (Figure 3.1A). Second, Gtn-HPA matrix is
prepared by covalently cross-linking the Gtn-HPA conjugate with horseradish peroxidase (HRP)
56
and hydrogen peroxide by an HRP-catalyzed crosslinking mechanism (Figure 3.1B). The gelatin
content, HRP & H2O2 concentration is chosen based on the application and desired properties of
the Gtn-HPA hydrogel in the second step.
Synthesis of Gtn-HPA conjugate: Gtn–HPA conjugates were prepared by a general
carbodiimide/active ester-mediated coupling reaction in distilled water. 3.32 g of HPA was
dissolved in 250 ml of mixture prepared with 3:2 ratio of distilled water and N,N-
dimethylformamide (DMF) respectively. To this, 3.20 g of N-hydroxysuccinimide and 3.82 g of 1-
ethyl-3-(3-dimethylaminopropyl)-carbo-diimide hydrochloride were added. The reaction mixture
was stirred at room temperature for 5 hours, and the pH of the mixture was kept at 4.7. Then, 150
ml of 6.25 wt% gelatin aqueous solution was added to the reaction mixture and continuously
stirred overnight at room temperature at pH 4.7. Subsequently, the solution was dialyzed against
Figure 3.1 Schematic of the two-step fabrication of Gtn-HPA gel (A) A non-crosslinked Gtn-HPA conjugate is prepared using gelatin (Gtn) and 3-(4-hydroxyphenyl) propionic acid (HPA). Gtn-HPA conjugate can be lyophilized to be used for later experiments (B) Gtn-HPA gel is prepared by enzymatic HRP-catalyzed crosslinking mechanism using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). Figure courtesy – Hu et al Biomaterials, 2009. 30(21): 3523-3531
57
100 mM sodium chloride solution for 2 days, 25% ethanol in distilled water for 1 day and distilled
water for 1 day, successively. The purified solution was lyophilized to obtain the Gtn–HPA
conjugate [10]. Lyophilized Gtn-HPA conjugate was obtained from Dr. Motoichi Kurisawa, Agency
of Science, Technology and Research (A*STAR), Singapore.
Synthesis of Gtn-HPA hydrogels: Gtn-HPA gels were prepared by first preparing a Gtn-HPA
conjugate solution. In this example, procedure to prepare 8% Gtn-HPA is discussed (Table 3.1).
12% Gtn-HPA solution was prepared by adding PBS to the lyophilized Gtn-HPA conjugate to a
final concentration of 120 mg/ml in PBS. The resulting solution was kept at 37 °C for an hour for
complete dissolution of the Gtn-HPA conjugate to prepare a homogeneous 12% Gtn-HPA
conjugate solution. 12% Gtn-HPA, 1x PBS, HRP and H2O2 was added in volumetric ratio of
20:8:1:1 to a final concentration of 8% Gtn-HPA, 0.1 U/ml HRP and 6.8 mM H2O2 in the specified
order. HRP and H2O2 solutions were kept at 4°C and away from the direct light.
3.1.3. Mechanical properties of Gtn-HPA: Mechanical properties should be considered
while developing an injectable hydrogel for in vivo applications because modulus mismatch has
been shown to induce strains at the interface impeding integration of the gel with the native tissue
[11]. Therefore, one of the noteworthy advantages of the Gtn-HPA gels is that the mechanical
properties are tunable based on the concentration of H2O2 and HRP used for synthesis of the
Gtn-HPA gel. Prior work in our laboratory showed that the gelation rate and shear modulus of the
Gtn-HPA matrix is independently tunable by controlling the concentration of HRP and H2O2
respectively (Figure 3.2AB) [12]. Moreover, concentration of H2O2 and the gelatin content of the
Gtn-HPA affects the degradation rate of the Gtn-HPA gels – an important property for the tissue
engineering applications (Figure 3.2C). Greater the H2O2 concentration, higher is the stiffness and
therefore slower is the rate of degradation of the Gtn-HPA gel. Increase in the concentration of
HRP reduces the gelation time. These studies and parameters were important in determining the
final Gtn-HPA formulation that was evaluated in vivo in the SCI models for this thesis work.
Formulation Gtn-HPA HRP H2O2 Gelation time and estimated modulus
8% Gtn-HPA 80 mg/ml 0.1 U/ml 6.8 mM ~ 3 min & ~ 2000 Pa
12% Gtn-HPA 120 mg/ml 0.1 U/ml 10.2 mM ~ 4 min & ~ 3000 Pa
Table 3.1 Gtn-HPA formulation with their constituent concentrations
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3.1.4. Prior in vitro studies with Gtn-HPA: Given the promise of Gtn-HPA hydrogels with
valuable tunable properties, various in vitro evaluation of the Gtn-HPA with different cell types
have assessed the permissiveness of the Gtn-HPA for cellular migration and effect on the fates
of the cells cultured onto the 2D or 3D Gtn-HPA hydrogels. Wang et al reported that the stiffness
of the Gtn-HPA gel strongly directed cell adhesion, proliferation and differentiation of the human
mesenchymal stem cells (hMSCs) for both 2D and 3D cell culture [10, 13]. The stiffer Gtn-HPA
showed greater spreading area, more stable cell attachment, faster migration and better
organized cytoskeleton than the softer Gtn-HPA gel. hMSCs cultured on the softer Gtn-HPA gel
were reported to express neurogenic markers while the stiffer Gtn-HPA gel upregulated the
myogenic markers in hMSCs without addition of any chemical or differentiation cues. Similar
evaluation was performed by Wang et al with human fibroblasts (HFF-1) to study the effect of the
stiffness of the Gtn-HPA gel. In 2D environment, the proliferation of HFF-1 increased when
cultured on stiffer Gtn-HPA gel. However, the proliferation decreased with stiffer 3D environment
[14]. The phenomenon of the stiffness-dependent differentiation of stem cells and proliferation of
cells have been widely accepted in the tissue engineering research as exhibited by Gtn-HPA [15].
Wang et al presented encouraging findings of preliminary in vivo evaluation of the Gtn-HPA
Figure 3.2 Dependence of HRP and H2O2 concentration on the gelation time, stiffness and degradation of the Gtn-HPA gels. Insert in C plots the rate of degradation (%/hr).
Figure modified from Lim, TC et al Biomaterials, 2012. 33(12): 3446-3455
59
hydrogels with varying stiffness for applications in cartilage repair in a rabbit model. The results
showed stiffness-dependent cartilage repair. Gtn-HPA hydrogels with medium stiffness were
shown to have greater hyaline cartilage formation compared to low or high stiffness Gtn-HPA
matrices. Moreover, medium stiffness Gtn-HPA hydrogels also promoted better integration with
the surrounding unaffected tissue promoting osteochondral repair [16]. Various studies have since
employed Gtn-HPA in several applications including microfluidic cell culture, bone regeneration
and personalized tissue engineering [17-19].
Prior work in our laboratory by Lim et al commenced in vitro evaluation to study the effects
of Gtn-HPA hydrogels on the migration and differentiation of adult neural stem cells (aNSCs).
Results showed high viability of the aNSCs on the Gtn-HPA matrix with good cell adhesion. Gtn-
HPA promoted proliferation and migration of aNSCs. Interestingly, viability of the aNSCs was
higher than collagen scaffolds even with as high concentration as 500 µM H2O2 indicating that
Gtn-HPA cross-linking mechanism aided in increasing the resistance to oxidative stress by
preconditioning aNSCs to sub-cytotoxic levels of oxidative stress. Remarkably, Gtn-HPA
modulated the differentiation of aNSCs towards astrocytic and neuronal lineage – however, with
mixed conditions for astrocytic and neuronal fate, Gtn-HPA showed augmented levels of βIII
tubulin compared to GFAP indicating that Gtn-HPA promoted differentiation of aNSCs towards
neuronal fate [7, 20]. Moreover, SDF-1α encapsulated in Gtn-HPA promoted chemotactic
recruitment of adult rat hippocampal neural progenitor cells into the Gtn-HPA gel confirming that
Gtn-HPA is permissive of cellular migration in addition to providing a cytocompatible matrix [12].
3.1.5. Gtn-HPA - promising biomaterial for spinal cord injury repair: Several promising
biomaterials were evaluated in our laboratory for tissue repair and functional recovery after spinal
cord injury in rodent models. Spilker et al used pre-formed collagen and collagen-GAG tubes in a
T7-T10 5-mm complete transection rodent model to evaluate tissue regeneration after spinal cord
injury. Although the collagen-based tubes resulted in oriented axonal and connective tissue
constituents with reduced scar formation, much of the collagen-based tube was absorbed by 30
days. Additionally, the collagen-based matrices and tubes did not induce migration of endogenous
reparative cells. Moreover, pre-fabricated tubes would not be clinically feasible given that the
spinal cord cavities are irregular in shape and heterogeneous in volume [21, 22]. Subsequently,
Cholas et al evaluated pre-formed collagen scaffolds delivering chondroitinase ABC (chABC) and
mesenchymal stem cells (MSCs) in a 3-mm and 5-mm hemi-section model of spinal cord injury.
The collagen-based scaffold with the therapeutic agents induced angiogenesis, cellular infiltration
and improved functional recovery in collagen-treated animals. However, there were two major
60
limitations of the study – the collagen scaffolds resorbed almost completely by four weeks and
pre-formed scaffolds would not be clinically viable to treat spinal cord injury [23]. However, it
became increasingly clear that pre-formed scaffolds are not suitable for future evaluation.
Therefore, Macaya et al evaluated a promising injectable collagen-genipin gel incorporating
fibroblast growth factor (FGF-2) in a 3-mm hemi-resection model of spinal cord injury in Lewis
rats based on the exciting in vitro results [24]. The in vivo evaluation after four weeks post
implantation of the injectable collagen-genipin gel displayed greater astrocyte migration into the
defect site, some GAP-43 positive axons, and correlation between the functional score and
spared tissue. However, collagen-genipin gel degraded as early as two weeks after implantation
limiting the long-term delivery of FGF-2 for any potential therapeutic effect. Increasing the genipin
concentration, to obtain greater stiffness of the collagen-genipin gel with greater cross-linking,
was not feasible because of toxicity [25]. Due to these limitations and our prior work with
biomaterials for spinal cord injury repair, we devised specific comprehensive criteria for the
biomaterial to be evaluated in vivo for this thesis work as follows [26] and Gtn-HPA met these
criteria making it a promising biomaterial for spinal cord injury applications:
1) It should be biocompatible without inducing more than typical inflammatory response
2) It should be injectable so that it can be delivered to the injured cavity in minimally invasive
manner to abate surgical damage during implantation
3) It should be able to gelate in situ to conform to the heterogeneous spinal cord cavities
4) It should be non-swelling not to apply pressure to the surrounding tissue, which is known to
initiate further secondary damage after the injury
5) It should have similar mechanical properties as that of the spinal cord to minimize secondary
damage and non-shrinking so that it can integrate well with the surrounding tissue
6) The constituents of the biomaterial should not induce cytotoxicity in the tissue
7) It should serve as a viable replacement of the disappearance of stroma lost due to spinal cord
injury and be permissive of cellular migration, proliferation and differentiation into the defect site
to promote neural repair
8) It should have tunable physical properties to control gelation time, stiffness and degradation
characteristics. Specifically, for experimental models, it should be able to complete gelation in
two-four minutes to visually confirm the gelation in the surgical hemi-resection model
9) It should have chemical moieties such as RGD sequences as ligand for cell integrins
10) It should be able to persist in vivo for timeline of our experiment (i.e. four weeks) and longer
11) It should be able to incorporate and locally deliver signaling molecules, growth factors, and
cells in the injury site without the particular use of vehicles
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Based on the promising in vitro and preliminary in vivo evaluation of Gtn-HPA matrices
in the intracerebral hemorrhagic (ICH) stroke rodent model, we examined the Gtn-HPA in greater
detail to assess if it would be a potential candidate biomaterial that can overcome the limitations
of previous work and meet the devised criteria above. We found Gtn-HPA to be very promising
given its properties. First, both in vitro and in vivo evaluation of the Gtn-HPA have found it be a
biocompatible material that does not elicit a severe inflammatory reaction. Moreover, it is an
injectable biomaterial capable of undergoing covalent cross-linking after being injected as a liquid
[10, 20]. Therefore, it can provide conformational filling of the heterogenous spinal cord injury
cavitary lesions. In addition, HRP and H2O2 allow for independent tuning of rate and degree of
cross-linking, respectively by choosing a desired concentration. This independent tunability is
essential in obtaining an optimal degradation rate, and in matching the physical properties (elastic
modulus) of the gel and of the spinal cord tissue [7]. The shear modulus of spinal cord is reported
to be an average of 1500-2500 Pa [25]. The concentration of 1.7 mM H2O2 per 2wt% Gtn-HPA
gel would fabricate Gtn-HPA in the range of reported values of the spinal cord (Figure 3.2B).
Moreover, desired gelation time of two-to-four minutes was achieved using HRP concentration
between 0.1 and 0.15 U/ml using quick bench-top Gtn-HPA fabrication. Furthermore, Gtn-HPA
has been shown to be permissive of cellular migration as reported in several studies [7, 12, 20].
Gtn-HPA was found to be non-shrinking and non-swelling based on the prior in vitro evaluation
by Wang et al [10]. In vivo evaluation of Gtn-HPA for intracerebral hemorrhagic stroke model
showed persistence of the Gtn-HPA gel two weeks after implantation in our laboratory work.
Therefore, these collective prior studies show that the Gtn-HPA meets all the criteria of an ideal
biomaterial for implantation in the cavity created in the spinal cord injury to assess the tissue
repair, regeneration and functional recovery in SCI model (Table 3.2). Our work will evaluate the
persistence of the Gtn-HPA for four weeks in 2-mm complete and hemi-resection spinal cord
injury rat models. Considering the results from prior in vitro and in vivo evaluation of Gtn-HPA, we
propose to evaluate the effect of injectable Gtn-HPA gel incorporating epidermal growth factor
(EGF) alone or in combination with stromal cell-derived factor (SDF-1α) because it is a very
promising biomaterial for applications in spinal cord injury.
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Property Function Gtn-HPA characteristics
Reference
Porosity Tunable porosity, cell encapsulation and migration
Tailorable porosity, hMSCs in 3D Gtn-HPA culture, monocytes
Al-Abboodi et al, 2014 [19]; Wang LS et al, 2010 [10]; Bystronova et al, 2018 [27]
Tunable mechanical properties
Control of stiffness to match the host tissue modulus and gelation rate
Concentration of H2O2 (stiffness) and HRP (gelation time)
Lim TC et al, 2012 [7]
Degradation rate Persistence for long duration in vivo
Concentration of H2O2 and % gelatin content
Wang LS et al, 2010 [13]
Maintain cell viability
Provide non-cytotoxic environment
High viability for hMSCs, HFF-1, aNSCs
Wang LS et al, 2012 [14]; Lim TC et al, 2013 [7, 12]
Permit cell adhesion
Promote cell proliferation and migration
RGD sequences Hu et al, 2009 [9]
Permissive of cell migration, proliferation and differentiation
Repopulate the cells and promote tissue repair and regeneration of the tissue
Stiffness-dependent differentiation of hMSCs, migration of aNSCs
Wang et al, 2012 [28], Lim et al, 2013 [12]
Shrinking Integrate well with surrounding host tissue
Non-shrinking Lim TC, 2014 [20]
Swelling Minimize additional pressure on the host tissue
Non-swelling Wang LS, 2010 [10]
Oxidative stress Reduced oxidative stress to minimize cytotoxicity
Confers resistance to oxidative stress
Lim TC et al, 2012 [7]
Formulations Fabrication in different forms
Electrospun fibers, hydrogels
Hu et al 2009 [9]; Wang LS et al
In vivo injectable implantation
Pre-clinical and clinical applications
Bone regeneration, cartilage repair, spinal cord injury, stroke injury, retinal pathologies
Chun et al, 2016 [18]; Wang et al, 2014 [16]; Lim TC, 2014 [20]; Shah, A 2018 (unpublished), Love, C (ongoing), Colombe, P (ongoing)
Table 3.2 Gtn-HPA exhibits various important properties making it an ideal biomaterial for spinal cord application
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3.2. Growth factor: Epidermal growth factor (EGF)
3.2.1. Introduction: Epidermal growth factor (EGF) is shown to induce cell growth,
proliferation, differentiation and signaling modulation in mesodermal and ectodermal cells.
Specifically, in addition to other organs such as kidney, thyroid gland and pancreas, it is also
produced in the nervous system. EGF has been isolated from central nervous system (CNS),
peripheral nervous system (PNS) and cerebrospinal fluid (CSF) although the concentration of
EGF varies depending on the location in the nervous system [29]. Immunohistochemistry
demonstration of EGF and EGFR showed the presence of both EGF and EGFR in spinal cord
tissues specifically in the anterior horn cells [30]. CNS has abundance of epidermal growth factor
receptors (EGFR) and has been shown to play critical role in the development of the brain. EGFR
is activated predominantly by EGF but also by other factors such as heparin-binding epidermal
growth factor (hb-EGF) and transforming growth factor-α (TGF- α) [31]. EGF and its family of
proteins have been shown to play a critical role during embryonic development of the neural
tissue. Neuregulin-1 (NRG1) contains EGF-like domain and is widely studied and characterized
to play a vital role during neural development. Interestingly, NRG1-mutated mice show profound
changes in the neural development at various temporal stages [32]. Moreover, NRG1 is released
from the mature neurons to promote the formation and maintenance of glial cells, which are
essential in directing the migration of neurons from the ventricular regions to the developing
tissue. Furthermore, study by Nelson et al reported that the high concentrations of EGF leads to
greater growth and survival of neurogenic radial glial cells in human neurosphere culture including
higher expression of glial markers [33].
EGF and NRG1 plays a major role in axon guidance, axon sprouting, axonal myelination
and synapse formation [32, 34, 35]. EGF aids enhanced neural cell proliferation in the sub-
ventricular zone in brain. Moreover, EGF has shown great promise by promoting neurogenesis in
brain interneurons as well [36]. In an in vitro study, cells isolated from embryonic brain and adult
sub-ventricular zone differentiated into radial glial cells and supported neuronal cell migration [37].
One of the most profusely reported roles of EGF in the nervous system regulating the cell
recruitment, proliferation and eventual differentiation of neural stem cells (NSCs). NSCs are highly
responsive to EGF receptors stimulating their proliferation [38]. Due to its pivotal role in neural
tissue development and growth, EGF has been studied in SCI experimental models to recruit
endogenous NSCs and promote tissue repair post SCI. For example, intrathecal administration
of EGF resulted in significantly greater ependymal cell proliferation in the central
canal immediately rostral and caudal to the lesion and greater white matter sparing compared to
controls [39]. Several other studies have also shown axonal regeneration and functional recovery
64
in SCI using EGF [40, 41]. Therefore, EGF was considered as a potential factor to be incorporated
in Gtn-HPA.
3.2.2. Prior relevant in vitro studies with EGF: There are several relevant and promising
in vitro investigations that prompted the delivery of EGF in the injury site created by the spinal
cord pre-clinical rat model in our work. There is evidence that multipotent neural stem cells are
present in the ependymal layer near the central canal of the adult mammalian spinal cord. These
cells were confirmed to be NSCs by Nestin, Sox2, and CD-133 [42]. In addition, these cells are
highly responsive in proliferation and migration to EGF treatment [43]. Several studies have
supported the findings of these studies. Based on the prior work demonstrating higher levels of
EGF in the region of injury after spinal cord injury [44], Kang et al studied the effect of EGF on the
proliferation of neural progenitor cells isolated from adult mice spinal cord. As early as 3-6 days
of culture in EGF-containing medium, the authors reported significantly greater proliferation of
neural progenitor cells compared to the controls indicating that EGF truly promotes the cellular
growth of neural progenitor cells present in the spinal cord via JAK2/STAT3 and MAPK signaling
pathway. These pathways are reported to be of critical importance in the regeneration after spinal
cord injury in various experimental models [45].
Lam et al studied the effect of EGF on the neural differentiation of embryonic stem cells
and axon growth in ESCs cultured on PLLA nanofibrous scaffolds [46]. They also assessed the
effect of adsorbed and soluble EGF. The results showed that EGF treatment significantly
increased the expression of neuronal markers NES and NEF3 in ESC-derived neural cells
compared to the controls suggesting that EGF promotes differentiation into the neuronal lineage.
In addition, EGF promoted greater axonal outgrowth from the surface of the nanofibers when they
were functionalized with heparin compared to the controls. Moreover, directly adsorbed EGF on
the nanofibers were not effective in promoting axonal growth on the nanofibers. These results
suggest that soluble EGF delivery would be effective in Gtn-HPA compared to its delivery in a
vehicle to which EGF would be adsorbed. Yet another interesting in vitro assessment of the role
of EGF was done by Garcez et al. Neural crest stem cells were isolated and were exposed to
EGF for six days. After 6 days of culture, EGF seemed to increase the number of melanocytes
and neurons in the neural crest stem cell culture [47]. These results further support that the theory
that EGF promotes cell growth, proliferation, migration of the neural stem cells and differentiation
of these neural stem cells into neuronal lineage. Therefore, we propose to deliver soluble EGF
via Gtn-HPA in the injury site in our 2-mm complete and hemi-resection preclinical spinal cord
injury rat model to recruit endogenous neural stem cells into the injury site and promote their
65
differentiation into neurons and glial cells. Our proposition also found rationale from the results of
various in vivo studies with EGF.
3.2.3. Prior in vivo studies incorporating EGF in SCI models: Encouraging
preliminary in vitro studies investigating the role of EGF have led to pre-clinical examination of
the role of EGF in promoting tissue repair and recovery post spinal cord injury. EGF has been
shown to have neuroprotective effect in spinal cord after SCI in a rat model [48]. Moreover, it has
shown promise as a therapeutic agent for preventing blood-spinal cord barrier interruption after
spinal cord injury [49]. Considering these beneficial effects of EGF, a recent study by Ozturk et al
investigated the effect of EGF in a rat model of spinal cord injury in the acute and subacute phase
by delivering EGF in the injury site with a sterilized pipette with a Hamilton syringe. Injection of
EGF was shown to reduce the apoptosis-related marked in the injury site as early as 8 days post
injury. Moreover, EGF also increased the levels of antioxidants suggesting that EGF plays a role
in regulating apoptosis and promoting reduction in the oxidative stress in the injury site, thereby
promoting reparative and regenerative mechanism. Lastly, EGF also showed better functional
recovery than the controls. However, 8-day timepoint is too early to evaluate functional recovery
[50]. Recently, Sun et al explored the benefits that tacrolimus (FK506) could provide in tissue
repair after spinal cord injury. Tacrolimus is an FDA-approved immunosuppressant and typically
used to mitigate the acute rejection after transplant surgeries. Remarkably, tacrolimus increased
the levels of EGF expression both in vitro and in vivo, which led to longer neurite length suggesting
that EGF plays a vital role in promoting neurite outgrowth after injury. Moreover, better functional
recovery was observed suggesting that EGF plays a key role in tissue repair post SCI [51]. Alluin
et al investigated a combinatorial approach with EGF as one of the key growth factors in the
cocktail of factors containing FGF-2, PDGF-AA, and chondroitinase ABC (chABC) in a clip spinal
compression model. This combination was injected into the injury site via subarachnoid catheter
connected to an osmotic pump. The combination showed reduced GFAP presence in the
perilesional area indicating reduction of astrogliosis near the injury site. Interestingly, even the
macrophages response was attenuated in the defect area due to the administration of the
combination of chABC and growth factors. One of the key findings was collateral sprouting of the
corticospinal tract using BDA tracing. There was greater spared tissue observed in the cord due
to implantation of the chABC with growth factors compared to the controls. Lastly, this intervention
also shown improved functional recovery suggesting augmentation of neuronal plasticity [40].
Given the complexity of the damage after spinal cord, more combinatorial studies such as by
Alluin et al should be investigated to promote repair after spinal cord injury.
66
Based on the finding that EGF and FGF-2 induces proliferation of the stem cells residing
near fourth ventricle in the brain and spinal cord, Martens et al pursued an in vivo investigation in
a mouse model. They injected a combination of EGF and FGF-2 in the fourth ventricle in the brain
and central canal of adult male CD1 mice. Just six days post injection of EGF and FGF, greater
proliferating cells were observed in both the cervical spinal cord and near fourth ventricle in the
brain. Specifically, in the spinal cord, these cells were in the ependymal and subependymal tissue
closer to the central canal. Indeed, these cells were nestin positive indicating the presence of
neural stem cells. The results from this study indicated that endogenous neural stem cells near
the central canal in the spinal cord proliferate in response to EGF and FGF-2 [52]. Niche of these
neural progenitor cells has previously been shown as well [42]. Combination of EGF and FGF-2
was investigated by Kojima et al in an adult rat spinal cord. EGF and FGF-2 was intrathecally
delivered over 14 days using an osmotic pump. EGF groups showed the two highest BrdU+ cells
in the ependymal tissue were found in EGF alone and EGF+FGF-2 group. These results suggest
that EGF has a mitogenic effect on the migration and proliferation of the ependymal precursor
cells, which are shown to be closely related to neural stem cells. As anticipated, these cells also
stained positive for nestin confirming that these ependymal precursor cells are neural stem cells
(NSCs) [41]. In a more comprehensive in vivo study by Hamann et al investigating the same EGF
and FGF-2 combination in a clip compression rat model. EGF and FGF-2 were delivered in a
highly concentrated collagen solution to localize the growth factors in the injury site. Interestingly,
they labeled the injected EGF and FGF-2 with markers. The results indicated that EGF readily
penetrated the injury site in the spinal cord while FGF-2 was predominantly observed in the dura.
Furthermore, significantly less cavitations were seen in the growth factors group indicating that
EGF promoted tissue sparing including greater white matter sparing in the injury site. Moreover,
as reported by Kojima et al, they also observed greater BrdU+ cells in the EGF and FGF-2 group
than the controls in the ependymal region [39].
All these studies validated a critical mitogenic role of EGF in proliferation of the neural
stem progenitor cells found near the central canal. However, ability of EGF to chemotactically
recruit endogenous NSCs has not been studied in vivo. Prior work in our laboratory showed
chemoattractive effect of EGF towards NSCs in vitro. Injection of Gtn-HPA/EGF in the
intracerebral hemorrhage model of the stroke showed the ability of EGF to repopulate the injury
area with NSCs (manuscript in publication). Considering encouraging results of the in vivo
evaluation of EGF as discussed above, we propose to provide evidence of EGF-induced migration
of the NSCs to the injury site in a rodent SCI model.
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3.3. Chemokine: Stromal cell-derived factor-1α (SDF-1α)
3.3.1. Introduction: SDF-1α, also known as CXCL12, was one of the first α-chemokines to
be discovered and has the closest homology to β-chemokines. SDF-1α is regarded as a
chemoattractant that is synthesized by bone marrow stromal cell lines. CXCR4, one of the
receptors for SDF-1α, along with SDF-1α have been highly expressed in a developing embryo
indicating the crucial role SDF-1α plays in the development process [53]. SDF-1α has been widely
studied because of its ample secretion and rich expression in nearly all the organs. SDF-1α has
been shown to be a potent chemoattractant for monocytes, T cells, pre-B cells, hematopoietic
progenitor cells, and dendritic cells. SDF-1α has been shown to play a prominent role in various
processes such as implantation of the embryo, germ cell migration, and cerebellar development
[54]. Specifically, exploratory research over the last two decades have shown critical role of SDF-
1α/CXCR4 signaling pathway in the development of cerebellum, dorsal root ganglion and cerebral
cortex [55, 56]. The common aspect of SDF-1α in all these processes defined the role of SDF-1α
in the migration of cells such as interneurons and neuronal precursors. Strangely, Tiveron et al
demonstrated that the motor neuron progenitor cells of the cortex express SDF-1α to regulate the
migration of the interneurons near the subventricular zone [57].
In addition to the migration of precursors and interneurons, SDF-1α has also been shown
to act as a cue for axonal guidance during neural development. Lieberam et al showed that the
gradient of SDF-1α directs the axonal guidance in embryonic development of the spinal cord. The
growth cones are guided towards their final peripheral targets based on the varying expression of
SDF-1 along the axon path. Specifically, initial axonal trajectory of motor neurons towards their
peripheral target in the spinal cord is regulated by SDF-1α. The absence of SDF-1α signal was
reported to drive the axon dorsally as opposed to ventrally [58]. In addition to guiding the axons,
SDF-1α modulates the concentrations of known growth cone repellants such as semaphorins in
the growth cones of target axons of neurons [59]. These studies suggest that SDF-1α controls
numerous facets of CNS development. Furthermore, there is a growing evidence that SDF-1α is
found to play important role in axonal pathfinding, outgrowth and branching. In addition, SDF-1α
seem to be involved in promoting nerve regeneration by regulating synaptic function and
influencing the release of cytokines for tissue repair after CNS injury [60, 61]. In the context of our
work, the primary role of SDF-1α in influencing the migration, proliferation and differentiation of
neural stem progenitor cells (NSCs) found in the ependymal region around the central canal of
the spinal cord was of utmost promise. Validating its role, CXCR4 is strongly expressed in
ependymal layer of the central canal in spinal cord post SCI [62].
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3.3.2. Prior relevant in vitro studies with SDF-1α: Various in vitro investigations have
shown the promise of SDF-1α to recruit interneurons, NSCs and other glial cells. Several studies
have shown the chemotactic response of interneurons towards SDF-1α in a chain migration model
[63, 64]. Specifically, Lysko et al performed an insightful in vitro mouse explant system experiment
studying the migration, speed of migration and branching of the interneurons when they were
exposed to SDF-1α. They were able to show that decreasing the SDF-1α levels led to slower
migration rate and decrease in the number of migratory interneurons. Moreover, decreasing SD-
1α also led to profuse branching of interneurons suggesting that the directional guidance was
affected as well. Moreover, the results also showed that SDF-1α is necessary for faster and proper
migration of interneurons [65]. In addition to interneurons, glial cells such as oligodendrocyte
precursor cells (OPCs) and astroglia are also responsive to the SDF-1α. Kadi et al investigated
the effects of various growth factors in the migration regulation of OPCs 48 hours after being
exposed to the mitogens. They used OPC-like cell line known as Oli-neu. A proliferation
developed by the authors showed that SDF-1α induced proliferation and migration of Oli-neu cells
compared to the untreated controls. Interestingly, the concentration of SDF-1α had significant
effect on the proliferation of these cells. As a validating method, they also blocked the CXCR4
receptors in these cells and exposed these cells to SDF-1α. This modification reduced the
proliferation rate of the cells indicating the role of SDF-1α/CXCR4 mediated chemoattraction of
OPCs. In addition to proliferation, SDF-1α also increased the levels of myelin basic protein
expression suggesting that it promotes myelin sheath formation in vitro [66]. Odemis et al
investigated the role of SDF-1α in the chemotaxis of cortical astroglia. The authors reported
significantly higher migration index using SDF-1α mediated by IL-6 and CAMP. The authors
demonstrated that IL-6 and cAMP increased the CXCR4 expression on the cell surfaces of cortical
astroglia making them more responsive to SDF-1α. This could be a possible mechanism by which
IL-6 and cAMP induces SDF-1α-dependent chemotaxis of the astrocytes after injury in the
nervous tissue [67]. In the context of our work, we aim was to recruit endogenous neural stem
cells to the spinal cord injury site to promote repair and regeneration post SCI. Several in vitro
studies have shown strong mitogenic and chemoattractive role of SDF-1α towards NSCs [60, 68,
69].
A recent study by Stewart et al engineered the mesenchymal stem cells (MSCs) to
overexpress SDF-1α. In a transwell migration assay, these SDF-1α-expressing MSCs were
seeded in the migration chamber. The results showed significantly higher migration of the neural
stem cells into the well seeded with SDF-1α-expressing MSCs compared to the normal MSCs-
seeded chamber. This study indicates the chemotactic response of NSCs towards SDF-1α [70].
69
These results were supported by the findings in our laboratory by Lim, TC. In a 3D migration
assay, cell-free SDF-1α incorporated Gtn-HPA gel was placed in the core surrounded by aNSCs
seeded in the type I collagen gel annulus. After 7 days of culture and migration, Gtn-HPA/SDF-
1α cores showed increased accumulation of the NSCs at the interface between the Gtn-HPA and
type I collagen construct. Moreover, administration of AMD3100, a well-known antagonist of the
CXCR4, in the medium showed reduction in the number of cells accumulated at the interface.
Both individual cell migration and chain migration was observed in the assay. In addition, SDF-
1α also induced increased migration distance of the neural stem cells into the Gtn-HPA core. The
mitogenic role of SDF-1α was also confirmed with greater proliferation of aNSCs observed in the
Gtn-HPA/SDF-1α core [20, 71]. Considering the multifunctional role of SDF-1α in attraction,
proliferation and differentiation of various cells such as OPCs, NSCs and other glial cells that are
important for neural tissue repair, we were encouraged to evaluate these characteristics of SDF-
1α in vivo in our spinal cord injury model. Our proposition is that sustained localized delivery of
SDF-1α will enhance the recruitment of the precursor cells by EGF to the injury site repopulating
the injury site with reparative cells that can differentiate into neuronal and glial fate and can
stimulate the release of pro-regenerative molecules.
3.3.3. Prior in vivo studies incorporating SDF-1 α in SCI models: SDF-1α is expressed
in the adult spinal cord predominantly in dorsal corticospinal tract and the meninges. Moreover,
its receptor, CXCR4, has high levels of expression in the ependymal layers of the central canal
probably in the neural stem progenitor cells niche [62]. There is strong evidence that the levels of
SDF-1α and its receptor, CXCR4, drastically increase as early as 2 days after inducing a traumatic
spinal cord injury in rats. These upregulated levels can also be detected 42 days post SCI [72].
Similar upregulation of SDF-1α is also observed after stroke injury and ischemic injuries of the
central nervous system pointing to the ubiquitous role of SDF-1α in the repair and potential
regenerative response in the CNS [73]. After spinal cord injury, several cells such as reactive
astrocytes, macrophages, endothelial progenitors, oligodendrocyte precursor cells and neural
progenitor cells exhibit increased expression of SDF-1α and its receptor CXCR4 [68]. Given the
pervasive role of SDF-1α, a wide range of pre-clinical studies have investigated the role of SDF-
1α in promoting repair and functional recovery after spinal cord injury. Jaerve et al infused SDF-
α in the dorsal hemi-resection injury site via a intrathecal catheter placed in the subarachnoid
space. The catheter was connected to a osmotic minipump that was loaded with 10µM SDF-1α
ensuring sustained local release. Infusion took place over 7 days and then the catheter was
removed. Five weeks after the injury, they showed rostral axonal regrowth and reduced scarring
due to the infusion of SDF-1α. They employed both young and geriatric rats to show that the
70
axonal regeneration capacity is not hampered in geriatric rats suggesting that although the axonal
sprouting might be diminished in geriatric rats, the potential of regeneration in CST fibers was
comparable between the young and the geriatric rats [74]. Stewart et al utilized another approach
to evaluate the effect of SDF-1α in a spinal cord injury model. They transplanted engineered SDF-
1α-overexpressing MSCs into the spinal cord injury created in a contusion model. The result
showed significantly greater functional recovery assessed using the BBB scale in SDF-1α/MSCs
group compared to the untreated controls. Moreover, intervention of SDF-1α/MSCs reduced the
astrogliosis at the border outline the defect suggesting a scar-inhibitory role of SDF-1α. Moreover,
the injured area was also reduced in SDF-1α/MSCs groups suggesting a neuroprotective effect
of SDF-1α after spinal cord injury. Probably as a result of neuroprotection of unaffected tissue,
greater white matter sparing was observed in the SDF-1α/MSCs group a mm away from the injury
site. Interestingly, SDF-1α also promoted greater presence of GAP43-positive axons in the injury
site – marker for axonal cone growth. These collective results direct potential role of SDF-1α in
neuroprotection, functional recovery and axonal regeneration after spinal cord injury [70].
The role of SDF-1α was also reported in another great examination by Zendedel et al. In
the study, SDF-1α was intrathecally administered via an osmotic pump for 14 days. After 28 days,
rats with SDF-1α concentration of 500-1000 ng/ml showed significantly higher behavioral scores,
decreased numbers of apoptotic cells, increased astroglia and microglia responses, and induced
angiogenesis by intrathecal injection of SDF-1α in SCI contusion model suggesting the
recruitment of various vital cells of the spinal cord tissue repopulating the injury site [75].
Moreover, encouraging in vivo studies in our laboratory have shown increased presence of the
neural stem cells in the stroke lesion in the groups where SDF-1α was locally delivered using Gtn-
HPA matrix in an intracerebral hemorrhagic model of stroke. Significantly high DCX+ cells were
observed around the Gtn-HPA gel. In the same evaluation, Gtn-HPA/ SDF-1α promoted
neovascularization as well as neutrophilic response [20]. Given the prominent role of SDF-1α
reported in promoting repair in spinal cord injury and central nervous system, we propose to utilize
the synergistic effects of EGF and SDF-1α to attract endogenous stem cells and glial cells in
hemi-resection model of SCI.
71
3.4. Two formulations of Gtn-HPA for injection in the SCI models: Based on the rationale
provided for Gtn-HPA, EGF and SDF-1α in this chapter, our goal was to evaluate the synergistic
effects of EGF and SDF-1α locally delivered by an injectable Gtn-HPA that is capable of
undergoing covalent crosslinking in situ after being injected as a liquid in the cavity created by
surgical excision rat model of SCI after four weeks. Based on the positive results from the prior
work in the intracerebral hemorrhagic stroke model of stroke injecting Gtn-HPA with SDF-1α
encapsulated in polyelectrolyte complex nanospheres (PCN), we chose 8% Gtn-HPA gel to be
injected into the 2-mm complete and hemi-resection models of spinal cord injury. Since a
preliminary study showed significant inflammatory response around the Gtn-HPA probably due to
the PCNs, we wished to evaluate the effect of incorporating soluble growth factors EGF and SDF-
1α without any delivery vehicle.
The first formulation was as follows: 20 µl of 8% Gtn-HPA + 6 µg/rat EGF + 6 µg/rat SDF-
1α was injected in the 2-mm complete resection SCI model. The methods and the results are
described in Chapter 4 of this thesis. However, we did not see the persistence of Gtn-HPA in all
the animals after four weeks. Several reasons were considered for the disappearance of the Gtn-
HPA gel from the injury site: 1) the degradation rate of 8% Gtn-HPA is high leading to complete
disappearance by four weeks, 2) the Gtn-HPA gel was dislodged from the injury site due to
mechanical forces experienced by the Gtn-HPA gel during the movements post-operation, 3)
significant migration of the cells around the Gtn-HPA gel led to more secretion of degrading
enzymes such as MMP2, and 4) Injection of Gtn-HPA into a bleeding cavity would lead to
disruption of gelation procedure of the Gtn-HPA and therefore, the stiffness of resultant Gtn-HPA
is not as anticipated. Since we were able to see substantial Gtn-HPA in the groups with Gtn-HPA
incorporating both EGF and SDF-1α, the third reason was unlikely. Therefore, we designed a
second formulation of the Gtn-HPA gel to address these concerns. The gelatin content was
increased to 12% to reduce the degradation rate of the Gtn-HPA gel.
The second formulation was designed as follows: 20 µl of 12% Gtn-HPA + 6 µg/rat EGF
+ 6 µg/rat SDF-1α was injected in the 2-mm complete resection SCI model. The dosage of the
growth actors was kept the same based on the evaluation in both the models. The second
formulation along with an improved dural replacement technique (to better contain the Gtn-HPA
gel in the cavity) as described in Chapter 6 was evaluated in the 2-mm hemi-resection model. As
a result, all the animals in the groups showed presence of Gtn-HPA gel in the injury site. In an
attempt to characterize the release of growth factor from these Gtn-HPA formulations, release
profile of EGF from 8% and 12% Gtn-HPA was examined.
72
3.5. Release profile of EGF from Gtn-HPA formulations for four weeks
3.5.1. Methods: Given the encouraging results from prior work with EGF delivery in
intracerebral hemorrhage stroke model, studying the release of EGF from Gtn-HPA was
important. Four experimental groups were studied for the release of EGF with n = 4 for each
group: 1) 8% Gtn-HPA only, 2) 12% Gtn-HPA only, 3) 8% Gtn-HPA/EGF, and 4) 12% Gtn-
HPA/EGF. For the EGF groups, 6 µg of EGF was encapsulated in both 8% and 12% Gtn-HPA
formulations. The concentrations of each constituent are shown in Table 3.2.
Formulation Gtn-HPA HRP H2O2 EGF
8% Gtn-HPA 80 mg/ml 0.1 U/ml 6.8 mM 0
8% Gtn-HPA/EGF 80 mg/ml 0.1 U/ml 6.8 mM 6 µg
12% Gtn-HPA 120 mg/ml 0.1 U/ml 10.2 mM 0
12% Gtn-HPA/EGF 120 mg/ml 0.1 U/ml 10.2 mM 6 µg
Table 3.3 Experimental groups for EGF release study with their concentration constituents
After fabrication of the Gtn-HPA gel in 2 ml cryotubes, gels were submerged in 1.5 ml PBS release
buffer. 0.75 ml of supernatant was collected at the following time points to study the release of
EGF: 1 hr, 1d, 2d, 3d, 5d, 7d, 9d, 12d, 15d, 18d, 22d and 28d followed by addition of 0.75 ml PBS.
four-week endpoint was chosen because of the four-week evaluation timepoint in the in vivo
evaluation. After four weeks, Gtn-HPA gel was degraded using Collagenase IV solution for 3
hours at 37 °C. The concentration of EGF at these time points was assessed using EGF ELISA
kit (R&D Systems). For unknown samples, 1:600 dilution was used for all the time points.
3.5.2. Results and discussion: The standard curve fit was calculated using 4-parameter
logistic regression curve. The concentration and cumulative EGF release amount over four weeks
were calculated based on the best fit equation using the 4-parameter standard curve for all four
groups. We observed that no EGF was released from the 8% and 12% Gtn-HPA groups as
expected. However, the results show that there was no statistical difference in the amount of EGF
released up to 9 days possibly due to the initial burst effect typically observed in the drug-delivery
systems (Figure 3.3). However, greater EGF was released from 8% Gtn-HPA after 9 days
compared to the 12% Gtn-HPA gel (Two-way ANOVA, p<0.05). However, the differences in the
amount of EGF was small probably due to the ability of small molecules such as EGF to diffuse
from the dense Gtn-HPA network. The EGF release curve indicates that Gtn-HPA is able to locally
deliver EGF in a sustained manner. Moreover, after four weeks, all EGF is not released for both
8% and 12% confirming the presence of EGF in the degraded gel after four weeks. Further in vivo
73
evaluation employing immunohistochemistry was performed to validate the presence of EGF after
four weeks.
Figure 3.3 Release profile of EGF from 8% and 12% Gtn-HPA gel over four-week duration to mimic the in vivo evaluation. Greater EGF was released from 8% Gtn-HPA after 9 days. However, the difference of EGF released was small between 8% and 12% groups after four weeks
74
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68. Jaerve, A., J. Schira, and H.W. Müller, Concise Review: The Potential of Stromal Cell-Derived Factor 1 and Its Receptors to Promote Stem Cell Functions in Spinal Cord Repair. Stem Cells Translational Medicine, 2012. 1(10): p. 732-739.
69. Filippo, T.R.M., et al., CXCL12 N-terminal end is sufficient to induce chemotaxis and proliferation of neural stem/progenitor cells. Stem Cell Research, 2013. 11(2): p. 913-925.
70. Stewart, A.N., et al., SDF-1 overexpression by mesenchymal stem cells enhances GAP-43-positive axonal growth following spinal cord injury. Restor Neurol Neurosci, 2017. 35(4): p. 395-411.
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72. Knerlich-Lukoschus, F., et al., Chemokine expression in the white matter spinal cord precursor niche after force-defined spinal cord contusion injuries in adult rats. Glia, 2010. 58(8): p. 916-31.
73. Hill, W.D., et al., SDF-1 (CXCL12) is upregulated in the ischemic penumbra following stroke: association with bone marrow cell homing to injury. J Neuropathol Exp Neurol, 2004. 63(1): p. 84-96.
74. Jaerve, A., et al., Differential effect of aging on axon sprouting and regenerative growth in spinal cord injury. Experimental Neurology, 2011. 231(2): p. 284-294.
75. Zendedel, A., et al., Stromal cell-derived factor-1 alpha (SDF-1alpha) improves neural recovery after spinal cord contusion in rats. Brain Res, 2012. 1473: p. 214-26.
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Chapter 4:
Investigating the effects of Gtn-HPA
matrix with EGF and SDF-1α in a T8 2
mm complete resection rat model of
spinal cord injury after four weeks
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4.1. Introduction and clinical motivation
There are various animal species and models that are used to study functional recovery
and repair after spinal cord injury (SCI). Rat models are typically used to assess potential
treatments for spinal cord injury due to their closely described pathophysiology post SCI, easiness
for post-operative care, well established functional recovery analysis procedures, low cost and
low incidence of infections [1]. Well-established and validated animals models include contusion-
compression models, ischemic models, full or partial transection models and chemical models [2].
The specific SCI models are chosen based on the therapy being evaluated and the clinical
motivation for the evaluation. As reported in the introduction, there are various causes for the
spinal cord injury – vehicular accidents, falls, violence and sports activities. Violence-related
injuries account for as high as 14% of total spinal cord injuries in the USA [3]. The causes range
from gun wounds, knives, and Improvised Explosive devices (IEDs). Typically, violence-related
injuries result in complete or partial transection of the cord. These incidences are higher in the
veterans’ population. Out of 250,000 people living with spinal cord injury, 42,000 were veterans
as per the 2009 report of the Veterans Affairs (VA) Department – a staggering 17%. This makes
VA the single-largest network to provide quality care to the veterans suffering from spinal cord
injury across the United States. Therefore, it was critical for us to evaluate our proposed treatment
in the context of military applications – motivation for our work in this chapter [4, 5].
Given the incidence of spinal cord injury in veterans population and major cause of these
injuries being the gun wounds, knife laceration, and IEDs, we were motivated to evaluate the
effect of EGF and SDF-1α locally delivered by the biomaterial Gtn-HPA in promoting spinal cord
injury repair and functional recovery in a 2 mm T8 complete resection rat model. The formulation
of the Gtn-HPA gel and the dosage of the EGF and SDF-1α was chosen based on the in vitro
studies presented in Chapter 3. The evaluation of the performed after four weeks of inducing the
complete resection spinal cord injury and injecting the Gtn-HPA with appropriate growth factors.
The results presented in this chapter provided proof of principle for further testing and were
promising to deduce significant conclusions about the proposed therapy. The work gave a
valuable insight into the interaction of the proposed biomaterial and its interactions with the
surrounding tissue at the injury site. Learnings from this study were incorporated in designing a
second generation Gtn-HPA gel formulation and incorporating more assessment methods to
reliably evaluate the injury site with its population, which are discussed in Chapter 5 and Chapter
6.
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4.2. Overall goal and hypotheses of this chapter
The overall goal of this chapter was to investigate the effect of epidermal growth factor
(EGF) and stromal cell-derived factor-1α (SDF-1α) locally delivered by injectable gelatin-
hydroxyphenyl propionic acid (Gtn-HPA) in promoting healing and functional recovery in a 2 mm
standardized T8 full-resection rat model of SCI.
Chapter-specific hypotheses:
1) Gtn-HPA gel is able to fill in the cavity created by the 2 mm resection during injection.
2) Gtn-HPA is biocompatible and does not elicit major inflammatory response.
3) 8% Gtn-HPA is present in the injury site after four weeks and is permissive of cellular migration
(e. g. stiffness would not be very high to impede cellular migration)
4) EGF can recruit endogenous neural stem cells (NSCs) to the injury site while SDF-1α can
recruit endogenous glial cells to the injury site.
5) Better functional recovery is observed because of the intervention of Gtn-HPA injection with or
without EGF and SDF-1α.
6) SDF-1α enhances the response in all assessment metric compared to EGF delivery.
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4.3. Methods
4.3.1. Gtn-HPA gel fabrication: Raw material of gelatin-hydroxyphenylpropionic acid
conjugate was obtained from Dr. Motoichi Kurisawa at Agency of Science, Technology and
Research (A*STAR), which was prepared as per the protocol described previously [6]. Briefly,
Gtn–HPA conjugates were prepared by a general carbodiimide/active ester-mediated coupling
reaction in distilled water. 3.32 g of HPA was dissolved in 250 ml of mixture prepared with 3:2
ration of distilled water and N,N-dimethylformamide (DMF) respectively. To this, 3.20 g of N-
hydroxysuccinimide and 3.82 g of 1-ethyl-3-(3-dimethylaminopropyl)-carbo-diimide hydrochloride
were added. The reaction mixture was stirred at room temperature for 5 hours, and the pH of the
mixture was kept at 4.7. Then, 150 ml of 6.25 wt% Gtn aqueous solution was added to the reaction
mixture and continuously stirred overnight at room temperature at pH 4.7. Subsequently, the
solution was dialyzed against 100 mM sodium chloride solution for 2 days, 25% ethanol in distilled
water for 1 day and distilled water for 1 day, successively. The purified solution was lyophilized to
obtain the Gtn–HPA conjugate.
8 wt% Gtn-HPA gel was fabricated as per the procedure described in Chapter 3.
Specifically, 12% (w/v) Gtn-HPA conjugate solution was prepared fresh under sterile conditions
and kept on ice. Recombinant rat EGF (Peprotech, Cat# 400-25) and recombinant rat SDF-1α
solutions (Peprotech, Cat# 400-32A) were aliquoted as per the recommended concentration of
1.0 mg/ml and stored at -20°C. Depending upon the experimental group, EGF and SDF-1α were
added to the freshly prepared 12% Gtn-HPA conjugated solution at room temperature. The final
dosage of the EGF and SDF-1α was 6 µg/rat. To this mixture, 2 µl of Horseradish peroxidase
(HRP) (Wako Pure Chemical Industries, Japan) was added with final HRP concentration of 0.1
U/ml. Then, 2 µl of H2O2(Sigma Aldrich) was added to give final H2O2 concentration of 6.8 mM.
Addition of H2O2 initiated the cross-linking and gelation process. The solution was mixed
thoroughly for homogenous gelation and immediately injected into the cavity before the solution
turned into a hydrogel with an appropriate gelation time.
4.3.2. Animal surgical procedure and Gtn-HPA gel injection: Adult female Lewis rats
(Charles River Laboratories, Willington, MA) weighing 201-225 grams were chosen as the rat
species in this study based on the previous work in our laboratory. Animals were given at least
48 hours to acclimatize before the major survival surgery of inducing a spinal cord injury. Before
the surgery, animals were singly housed in a cage as per the housing requirements after the
spinal cord injury to minimize the pain. Survival surgery was performed as per the approval from
Veterans Affairs Boston Healthcare system Institutional Animal Care and Use Committee
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(IACUC) on the protocol #378-J. Animal surgeries were performed at the Animal Research
Facility located in the research building of the Veterans Affairs Jamaica Plains, MA campus.
Animal surgery was performed by the skilled and experienced Dr. Hu-ping Hsu,
Department of Orthopedic surgery, Brigham and Women’s Hospital. The complete resection
model is based on the work done by Cholas et al [7]. After recording their weight immediately
before the surgery, Lewis rats were anesthetized using 2-4 % isoflurane in oxygen through a nose
mask for induction followed by 1-3 % isoflurane for maintenance of anesthesia, during the surgical
procedure. On average, the procedure lasted for 30-45 mins and isoflurane was administered for
the entire duration. After confirming that the rat is unconscious, rats were placed in the prone
position on a flat operating board and all the limbs were gently fixed using rubber bands to stabilize
the rat. The hair on the back of the rat were shaved and the skin was thoroughly cleaned with
betadine. A 4-cm skin incision was made on the thoracic spine portion. The skin and musculature
overlying the thoracic spine was incised along the midline and retracted laterally from the vertebral
column. A laminectomy was performed removing the dorsal aspect of T7-T10 using small bone
rongeurs and microscissors, exposing approximately 10 mm of spinal cord. Bleeding from the
muscle was controlled by Gelfoam® (Pfizer, Andover, MA). The dura will be incised in the midline
and the dorsal spinal artery will be coagulated with a bipolar cautery. A sterile 2 mm plastic
template was placed in the center of the exposed spinal cord and a lateral complete resection of
the spinal cord was created by making two lateral cuts (2 mm apart) using ultra fine surgical
scissors using the 2 mm template as guide (Figure 4.1). A 2 mm gap was created by removing
the tissue between the two lateral cuts. Bleeding was controlled using Gelfoam® placed into the
defect site. Once hemostasis was achieved, the Gelfoam® was removed from the defect and Gtn-
HPA gel was immediately injected that was freshly fabricated immediately before the injection. In
control animals [N], no treatment was injected into the defect. However, 8 wt% Gtn-HPA gel was
injected in the experimental groups as per the dosage mentioned above. In 8wt% Gtn-HPA gel
only [G8] group, Gtn-HPA gel was fabricated and injected without addition of any factors.
However, appropriate growth factors were added to the Gtn-HPA conjugate solution prior to the
gelation for local delivery of EGF and SDF-1α [GE8 and GES8]. In all the groups that involved
injecting the Gtn-HPA gel into the cavity, 20 µl of the 8 wt% Gtn-HPA gel, with or without the
factors were injected into the cavity using a pipette such that gelation will occur rapidly within in a
few minutes to confine the material to the lesion site. The injury was closed after visual
confirmation of complete gelation and filling of the complete 2 mm defect with the Gtn-HPA gel.
Following gel injection, a thin collagen membrane was placed over the top of the spinal cord
wound as dural replacement, to maintain positioning of the scaffold and separate the wound site
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from the overlying tissue. Following treatment and placement of the collagen membrane, the
overlying musculature was closed using 4-0 vicryl sutures (Johnson and Johnson, Sommerville,
NJ) and the skin was closed with wound clips.
4.3.3. Experimental design: There were three experimental groups evaluated in this study
with the fourth group being the control group that received no injection treatment. The animals
were randomly assigned to the groups and were blinded to the surgeon during injection. I was
blinded to the treatment group and animals were identified using their serial number only. The
following table shows the groups and the number of animals in each group.
Experimental treatment Group Duration of
evaluation
Study size
(n)
Control – no injection N 4 weeks 5
8 wt% Gtn-HPA gel only G8 4 weeks 6
8wt% Gtn-HPA gel with EGF GE8 4 weeks 3
8wt% Gtn-HPA gel with EGF and SDF-1α GES8 4 weeks 5
Table 4.1. Experimental Design for the study to evaluate the Gtn-HPA along with EGF and SDF-1a in 2
mm complete section rat model
Figure 4.1. (left) 2 mm complete resection cavity created by laminectomy at T8 level shown with the 2 mm template after achieving hemostasis (right) Cavity injected with the Gtn-HPA gel and covered with collagen membrane as dural replacement
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4.3.4. Animal care post-surgery: Post-surgery, animals were carefully brought to the pre-
OR room and placed in a heated cage with constant supply of oxygen at 1 L/min to maintain body
temperature and breathing. Rats were subcutaneously injected with warm 3 ml of lactated
Ringer’s solution to compensate for the blood loss during the surgery. They were subcutaneously
administered Meloxicam (1 mg/kg) as an analgesic and Cephazolin (35 mg/kg) as an antibiotic to
manage pain and infections, respectively. Rats were monitored for breathing every ten minutes
for an hour after surgery, every half an hour subsequent two hours and every hour for the last
three hours. Continuous oxygen supply was maintained until they were transported to their
housing room in the animal research facility. 6 hours post-surgery, their bladders were expressed
manually and placed in the cage with wood-chip bedding with free access to food and water. Dry
food was kept on the flood of the cage in the initial days for easier access. Rats were kept in a
light/dark automated cycle in the housing room as per the research facility guidelines. Due to the
loss of bladder control, their bladders were manually emptied using Crede’s technique twice a
day with utmost care to minimize the pain. Bladder volume was recorded in three categories –
small, medium and large during manual expression. Rats were subcutaneously administered
analgesic Meloxicam (1 mg/kg) once every 22-24 hours for four days post-surgery while antibiotic
Cephazolin (35 mg/kg) was administered twice in a day for a week post-surgery. They were also
administered lactated Ringer’s solution as necessary based on their hydration status. One week
after the surgery, suture clips were removed after confirming suturing of the skin. Animals were
anesthetized using 2-3% isoflurane for painless removal of the wound clips. They were weighed
once a week and their record was meticulously kept in the housing room as per the VA animal
research facility requirements.
4.3.5. Behavioral assessment – BBB scale: In order to assess the functional recovery of
the animals after the surgery, open-field locomotor test was conducted once every week until
sacrifice. Animals walked freely on the table with absorbent chute. Their movement, especially of
the hindlimbs, was digitally video recorded for three minutes. The animals were scored using the
well-established 21-point Basso-Beattie-Bresnahan (BBB) locomotor rating scale [8]. The scale
ranged from 0 (complete paralysis) to 21 (normal gait) as listed in Table 4.2. The evaluator was
blinded to the treatment group during scoring of the both hindlimbs. Separate BBB score was
assigned to the left and the right hindlimb each week.
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BBB score Description
0 No observable hindlimb movement
1 Slight movement (<50% of the range of motion) of one or two of the three joints – hip, knee and ankle
2 Extensive movement (>50% of range of motion) of one joint and slight movement of another joint
3 Extensive movement of the two joints
4 Slight movement of all three hindlimb joints
5 Slight movement of the two joints and extensive movement of the third joint
6 Extensive movement of two joints and slight movement of the third joint
7 Extensive movement of all three hindlimb joints
8 Sweeping movement (rhythmic movement of all three hindlimb joints) without any weight bearing (elevation of the hindlimb during movement) by the hindlimbs
9 Plantar paw placement with some weight bearing in stationary phase or minor weight bearing with dorsal stepping of the paw
10 Occasional (<50% of the times) weight bearing with plantar placement of the paw without any hindlimb-forelimb (HL-FL) coordination
11 Occasional weight bearing with plantar placement of the paw and occasional FL-HL coordination
12 Frequent (>50% of the times) weight wearing with plantar placement of the paw with occasional FL-HL coordination
13 Frequent weight bearing with the plantar placement of the paw and frequent Hl-FL coordination
14 Consistent (~100%) weight-bearing with plantar steps, consistent FL-HL coordination with rotational movement of the paw during stepping
15 Consistent FL-HL coordination with consistent dragging of the toes and rotational movement of the paw during stepping
16 Consistent FL-HL coordination with frequent dragging of the toes and rotational movement of the paw during stepping
17 Consistent FL-HL coordination with occasional dragging of the toes and rotational movement of the paw during stepping
18 Consistent FL-HL coordination with no dragging of the toes with parallel paw position during liftoff and initial contact
19 Consistent FL-HL coordination, no dragging of the toes, parallel paw position during liftoff and initial contact and tail is down part or all the time
20 Consistent FL-HL coordination, no dragging of the toes, parallel paw position during liftoff and initial contact and tail is consistently up
21 Coordinated gait, consistent toe clearance, predominant paw position is parallel during movement and tail consistent up with full trunk stability
Table 4.2 BBB scale implemented to assess the functional recovery for the rats after the spinal cord injury with the BBB score and corresponding functional description of the hindlimb
Adapted from the Basso et al, J Neurotrauma 1995. 12(1): p. 1-21.
4.3.6. Animal sacrifice and harvesting of the spinal cord: After four weeks, animals were
sacrificed by transcardial perfusion. Rats were administered a dose of 150 mg/kg of sodium
pentobarbital and anesthetized. The level of anesthesia was checked by pinching their tail.
Quickly, a thoracotomy was performed to expose the heart and the needle attached to the
peristaltic pump was inserted into the left ventricle and into the ascending aorta. The pump was
turned on at the speed of four initially and then the right atrium was quickly cut to allow open
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system for the flow of perfusion solution. The speed of the pump was increased to 7. A total of
120 ml of heparinized saline (20 U/ml) was circulated through the animal, followed by 120 ml of
4% paraformaldehyde (PFA) without introducing any bubbles into the line. Both the solutions were
kept on ice during the perfusion. After completing the sacrifice, the spinal column along with the
musculature was cut from the neck region to pelvic region. The entire spine was placed in 50 ml
4% PFA at 4°C overnight. The following day, the spine was trimmed of the overlying musculature
while leaving the vertebral bone and fascia over the defect intact. The trimmed spine was kept in
4% PFA for additional 2 days at 4°C. After three days, they were kept in 60 ml of PBS for three
days at 4°C. Then, the spinal cord was carefully isolated and harvested from the spinal column
using bone rongeurs, surgical scissors, and a scalpel.
4.3.7. Tissue embedding procedure and cryosection: Immediately after harvesting the
cord from the spine column, the spinal cord with the injury site was embedded in Tissue Tek OCT
compound solution kept in a rectangular tinfoil mold (Figure 4.2). The spinal cord was then flash-
frozen in the OCT compound solution using isopentane cooled in liquid nitrogen. The frozen block
with proper labeling with animal information was kept on dry ice until they were stored at -80°C.
The blocks were serially cryosectioned using Leica Cryostat CM 3050 (Leica Biosystems,
Wetzelar, Germany). 30 µm thick coronal sections of the spinal cord were cut and were mounted
on a pre-treated Superfrost Gold glass microscope slides (Fisher Scientific, Hampton, NH).
4.3.8. Histology and histomorphometric analysis: Tissue sections were warmed to room
temperature and then baked at 60 °C for 3 hours prior to histological staining to improve the
adhesion to the glass slides. The tissue sections roughly in the middle of the coronal plane were
chosen to best represent the tissue morphology for each rat. Then, after they reached room
temperature, tissue sections were rehydrated in PBS for 45 mins to dissolve the OCT on the
slides. Tissue sections were stained with Hematoxylin and Eosin (H&E) method to visualize the
Figure 4.2 Embedding the spinal cord (left) gross image of the Spinal cord with the injury site carefully harvested from the spinal column (middle) spinal cord kept in Tissue Tek OCT compund prior to flash-freezing in isopentane cooled in liquid nitrogen (right) Frozen block with spinal cord mounted on the object holder in the cryostat for coronal cryosectioning
87
tissue morphology and the implanted gel at the injury site. After being rehydrated in PBS, the
tissue sections were permeabilized in cold methanol for 10 min followed by immersion in water
for 5 min, rinsed in PBS for 10 min and again immersed in water for 5 min. Then, they were stained
with hematoxylin Gill #2 solution for 4 min followed by thorough wash in running tap water for 5
min. Then, the tissue sections were decolorized with 3 quick drops in acid alcohol and then were
thoroughly washed in running tap water for 5 min. Furthermore, they were stained with eosin for
40 seconds. Tissue sections were dehydrated with 100% alcohol (2 x 3 min) and processed in
xylene (2 x 3 min). Coverslip was mounted on using cytoseal and then the tissue sections were
air dried in a chemical hood overnight.
The brightfield microscopy images were acquired using Olympus microscope. In addition,
Zeiss automated slide scanner was used to scan the entire tissue sections (Zeiss, Germany). For
each animal, gel area and cellular migration area immediately next to the gel islands were
calculated using freehand option in ImageJ (NIH, Bethesda, MD). Two analysis methods were
used to validate the findings. The first method (Method 1 – total area method) measured the total
Gtn-HPA gel area in all the gel islands and total cellular migration immediately around all the gel
islands in the injury cavity were measured in ImageJ using ROI manager tool. The second method
(Method 2 – standardized ROI method) measured the Gtn-HPA gel area and cellular migration
immediately surrounding the gel in three standardized 0.3 mm2 ROI at the interface using ImageJ
in three randomly chosen images. Average gel area and migration area from each animal was
calculated using both the methods and compared among the groups. All histological analyses
were conducted blindly with respect to the treatment group.
4.3.9. Immunofluorescent staining and analysis: Tissue sections were stained with a
biochemical marker to obtain more specific details regarding the cellular population in the injury
site and immediately surrounding the gel islands. Tissue sections were warmed to room
temperature and then baked at 60 °C for 3 hours prior to immunofluorescent staining to improve
the adhesion to the glass slides. Primary antibodies, dilutions and their antigen retrieval were
performed as shown in Table 4.3. Then, after they reached room temperature, tissue sections
were rehydrated in PBS for 45 mins to dissolve the OCT on the slides. Subsequently, antigen-
retrieval was performed for the tissue sections in low pH citrate buffer (Vector laboratories, CA,
USA) at 97 °C for 20 min. Then, tissue sections were cooled to room temperature in the antigen
retrieval solution and then immersed in 0.3% Triton-X in PBS for 30 min. Next, they were washed
with quick dips in PBS and then permeabilized in cold methanol for 10 min followed by PBS wash
for 5 min. Then, the slides were cleaned and marked with an IHC marker around the section to
contain the staining solutions to the sections. The tissue sections were blocked with serum-free
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protein block (Dako, Agilent, USA) for 1 hr. Then, they were washed with 0.05% Tween20 in PBS
solution (5 min) and then in PBS (2 x 5 min). Then, they primary antibodies diluted in Dako
antibody diluent solution were added to the sections in a black staining box with ample moisture.
The slides were then kept overnight at 4 °C. Next day, slides were washed with 0.05% Tween20
in PBS solution (5 min) and then in PBS (2 x 10 min) followed by addition of secondary antibodies
diluted in Dako antibody diluent. The slides were incubated with secondary antibodies for 2 hours
at room temperature in the black staining box. Subsequently, they were washed with 0.05%
Tween20 in PBS solution (5 min) and then in PBS (2 x 10 min). After 3 quick dips in deionized
water, coverslips were mounted with DAPI-containing Fluoro Gel-II mounting medium (Electron
Microscopy Sciences, Hatfield, PA) and stored at 4 °C.
Fluorescent images were acquired using epifluorescence microscope (Olympus BX60)
and confocal laser microscope (Nikon). The images were analyzed using ImageJ to quantify the
signal from the biomarker of interest. Three random images were selected and analyzed with a
standardized 0.3 mm2 ROI. Images were converted to 8-bit images followed by a standard
threshold optimized for each antibody. Then, the percent area covered by the fluorescent signal
in the standardized 0.3 mm2 ROI was measured using ImageJ. The average percent area was
calculated and used for analysis for comparison among the treatment groups.
Primary antibody Dilution Antigen retrieval
Secondary antibody Cells/molecules identified
Rabbit poly clonal anti-GFAP (abcam #ab7260)
1:400 Citrate buffer pH 6 (97 °C at 20 min)
Dylight 488 Donkey anti-rabbit IgG (Jackson Immunoresearch)
Astrocytes
Rabbit monoclonal anti-NeuN (abcam #ab177487)
1:400 Citrate buffer pH 6 (97 °C at 20 min)
Cy3 Red Donkey anti-rabbit IgG (Jackson Immunoresearch)
Mature neurons
Rabbit polyclonal anti-Iba1 (Wako #019-19741)
1:400 Citrate buffer pH 6 (97 °C at 20 min)
Dylight 488 Donkey anti-rabbit IgG (Jackson Immunoresearch)
Microglia/ macrophages
Mouse monoclonal anti-Nestin (Millipore #MAB353)
1:200 Citrate buffer pH 6 (97 °C at 20 min)
Cy3 Red Donkey anti-mouse IgG (Jackson Immunoresearch)
Neural stem cells
Rabbit polyclonal anti-EGF (Peprotech #500-P277)
1:500 Citrate buffer pH 6 (97 °C at 20 min)
Dylight 488 Donkey anti-rabbit IgG (Jackson Immunoresearch)
Rat EGF
Mouse monoclonal anti-MMP2 (Millipore #MAB3308)
1:300 Citrate buffer pH 6 (97 °C at 20 min)
Cy3 Red Donkey anti-mouse IgG (Jackson Immunoresearch)
Gelatinase MMP2
Table 4.3 Immunofluorescent staining of the cells and molecules with their staining protocol
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4.3.10. Statistical analysis: Before the experiment, our power calculation for sample size
determination is based on the desire to determine significant a 30% difference in a selected
outcome variable with a 15% standard deviation, and with α=0.05 and β=0.05. The statistical
significance among the experimental groups for histomorphometric and IHC staining results were
determined by one-way ANOVA analysis using Tukey Post-hoc tests. Non-parametric data such
as BBB scores were analyzed for statistical significance using the Krusal-Wallis Test, Fisher’s
Exact Test or Chi squared test. SPSS 25 (IBM) and Graphpad Prism 8.0 (Graphpad) were used
for the statistical analysis.
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4.4. Results
4.4.1. Qualitative observations of the animals and Gtn-HPA injection: The animals
tolerated the surgery well and had no major complications immediately after the surgery. One
animal from the Gtn-HPA with EGF (GE8) group could not survive due to surgical complications.
Gtn-HPA was injected into the cavity after achieving hemostasis. However, achieving complete
hemostasis was difficult despite the use of Gelfoam® in 6 out of 19 animals. We observed some
bleeding from the cavity during the Gtn-HPA gel injection. Visually, the Gtn-HPA gel achieved
complete gelation in 2-3 minutes. Post-operation, animals gained consciousness in roughly half
an hour after the surgery and were completely paralyzed in their hindlimbs. They had lost the
ability to empty their bladder due to loss of micturition reflex indicated by their full bladders, which
were manually expressed twice a day. All animals showed good markers of recovery in terms of
weight gain and rehydration. No animals were suspected of any infections after surgery. By four
weeks, they had partial return of the function in their hindlimb movement and bladder function.
They were well acclimatized with the open field locomotor test to assess functional recovery.
4.4.2. Functional evaluation using BBB scale: In order to assess the functional recovery
after the spinal cord injury, animals underwent open field locomotor test in which they freely
walked on the table. Prior to surgery, all animals displayed normal gait. However, immediately
after the injury, they suffered from complete paralysis for both the right and left hindlimb. Even
after one week, most animals were scored to be zero (complete paralysis) on the BBB scale in
both hindlimbs. During the course of four weeks, animals showed modest improvement in both
left and right hindlimb (Figure 4.3). As can be seen in the Figure 4-3, the average BBB scores
were less than 7 for all the four groups. After four weeks, all four groups showed functional
improvement compared to the first week. However, the treatment groups containing the Gtn-HPA
gel with the growth factors EGF and SDF-1α did not show better functional recovery compared to
the control (Krusal-Wallis test). To account for the improvement after four weeks from the first
week, each rat was treated as its own control and the improvement in the BBB score was
calculated to assess whether the treatment groups showed better improvement in the functional
score. Although the trend suggests that the treatment groups show more improvement in the BBB
score compared to the control especially in the left hindlimb, there was no statistical significance
observed (Figure 4.4). In addition, the improvement in the BBB scores were categorized in two
types – poor improvement (∆BBB = 0-2) and good improvement (∆BBB = 2+). In order to evaluate
the effect of our intervention of injecting Gtn-HPA gel, the number of animals showing poor and
good improvement were tabulated for both right and left hindlimb after four weeks in form of a
contingency table (Table 4.4). Interestingly, all the rats showed poor improvement in the control
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group while 10/14 rats showed good improvement after the injection of the gel in the cavity for the
left hindlimb. Fisher’s exact test analysis showed significant association between the degree of
improvement and the type of intervention in the left hindlimb indicating that injection of gel shows
better improvement in the BBB score compared to the control (** p<0.01).
Figure 4.3 Animals showed functional recovery after the spinal cord injury in both the right and left hindlimbs (Increase in the BBB score indicates functional recovery). However, treatment groups did not have better recovery compared to the control group after four weeks
Figure 4.4 Improvement in the BBB score was plotted in both the right and left hindlimbs after four weeks using each rat as its own control. Trend seem to indicate more improvement in the treatment groups with Gtn-HPA injection, but no statistical significance was observed. Mean ± SEM
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Table 4.4 Contingency table showing the number of animals showing poor and good improvement in the BBB score after four weeks in the controls versus the treatment group. Fisher’s exact test showed that there is better improvement in function when gel is injected in the cavity in the left hindlimb recovery
4.4.3. Gross histological outcomes: After four weeks, histological images showed
important characteristics of the injury site and the response to the Gtn-HPA gel. Across all the
groups, complete resection of the unaffected tissue was observed in histology and the injury site
was either filled with the fibrous tissue or Gtn-HPA gel. In the control [N] group where no material
was injected in the cavity site, the cavity was covered with dense fibrous tissue with empty areas,
which were presumably filled with cystic fluid prior to the tissue processing (Figure 4.5A).
Moreover, ferritin deposits were seen in the injury site indicative of blood clots. The injury site and
the unaffected spinal cord tissue was visibly different in terms of the morphology (Figure 4.6A).
On both proximal and dorsal ends of the injury, semi-circular stumps were observed, which are
typically observed in response to the severed axons [9]. Normal histological features of white and
gray matter were observed in the unaffected tissue proximal and distal to the injury site. In the
Gtn-HPA only [G8] group, injury site was substantially filled with the Gtn-HPA gel as opposed to
the dense fibrous tissue observed in the control group images after four weeks (Figure 4.5B).
However, dense fibrous tissue was also observed in the areas where the Gtn-HPA gel was not
present. The typical porous fibrillar structure of the Gtn-HPA gel was observed with weakly eosin
stained Gtn-HPA matrix as reported previously [10]. Moreover, usually Gtn-HPA gel was observed
in ‘gel islands’ throughout the injury site and not as a one contiguous structure. Gtn-HPA gel was
not physically continuous with the surrounding tissue. Implanted Gtn-HPA matrix was mostly void
of any cellular infiltration. However, some cells were seen at the boundary of the gel island (Figure
Intervention in the cavity
Poor improvement (∆BBB = 0-2)
Good improvement (∆BBB = 2+)
LEFT
Control – no injection 5 0
Treatment – gel injection 4 10
RIGHT
Control – no injection 2 3
Treatment – gel injection 4 10
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4.6B). The morphology of the Gtn-HPA with EGF [GE8] and Gtn-HPA with EGF + SDF-1α [GES8]
Figure 4.5 Representative H&E stained images of the four groups – (A) Control [N] (B) Gtn-HPA only [G8], (C) Gtn-HPA with EGF [GE8], and (D) Gtn-HPA with EGF and SDF-1α [GES8] showing Gtn-HPA islands and cellular migration around the Gtn-HPA islands in the EGF-delivered groups (i.e. GE and GES8). Scale bar = 1000 µm
gel
gel
gel
A
B
C
D
Gtn
-HP
A o
nly
[G8]
Co
ntr
ol
[N]
Gtn
-HP
A +
EG
F
[GE8
] G
tn-H
PA
+ E
GF
+ SD
F-1 α
[GES
8]
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sections was very similar (Figure 4.5C-D). Like the Gtn-HPA gel only group, histological sections
of these group showed the substantial presence of the Gtn-HPA gel in the injury site in form of
gel islands. However, contrary to the Gtn-HPA gel only group, there was a well-defined physically
continuous interface that was established with the surrounding tissue in the injury site as shown
by high magnification micrographs. In addition, qualitatively, many endogenous cells infiltrated
the injury site and migrated immediately surrounding the gel islands (Figure 4.6C-D). This was
evident by a blue band as a result of hematoxylin stained nuclei of the cells immediately around
the gel islands. The migratory pattern of the cells did not seem to be unidirectional – cells migrated
all around the gel islands. Remarkably, there were few sections in which cells were captured
A: Control [N] B: Gtn-HPA only [G8]
C: Gtn-HPA + EGF [GE8] D: Gtn-HPA+EGF+SDF-1α [GES8]
Figure 4.6 Micrographs showing important observations of the four groups (A) Control - dense fibrous tissue (B) Gtn-HPA only - morphology of Gtn-HPA, 'gel island', minimal cellular migration around the island, poor interface (C) & (D) presence of the Gtn-HPA gel, greater cellular migration around the gel, well-defined interface with the surrounding tissue, and migration of cells through the gel. Scale bar = 100 µm
95
through the gel islands in a somewhat linear arrangement, which could give insights into the
formation of the gel islands and degradation of the gel (Figure 4.6C).
It is important to note that the Gtn-HPA gel was not observed in in the injury site in all the
gel-injected groups. Table 4.5 shows the study size and number of animals that showed the
presence of the gel in the injury site. There are several reasons for the absence of the gel in
certain animals including incomplete gelation due to bleeding during injection, displacement of
the Gtn-HPA gel from the cavity due to animal movement or due to poor containment of the gel
by the collagen fascia membrane and degradation of the gel by four weeks.
Experimental treatment Group Duration of
evaluation
Study
size (n)
# of animals showing
presence of the gel
Control – no injection N 4 weeks 5 N/A
8 wt% Gtn-HPA gel only G8 4 weeks 6 5
8wt% Gtn-HPA gel with EGF GE8 4 weeks 3 3
8wt% Gtn-HPA gel with EGF
and SDF-1α GES8 4 weeks 5 4
Table 4.5. Table comparing the number of animals exhibiting the presence of the Gtn-HPA gel in the
injury site after four weeks in H&E staining
4.4.4. Histomorphometric evaluation of the injury site: Based on the observation that
there was substantial presence of the gel and cellular migration immediately around the gel
islands, there were two methods employed to measure the Gtn-HPA gel area and cellular
migration area immediately surrounding the gel islands in the injury site. The histological sections
evaluated to measure the areas represented the midplane coronal section of the cord.
The first method (Method 1 – total area method) measured the total Gtn-HPA gel area in
all the gel islands and total cellular migration immediately around all the gel islands in the injury
site in ImageJ using ROI manager tool. The average Gtn-HPA gel area was measured to be 0.25
± 0.10 mm2 for the Gtn-HPA only group [G8], 0.32 ± 0.18 mm2 for the Gtn-HPA with EGF group
[GE8] and 0.65 ± 0.36 mm2 for the Gtn-HPA with EGF and SDF-1α group [GES8]. Presence of
the growth factors did not affect the area of the gel present in the injury site (One-Way ANOVA,
Tukey post-hoc test, p > 0.05) (Figure 4.7A). The average cellular migration area immediately
surrounding the gel islands was measured to be 0.07 ± 0.01 mm2 for the Gtn-HPA only group
[G8], 0.49 ± 0.33 mm2 for the Gtn-HPA with EGF group [GE8] and 0.69 ± 0.10 mm2 for the Gtn-
HPA with EGF and SDF-1α group [GES8]. Local delivery of the EGF and SDF-1α with the Gtn-
HPA matrix significantly increased the number of the migrated cells immediately surrounding the
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gel islands compared to Gtn-HPA gel only group (One-Way ANOVA, Tukey post-hoc test, p <
0.05) (Figure 4.7B).
The second method (Method 2 – standardized ROI method) measured the Gtn-HPA gel
area and cellular migration immediately surrounding the gel in the standardized 0.3 mm2 ROI at
the interface using ImageJ in three randomly chosen images. The average Gtn-HPA gel area was
measured to be 0.062 ± 0.009 mm2 for the Gtn-HPA only group [G8], 0.062 ± 0.013 mm2 for the
Gtn-HPA with EGF group [GE8] and 0.053 ± 0.016 mm2 for the Gtn-HPA with EGF and SDF-1α
group [GES8]. Presence of the growth factors did not affect the area of the gel present in the
injury site (One-Way ANOVA, Tukey post-hoc test, p > 0.05) (Figure 4.8A). The average cellular
migration area immediately surrounding the gel islands was measured to be 0.039 ± 0.002 mm2
for the Gtn-HPA only group [G8], 0.112 ± 0.009 mm2 for the Gtn-HPA with EGF group [GE8] and
0.115 ± 0.013 mm2 for the Gtn-HPA with EGF and SDF-1α group [GES8]. Similar to the results
observed in Method 1 (total area measurement), local delivery of the EGF alone or EGF and SDF-
1α in combination with the Gtn-HPA matrix significantly increased the presence of the migrated
cells immediately surrounding the gel islands by 175% compared to Gtn-HPA gel only group
(One-Way ANOVA, Tukey post-hoc test, p < 0.05). However, the addition of SDF-1α did not
further increase the number of migratory cells compared to EGF alone (Figure 4.8B).
A B
Figure 4.7 Histomorphometric analysis performed on the entire injury site to calculate the total gel area and cellular migration immediately surrounding the gel based on Method 1 (total area method). (A) Delivering growth factors with the Gtn-HPA gel did not affect the extent of the gel present while (B) EGF and SDF-1α presence in the gel recruited significantly greater number of cells immediately surrounding the gel in the injury cavity four weeks post injection. * p < 0.05, Tukey post-hoc test and the bars showing Mean ± SEM
97
A B
Figure 4.8 Histomorphometric analysis performed in three random standardized ROI (0.3 mm2) to calculate the total gel area and cellular migration immediately surrounding the gel based on Method 2 (standardized area method). (A) Delivering growth factors with the Gtn-HPA gel did not affect the extent of the gel present while (B) EGF alone and EGF and SDF-1α in combination delivered with the recruited significantly greater number of cells immediately surrounding the gel in the injury cavity four weeks post injection. *** p < 0.001, Tukey post-hoc test and the bars showing Mean ± SEM
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4.4.5. Immunohistochemistry evaluation of the injury site: H&E staining provided general
morphology of the injury site. Next step was to identify the cell population that migrated into the
injury site and surrounding the gel islands. Sections were stained with various primary antibody
markers, results from which are reported below.
4.4.5.1. GFAP – Astrocytes: GFAP was used as a marker to study the response of the
astrocytes in and around the injury site. Numerous GFAP+ cells were observed around the injury
site and at the interface between the unaffected tissue and the injury site upon the qualitative
examination of the images (Figure 4.9). There was high density of the GFAP+ cells bordering the
injury site. The density of astrocytes was very high near the interface between the unaffected
B
C
D
Figure 4.9 Representative GFAP stained sections of the four groups (A) Control [N] (B) Gtn-HPA gel only [G8] (C) Gtn-HPA with EGF [GE8], and (D) Gtn-HPA with EGF and SDF-1α [GES8]. Scale bar = 1500 µm
A
Interface
99
tissue and the injury site, while it decreased towards the proximal and distal unaffected tissue.
Besides at the interface, the astrocytes were sparsely present in the injury site. Some GFAP+
cells had migrated into the injury site and had long thin cells with their astrocytic processes. In
order to assess the presence of astrocytes, GFAP+ cells were quantified at two different locations
– in the injury site and at the interface between the unaffected tissue and the injury site (Figure
4.9BC). Quantitative analysis of the area covered by GFAP+ cells in the injury site indicated no
significant difference among the treatment groups. However, the quantitative analysis of the area
covered by GFAP+ cells at the border of the unaffected tissue and the injury site showed
significant decrease in the presence of astrocytes in the three gel-injected groups (i.e. G8, GE8
and GES8) compared to the control [N] as shown in Figure 4.10 (One-Way ANOVA, Tukey post-
hoc test, p < 0.05). Furthermore, cells migrated immediately surrounding the gel islands did not
stain positive for the GFAP indicating that the cells recruited by EGF and SDF-1α possibly were
not astrocytes (Figure 4.11).
Figure 4.10. Quantitative analysis was performed to measure the %area covered by GFAP+ cells (astrocytes) at two locations - at the interface between the injury site and the unaffected tissue & in the injury site. Injection of Gtn-HPA gel with or without EGF and SDF-1α showed significantly less presence of astrocytes at the interface between the injury site and unaffected tissue while there were no differences among the groups regarding astrocytes present in the injury site. **** p <0.0001 and bars represent Mean ± SEM
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4.4.5.2. Iba-1 – microglia/macrophages: Sections were stained with Iba-1 biomarkers to
study the presence of the microglia and macrophages population. Iba-1+ cells were present
throughout the injury site across all the groups. They were sparsely present in the unaffected
tissue of the spinal cord in its typical morphology – small ramified cells with processes (Figure
4.12A). However, Iba1+ cells in the injury site had different distinct morphology – these cells were
enlarged spherical cells without any processes (Figure 4.12C). This enlarged morphology
suggests that these cells are activated microglia [11]. Interestingly, Iba1+ cells at the border of
the injury site showed both the types of morphology of Iba1+ cells (Figure 4.12B). The difference
in the morphology of the Iba1+ cells in the unaffected tissue and the injury site was consistent
across all the treatment groups. Quantitative analysis of the Iba1+ cells in the injury site showed
that the percent stained area occupied by Iba1+ activated microglia/macrophages was similar
across all the treatment groups (Figure 4.13).
Gtn-HPA+EGF+SDF-1α
*
* *
* *
*
Gtn-HPA+EGF+SDF-1α
Figure 4.11 The cells that have migrated immediately surrounding the gel block stained negative for GFAP indicating that these cells are not astrocytes (Top) GFAP stained section showing the presence of the gel marked by white arrow and the cellular migration around the gel block marked by red asterisks. (Bottom) The same animal H&E showing the presence of the gel and cellular migration for comparison with the GFAP stained section. Scale bar = 1000 µm
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4.4.5.3. NeuN – mature neurons: NeuN is a neuron-specific nuclear protein and is widely
used as a marker for mature neurons. NeuN staining was performed to assess if any newly
generated mature neurons are present either in the injury site or at the border of the injury site.
The staining protocol worked very well, and it showed cell bodies of the neurons stained with
NeuN. NeuN+ cells were present in the gray matter of the unaffected tissue on the proximal and
distal side of the injury site across all the treatment groups. However, very few NeuN+ cells were
present at the interface and arguably no NeuN+ cells were observed in the injury site. This result
was consistent across all the treatment groups (Figure 4.14).
Figure 4.12. Representative micrographs showing the Iba1+ cells present (A) in unaffected tissue with small ramified cells with processes (B) at the interface showing two types of morphologies - small ramified cells and enlarged spherical cells and (C) in injury site exhibiting enlarged spherical cells morphology. Scale bars = 200 µm
unaffected interface Injury site
A B C
Figure 4.13 Quantitative analysis of Iba1+ microglia/macrophages: percent area covered by the microglia/macrophages in the injury site is similar across all four groups. Bars represent Mean ± SEM
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4.4.5.4. MMP2 – Gtn-HPA gel degradation: It was of interest to study the mode of
degradation of the gel and Matrix metalloproteinases (MMPs) are speculated to be the enzymes
responsible for the degradation of ECM proteins. Representative sections were stained with
different anti-MMP antibodies. It was observed that there was strong signal of MMP2 presence at
the border of the Gtn-HPA gel for both Gtn-HPA gel only group [G8] and Gtn-HPA with EGF [GE8]
NeuN/DAPI NeuN/DAPI NeuN unaffected tissue near the interface injury site
G
ES
8
G
E8
G8
N
Figure 4.14. NeuN staining to stain for mature neurons for all four treatment groups - Control (N), Gtn-HPA gel only (G8), Gtn-HPA+EGF (GE8), and Gtn-HPA+EGF+SDF-1α (GES8). The image grid shows the representative images at three different locations – unaffected tissue, near the interface and injury site. There were no mature neurons present in the injury site while neuronal cell bodies were stained well in the unaffected gray matter. Scale bars = 200 µm
103
group (Figure 4.15). This result indicates that MMP2 is one of the molecules responsible for the
degradation of the Gtn-HPA gel in vivo. It is interesting to note the higher cellular density around
the Gtn-HPA gel for Gtn-HPA+EGF [GE8] group compared to Gtn-HPA only [G8] group, as noted
by the DAPI staining. This result is consistent with the observations noted in the H&E staining.
4.4.5.5. EGF – Gtn-HPA incorporated EGF: EGF was locally delivered to the injury site
using Gtn-HPA as a delivery biomaterial. The proposition is that as the gel degrades, EGF will be
released creating a concentration gradient to recruit endogenous neural progenitors and glial cells
in the injury site. In order to assess the presence of EGF in the Gtn-HPA gel, few representative
sections were stained with anti-EGF antibody, we found that wherever we see the presence of
the gel, EGF gave strong signal in the Gtn-HPA gel for the Gtn-HPA+EGF [GE8] group. However,
Gtn-HPA gel in the Gtn-HPA gel only group [G8] stained negative for the EGF, confirming the
presence of the EGF in the gel delivered with EGF four weeks post-injection (Figure 4.16). EGF
staining could be used as a method to reliably stain the Gtn-HPA gel in the EGF delivered groups.
MMP2 DAPI MMP2/DAPI
Gtn-HPA + EGF
gel
Gtn-HPA
gel
Gtn
-HP
A g
el
on
ly
Gtn
-HP
A g
el
+ E
GF
Figure 4.15 Representative MMP2 stained micrographs showing the strong signal of MMP2 at the border (marked by white dotted line) of the Gtn-HPA gel in the injury site as discovered in both the Gtn-HPA gel only [G8] and Gtn-HPA gel+EGF [GE8] group. Scale bars = 200 µm
Gtn-HPA + EGF
gel
Gtn-HPA
gel
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4.4.5.6. Nestin – Neural stem cells: Nestin is a type VI intermediate filament protein found
in neural stem cells. Nestin staining on the sections showed greater Nestin staining in the cells
present around the Gtn-HPA gel in the both the groups where chemoattractants, i.e. EGF and
SDF-1α were delivered with the Gtn-HPA gel [GE8 & GES8] compared to the Gtn-HPA gel only
group [G8] (Figure 4.17B-D). Control group [N] did not show the presence of Nestin positive cells
in the injury site (Figure 4.17A). Nestin staining showed elongated cells oriented perpendicular to
the border of the Gtn-HPA gel indicating the radial migratory pattern of the cells (Figure 4.17CD).
These Nestin positive cells were present predominantly in the cells migrated immediately
surrounding the Gtn-HPA gel in the injury site. It is important to note that these cells around the
gel were GFAP negative (Figure 4.11) and Nestin positive indicating that these are NSCs and not
astrocytes. Quantitative analysis of the Nestin positive cells showed that Gtn-HPA with EGF [GE8]
and Gtn-HPA with EGF+SDF-1α [GES8] groups showed significantly greater presence of NSCs
immediately surrounding the Gtn-HPA gel (One-Way ANOVA, Tukey post-hoc test, p < 0.05).
However, there was no statistical difference between GE8 and GES8 groups indicating that SDF-
1α did not enhance the recruitment of NSCs to the injury site. There was no statistical significance
between the Nestin positive cells between the control [N] and Gtn-HPA gel only [G] group
indicating that the gel alone is not able to recruit these cells to the injury site (Figure 4.18).
EGF DAPI EGF/DAPI
Gtn
-HP
A g
el
+ E
GF
G
tn-H
PA
ge
l o
nly
Gtn-HPA
Gtn-HPA + EGF
Figure 4.16 Representative EGF stained micrographs showing the strong signal of EGF in the Gtn-HPA gel in the Gtn-HPA gel+EGF [GE8] group while Gtn-HPA gel stained negative in the Gtn-HPA gel only group [G8]. Scale bars = 200 µm
105
A B
C D
Gtn-HPA [G8]
Gtn-HPA + EGF
GES8
Figure 4.17 Representative Nestin stained sections show that the (A) control and (B) Gtn-HPA gel only [G8] did not show Nestin+ cells in the injury site while (C) Gtn-HPA+EGF [GE8] and (D) Gtn-HPA+EGF+SDF-1α [GES8] showed significantly higher presence of Nestin+ cells around the Gtn-HPA gel islands (marked by white dotted line). Scale bars = 200 µm
GE8
106
Figure 4.18 Quantitative analysis of the Nestin+ cells showed that EGF and SDF-1 delivered by the Gtn-HPA gel were able to recruit significantly greater number of neural stem cells to the Gtn-HPA gel islands compared to the control and Gtn-HPA gel only group. Moreover, there was no significant difference between the GE8 and GES8 groups (***p<0.001, ****p<0.0001, Tukey post-hoc test; bars represent Mean ± SEM)
107
4.5. Discussion
In this chapter, we investigated the effects of injecting Gtn-HPA gel with or without EGF
and SDF-1α in a cavity created by complete resection of 2 mm of the spinal cord tissue at T8 level
in Lewis rat model. Since traumatic or contusion type of experimental model of spinal cord injury
do not completely sever the cord, surgical models such as complete or partial resection are
utilized to study the healing and neuronal regeneration in the field of tissue engineering [12].
Moreover, complete section models better mimic the most severe clinical ‘complete SCI’ inducing
complete break of the neuronal circuitry as observed in most violence-related SCIs such as gun
and knife wounds [13]. The most important advantage of the complete resection model is the
creation of a reproducible standardized defect at a pre-defined location on the cord. In addition,
complete resection is a model that has been used to study the ‘bridging gap’ therapies such as
scaffolds and similar devices [14]. Therefore, complete resection created a well-defined cavity in
which our proposed biomaterial Gtn-HPA can be injected. We studied four groups namely –
control [N], 8wt% Gtn-HPA gel only [G8], 8wt% Gtn-HPA gel with EGF [GE8], and 8wt% Gtn-HPA
gel with EGF and SDF-1α [GES8]. There were several assessment parameters we employed to
test our hypotheses mentioned in the ‘Overall goal and hypotheses’ section of this chapter.
4.5.1. Functional recovery assessment: Clinically, loss of function drastically affects the
quality of life of the patients. Therefore, improvement in the function is the most critical aspect of
pre-clinical studies that aim to treat SCI. Beattie, Basso, and Breshnahan (BBB) scale is a
validated, standardized and well-accepted behavioral scale that is used to assess the functional
integrity of rats’ hindlimbs post SCIs, usually with the underlying assumption that the cardiac and
respiratory functions are largely unaffected. BBB involves animals walking in an open field with
their hindlimb movement carefully observed. Various neurological functions such as joint
movements, weight bearing, fore/hind limb coordination, paw placement, and the directions of the
tail are judged based on the 21-point BBB scale [8, 15]. In our study, immediately after inducing
a 2 mm complete resection SCI at T8 level, both the right and left hindlimbs were completely
paralyzed. Even after a week, most animals were scored 0 (complete paralysis of hindlimbs) on
the BBB scale for both the hindlimbs. Rats showed very modest spontaneous recovery in the
function in the initial weeks, which is attributed to recovery from the spinal shock and ‘re-wiring’
to facilitate neuronal signal transmission [16]. After four weeks, animals in all four groups saw
modest recovery improving the BBB score by 2 (control) and 3-5 (for the treatment groups). The
extent of the recovery in BBB scores observed after four weeks post complete resection SCI in
our study is consistent with similar studies [1, 17-19]. For example, human dental pulp-derived
108
stem cells were implanted after inducing a complete transection injury in rats. After four weeks,
controls showed improvement in BBB score of 3 points while the treatment groups showed
improvement in the BBB score of 4-6 [18]. Although the trend suggested better improvement in
the treatment groups, our study did not indicate any statistical differences among the groups in
the functional recovery using the BBB scale after four weeks (Figure 4.3). Future study should
focus on repetition of the study with the same number of animals in each group. In order to
account for the differences in the baseline scores of the animals, each rat was treated as its own
control to compare the differences in the scores at 1 (acute) and 4 (chronic) weeks post injury. It
is important to consider that since the BBB scale is not linear, improvement from a score of 2 to
4 is different than the improvement in the score from 4 to 6 even though both would show net
improvement by two points. Interestingly, the number of animals that improved by more than 2
points were greater in the treatment groups compared to the control and were statistically
significant for the left hindlimb but not for the right hindlimb (Table 4.4). However, it is difficult to
pinpoint the exact underlying mechanism due to which more animals showed better improvement
in the Gtn-HPA gel injected groups compared to the controls, given highly sophisticated nature of
the neural tissue after an injury. Various explanations of recovery include the greater sparing of
the axons, intrinsic changes in the circuitry termed as neuroplasticity, and stimulation of the lower
motor neurons not directly affected by the injury to the upper motor neuron [20, 21]. Most probably
explanation is the neuroprotection of further secondary damage caused by the injury due to
implanted Gtn-HPA matrices. It could explain the better net improvement in BBB scores in the
Gtn-HPA injected groups compared to the controls observed in this study. This phenomenon has
been observed in various studies where the implantation of a biomaterial has shown
neuroprotective effects in preventing further degradation of the unaffected spinal cord tissue and
thereby showing better functional recovery in biomaterial treated groups [22-24]. However, longer
time period assessments with more animals would be required to assess potentially significant
benefit of the Gtn-HPA injection to improve functional recovery after complete resection SCI. In
addition, other behavioral tests and electrophysiological measurements should be considered for
functional assessment for future study.
4.5.2. Histological evaluation of the injury site: Histological evaluation was performed four
weeks after inducing SCI in coronal sections. In all the groups, it was evident that the surgery had
indeed created a complete break in the neuronal circuitry. The histological sections displayed
typical characteristics of central gray matter in both hemispheres in both the caudal and rostral
sides of the injury site. The length of the injury site was more than 2 mm supporting the well-
109
known phenomenon that the axons retract in both directions after complete transection and there
is secondary damage to the unaffected tissue progressing the injury [25, 26].
Although no treatment was applied to the control group [N], the injury site was filled two
kinds of morphology – ‘empty spaces’ and dense collagen fibrous tissue (Figure 4.5A). The empty
spaces probably represent the cystic cavitations, which are formation of fluid-filled cysts in
response to the injury. These cavities are usually filled with macrophages and the growth-
inhibiting molecules deposited in response to degradation of myelin [27]. These cavitations are
consistent in the chronology of the pathophysiological response because these cysts are
developed in the subacute and intermediate phase of the response – which coincides with the
four week evaluation of our study [28, 29]. In addition, ferritin deposits were observed in the control
group indicating the remains of the blood clot that was formed due to the bleeding either from
cord parenchyma or from surrounding muscles after creating the surgical injury. It is possible that
the blood clot provided a temporary matrix to the infiltrating inflammatory cells especially because
the dura membrane was compromised due to the surgical nature of the model. Both the
morphologies – cysts and dense fibrous collagen tissue is inhibitory to the cellular migration into
the cavity site for any potential reparative response. Therefore, goal of this study to replace the
growth-inhibitory environment with that of a biomaterial that would be permissive of the cellular
infiltration without eliciting major inflammatory response. Therefore, our proposed therapy
involves injecting a Gtn-HPA matrix in the defect site with chemoattractants, EGF and SDF-1α to
recruit endogenous neural stem cells and glial cells to the injury site.
After four weeks, Gtn-HPA gel covered the predominant area in the injury site in all the
treatment groups. It is important to note that this is the first time it has been shown that 8wt% Gtn-
HPA gel is present in the injury site after four weeks. This is a significant finding in the context of
previous work done in our laboratory with injectable materials for spinal cord repair. Previous work
aimed to develop injectable matrices to promote regenerative response in our laboratory included
the evaluation of collagen-genipin gels [30]. The results indicated that most of the injected
collagen-genipin gel was degraded as early as two weeks after injection. It is essential for the
biomaterial to persist for longer duration to facilitate the infiltration of cells into the defect, better
cell-substrate signaling, remodel the underlying ECM and deliver signaling molecules over long
period of time [31]. The presence of the Gtn-HPA matrix in most animals in all the treatment
groups was assuring that the Gtn-HPA could serve as a reliable scaffold to promote regenerative
response after spinal cord injury. However, we did not see the presence of the Gtn-HPA gel in all
the animals from a treatment group. We believe there are few potential explanations that account
110
for the absence of the Gtn-HPA gel in two out of 13 animals that were injected with Gtn-HPA
matrix in this study (Table 4.5). First, bleeding in the cavity site during the injection could cause
incomplete gelation or more porous Gtn-HPA gel leading to a less stiff Gtn-HPA gel than
anticipated. This would result in faster degradation of the gel in vivo. Second, the animals were
quick to gain consciousness after the surgery and did movements that could lead to dislodging of
the Gtn-HPA gel from the cavity because the dural replacement collagen membrane was placed
to cover the injury site and was not sutured to the cord. Third, some animals could show more
severe inflammatory response due to which the Gtn-HPA gel degraded quickly. These potential
explanations were considered in designing a second generation Gtn-HPA gel and in better
containing the gel in the cavity site.
Another interesting feature of the morphology of the Gtn-HPA gel was the shape of the
Gtn-HPA gel after four weeks. It was predominantly present in the injury site in form of multiple
‘islands’ of varying sizes (Figure 4.5B-C) as opposed to a continuous block (Figure 4.5D). These
islands could mimic the small cavitary lesions, which are common clinical manifestation of the
spinal cord injury due to various disease progressions [32]. We believe there could be several
mechanisms that could leads to the formation of ‘gel islands. First, heterogeneity in the modulus
of the Gtn-HPA gel during gelation could contribute to mechanical differences in the gel. These
differences could result in varying rates of the gel degradation leading to the break in the
continuous matrix resulting in formation of gel islands over time. Second, the number and the
types of cells present around the Gtn-HPA during acute phase of the pathophysiological response
could affect the extent of cellular migration into the Gtn-HPA matrix. For example, macrophages
are shown to occupy the injury site as early as a week after the spinal cord injury and release
various inflammatory cytokines and ECM degrading molecules, especially Matrix
metalloproteinases (MMPs) [33, 34]. MMP2 is a zinc-based gelatinase and we observed strong
MMP2 signal at the border of the Gtn-HPA gel islands in this study (Figure 4.15). Therefore, it is
possible that activated macrophages released significant amount of MMP2 at the border of the
continuous Gtn-HPA block resulting in formation of gel islands during the four weeks. Third,
animal movement immediately after the surgery and within initial weeks could mechanically break
the Gtn-HPA into physically separate entities leading to the formation of gel islands. More detailed
investigation for shorter evaluation duration should be done for future studies. For a proof of
principle, histological evaluation will be performed at earlier time points in few animals to
investigate the phenomenon of formation of gel islands in the study described in Chapter 6.
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Another feature important to discuss is the ability of the Gtn-HPA to establish a continuous
physical interface with the surrounding tissue to re-establish the lost framework due to the injury.
We observed that although Gtn-HPA alone was largely not able to establish an interwoven
physical structure with the surrounding tissue, Gtn-HPA with EGF [GE8] and Gtn-HPA with EGF
and SDF-1α [GES8] was able to form a well-defined continuous interface with the surrounding
tissue re-establishing continuous tissue throughout the injury site (Figure 4.6) This is in
accordance with the findings in various studies that matrices without any form of the molecular
cues failed at establishing a well-defined continuous framework with the surrounding tissue in
tissue repair approach [35, 36]. Therefore, signaling molecules such as EGF and SDF-1α are
critical to include in the injection of Gtn-HPA matrices.
One of the hypotheses of the study was that EGF and SDF-1α would promote the
recruitment of different cells types such as the endogenous neural stem cells and other glial cells
to the injury site. Qualitatively, greater number of cells had migrated to surround the Gtn-HPA gel
islands in both Gtn-HPA with EGF [GE8] and Gtn-HPA with EGF and SDF-1α [GES8] group
compared to the Gtn-HPA gel only [G8] group (Figure 4.5 & 4.6). Moreover, this finding in our
study is in accordance with the conclusion that Gtn-HPA gel is permissive of the cellular migration
both in vitro and in vivo brain ICH model as reported based on previous work in our laboratory
[10] . Quantitative analysis of the Gtn-HPA gel area and cellular migration immediately
surrounding the Gtn-HPA gel islands showed that EGF and SDF-1α were able to recruit
significantly greater number of cells to the Gtn-HPA gel islands compared to the Gtn-HPA gel only
group (Figure 4.7 & 4.8). We validated this result with two different quantification methods
measuring two parameters – gel area and cellular migration area immediately surrounding the
gel. The first method accounted for the all the gel islands and cellular migration immediately
around all of them present throughout the injury site. The second method measured both the
parameters in randomly chosen images in the injury site. Statistical analyses of these parameters
in both the methods showed significantly greater recruitment of cells by EGF and SDF-1α
compared to the control and Gtn-HPA only group (Figure 4.7 & 4.8). Therefore, we deduce that
Gtn-HPA gel alone would not be able to recruit cells to the injury site and therefore, signaling
molecules, i.e. EGF and SDF-1α, are essential. This is a critical result supporting our hypothesis
for this study. However, GES8 group did not show significantly greater cellular migration
compared to the GE8 groups indicating that presence of SDF-1α did not enhance the recruitment
of cells to the injury site. Subsequent to the promising histological observations and conclusions,
our goal was to identify the cells that migrated around the gel islands and in the injury site to study
the cell population of the injury site.
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4.5.3. Immunohistochemical evaluation of the injury site: Several biomarkers were
stained to identify the presence and the extent of various cell types and important molecules in
the context of this study. Specifically, GFAP (astrocytes), Iba1 (microglia/macrophages), NeuN
(mature neurons), Nestin (neural stem cells), EGF and MMP2 (Gtn-HPA gel degradation) were
stained to obtain a comprehensive composition of the injury site post Gtn-HPA implantation with
or without EGF and/or SDF-1α.
Astrocytes have been shown to play a multi-functional role in the context of repair and
regeneration after spinal cord injury. The multifaceted astrocytic response contributes to
neuroprotection of the spinal cord while inhibiting axonal regeneration [37]. Astrocytes are
typically categorized into three types in the CNS – naïve astrocytes, reactive astrocytes and scar-
forming astrocytes [38]. Naïve astrocytes help in maintaining the neuronal parenchyma and the
blood-brain barrier while the reactive astrocytes assist in limiting the spread of inflammatory cells
in response to injury promoting remodeling of the ECM and repair of the tissue [39]. These
reactive astrocytes convert to scar-forming astrocytes based on the environmental cues. They
form the scar by adhering to each other and releasing molecules like CSPG to produce a
penetrating glial scar that impedes axonal regeneration after spinal cord injury [40, 41]. We
observed the presence of GFAP+ cells in the unaffected tissue and in the injury site but
predominantly at the interface between the unaffected tissue and the injury site. Quantification of
GFAP+ cells showed no statistical differences between the groups in the injury site (Figure 4.10).
Moreover, the cellular migration immediately around the Gtn-HPA gel islands stained negative for
the GFAP indicating that those cells are probably not reactive astrocytes because GFAP is
considered to be the hallmark biomarker for reactive astrocytes (Figure 4.11) [42]. However,
quantification of the GFAP+ cells at the interface between the unaffected tissue and the injury site
showed significant reduction in the GFAP+ cells or astrocytes at the interface compared to the
control group indicating that implantation of Gtn-HPA matrix, with or without the growth factors,
into the cavity downregulates the astrocytic response to the injury thereby either delaying the
process of glial scar formation or reducing the extent of glial scar formation in the treatment groups
(figure 4.10). Interestingly, one hypothesis proposed by Macaya in his doctoral work stated that
the elongated astrocytic processes towards the center of the defect are growth-promoting while
the astrocytes bordering the injury site with dense processes are inhibitory [30]. Results of this
study show that although the treatment groups do not increase the presence of the growth
promoting astrocytes in the injury site, the treatment reduces the number of the inhibitory
astrocytes at the border. This is an important evidence to suggest that the treatment reduces the
glial astrogliosis, which could promote beneficial healing of the injury site.
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Post-injury, the resident microglia migrate to the injury site while the monocytes
differentiate into macrophages in the acute phase of the pathophysiological process after SCI.
Since they are phagocytic in nature, they contribute to the debris clearing in the injury site to
mitigate the secondary damage [11, 43]. Macrophages have been classified into two phenotypes
– M1 and M2. M1 is shown to be promoting the release of pro-inflammatory factors while M2
contributes in wound healing and repair. Interestingly, M1 have been shown to have detrimental
effect in the spinal cord injury but M2 macrophages have shown to promote regenerative
response after spinal cord injury [44]. Iba1 is a microglia/macrophage specific marker that was
used to assess the inflammatory response to the Gtn-HPA gel. The morphology of the Iba1+ cells
varied depending upon the location on the section (Figure 4.12). Unaffected tissue had typical
ramified phenotype cells with its processes indicating that these are resident microglia. Injury site
predominantly had enlarged macrophages indicating that these were the activated microglia or
macrophages responding to the injury and contributing the clearing the debris from the myelin
degradation and other necrotic tissue. Interface between the unaffected tissue and the injury site
had combination of both phenotypes possibly indicating that the resident microglia are being
recruited to the injury site in response to the cytokines released in the injury site. This observation
is consistent with the mechanism of the activation of microglia after spinal cord injury [45].
Quantitative analysis did not show statistical differences among the number of
microglia/macrophages present in the injury site among all four treatment groups (Figure 4.13).
This indicates that injection of Gtn-HPA, with or without the factors, did not elicit a severe
inflammatory response providing an in vivo evidence that Gtn-HPA is a biocompatible biomaterial.
Gtn-HPA and its constituents were well tolerated by the rats in the study. This is an important
finding in vivo for the Gtn-HPA material injected for the first time in the spinal cord injury models.
NeuN is a very specific biomarker for nuclear protein expressed in mature neurons. It also
has been used to study the differentiation of stem cells into neurons [46, 47]. Sections from all
treatment were stained with NeuN to assess whether any neuronal lineage was promoted by the
potential regenerative effects of the migrated cells in the injury site. Across all the treatment
groups, we found that there were no NeuN+ cells in the injury site while we were able to visualize
characteristic neuronal cell body stained with NeuN in the unaffected tissue and at the interface
of the unaffected tissue and the injury site. Qualitatively, the number of NeuN+ cells present in
the unaffected tissue were higher than at the interface (Figure 4.14). This finding is consistent
with our expected outcome of NeuN staining, especially four weeks after Gtn-HPA matrix
injection. However, future studies should quantitatively investigate any differences among the
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groups for the presence of neuronal differentiation in the injury site and near the interface for
longer duration post Gtn-HPA matrix injection.
Gtn-HPA is a gelatin-based biomaterial and has excellent tunable properties to control the
gelation time and stiffness by varying the concentration of its constituents [6]. In our study, Gtn-
HPA was used in an in vivo application for the spinal cord injury for the first time. Therefore, it was
important to comment on the potential mode of degradation of the Gtn-HPA in vivo. Although Gtn-
HPA was injected in the brain ICH model, it’s in vivo degradation behavior was not studied
previously [10]. Matrix metalloproteinases (MMPs) were reported to be involved into the cellular
migration of neural progenitor cells [48]. Out of various MMPs that were tested, MMP2 provided
strong signal specifically at the border of the Gtn-HPA gel island in both treatment groups G8 and
GE8 (Figure 4.15). Since the MMP2 signal was strong only at the border of the gel island, we
hypothesize that MMP2 is responsible for the degradation of Gtn-HPA gel to promote cellular
infiltration into the Gtn-HPA matrix. Remarkably, MMP2 is a gelatinase A enzyme and therefore
could be the agent to degrade gelatin-based Gtn-HPA biomaterial in vivo. More detailed
investigation should be performed in the future studies to assess the role of MMP2 with Gtn-HPA.
In addition to MMP2, EGF was the other protein stained with few preliminary sections to confirm
the presence of EGF in the Gtn-HPA gel with EGF [GE8] group. The stained micrographs
confirmed the presence of EGF exclusively in the Gtn-HPA gel island. However, the Gtn-HPA gel
island in the Gtn-HPA gel only group [G8] stained negative for the EGF serving as a negative
control (Figure 4.16). Although no statement can be made about the bioactivity of the EGF after
four weeks because the sections were processed with fixation, it is encouraging that Gtn-HPA
matrix is able to deliver EGF locally to the injury site at least over four weeks. Furthermore, it
could also provide a reliable method to locate the presence of the Gtn-HPA gel in groups where
EGF was delivered with the Gtn-HPA matrix. Specificity of the staining is high such that EGF
stained positive on a very small gel island as well (Figure 4.16, bottom). Future studies should be
designed to assess the bioactivity of the EGF in vivo. For example, EGF delivered by the Gtn-
HPA gel could be pre-labeled with a fluorophore prior to injection and could be longitudinally
followed over four or more weeks and investigate if the signal is limited to the Gtn-HPA gel
location. This experiment could offer interesting insight into the degradation mechanism and EGF
release mechanism in vivo. These preliminary findings will be thoroughly validated in more
animals in Chapter 6 by comparing EGF expression in different groups.
The identity of the cells migrated immediately surrounding the gel islands and into the Gtn-
HPA gel was determined after sections were stained with Nestin to evaluate the presence of
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neural stem cells in the injury site. Nestin is an intermediate type VI protein and is considered as
a biomarker for undifferentiated CNS cells prior to committing to a specific cell lineage, considered
to be the neural stem cells (NSCs) [49]. It is reported that NSCs are found in a niche at the poles
of the spinal cord ependymal layer near the central canal. Similar niche is observed in the SVZ
region in the brain [50]. These niches stain positive for Nestin and negative for GFAP confirming
the presence of NSCs [51]. There is a panel of biomarkers that can be used to further confirm the
cells to be NSCs and include Vimentin, Mushashi-1, and Sox 2 (R&D systems, SC205). In our
study, cells migrated around gel islands stained positive for Nestin for both Gtn-HPA with EGF
[GE8] and Gtn-HPA with EGF and SDF-1α [GES8] (Figure 4.17). We did not encounter any
Nestin+ cells in the injury site for the control group. There were some cells that stained Nestin
positive surrounding the gel islands in the Gtn-HPA gel only [G8]. Quantitative analysis of the
Nestin+ cells showed significantly greater number of NSCs that have migrated to the gel islands
in GE8 and GES8 groups compared to the control and G8 group (Figure 4.18). These results
show that Gtn-HPA gel alone is not capable of recruiting important cells such NSCs while EGF
and SDF-1α were able to recruit the endogenous NSCs from the spinal cord ependymal layer
niche near the central canal. Furthermore, there was no difference between the GE8 and GES8
group indicating that SDF-1α either does not play a role in NSCs recruitment or does not enhance
the recruitment of NSCs to the injury site. This is consistent with the proposed hypothesis that
NSCs are highly responsive to EGF [52]. Hemann et al reported that intrathecal administration
of EGF resulted in significantly greater ependymal cell proliferation in the central
canal immediately rostral and caudal to the lesion compared to controls and in greater white
matter sparing [53]. Similar behavior of the NSCs was observed in our study in response to EGF
four weeks post Gtn-HPA with EGF injection. Future studies should include longer duration of
evaluation and it would be interesting to find out the identity of the migrated cells and assess if
they differentiate into neural or glial fate to promote regenerative response and improve functional
recovery after the spinal cord injury.
In summation, a 2 mm complete resection of the spinal cord tissue at T8 level created a
severe spinal cord injury completely disrupting the neuronal circuity. 8wt% Gtn-HPA persisted in
the injury site four weeks post injection and was permissive of cellular migration, making Gtn-HPA
a promising biomaterial for future studies. Injection of the Gtn-HPA matrix decreased the presence
of growth-inhibitory astrocytes at the border of the injury site thereby reducing the extent of glial
scar formation. Gtn-HPA is well tolerated and did not elicit a severe inflammatory response.
Injecting the 8wt% Gtn-HPA matrix with EGF alone or in combination with SDF-1α promoted
recruitment of endogenous neural progenitor cells to the gel islands in the injury site. However,
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SDF-1α did not enhance the recruitment response. In a severe injury such as 2 mm complete
resection, injecting the Gtn-HPA matrix did not show better functional recovery compared to the
control four weeks post injection. However, left hindlimb shown better improvement in the
treatment groups between first and fourth week in the treatment groups compared to the controls.
The encouraging results in this study laid the foundation for designing a second generation Gtn-
HPA matrix formulation and for rigorous evaluation in a 2 mm hemi-resection animal model of SCI
in Chapter 6.
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Chapter 5:
Developing an ex vivo MRI protocol to
visualize the Gtn-HPA matrix in the
spinal cord
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5.1. Introduction and motivation
MRI plays a critical role in the clinical assessment and management of the spinal cord
injury clinically. Computed tomography (CT) is a widely used imaging modality to evaluate the
bone dislocations or fractures after a traumatic spinal cord injury. Although CT is a more
convenient and cost-effective method than MRI to assess the cause and the location of the injury,
it does not give information about the damage to soft tissues, such as spinal cord [1]. The advent
of MRI in 1980s led the neuroimaging community to realize the powerful capability of the MRI to
study the pathomorphic changes in the cord after injury [2]. In 1988, MRI was used to study
specific relevant pathophysiology for the diagnosis and prognosis after SCI. Specifically, for the
first time, MRI was utilized to identify and characterize the MRI signal patterns for hemorrhage in
the cord, edema in the cord and combination of both hemorrhage and edema [3]. Since then, MRI
has become the standard clinical tool used by the clinicians for neuroimaging. In current times,
MRI is widely used by the clinicians to evaluate the location of the injury, the severity of the injury,
extent of neurological deficit and presence of potential instability of the spine. MRI can serve as
a validation tool for the neurological deficit determined by the ASIA examination in the acute
setting [4]. However, there are certain trauma cases in which MRI is not advised due to logistical
issues such as patient stabilization and to prevent further injury [5]. With advances in the MRI
technology, many MRI sequences are available to the clinicians. Most widely used techniques
include sagittal T1 and T2 weighted imaging. However, short tau inversion recovery (STIR) and
gradient recall acquisition steady state (GRASS) area also implemented in applicable cases [4].
Although MRI is extensively used in the clinical setting, very few experimental SCI model
investigations include studying the injury using MRI. Histopathology is the most common method
used to assess the pre-clinical SCI models. However, histopathology techniques are labor-
intensive, invasive, and destructive for the cord. Moreover, it provides information in 2D plane
only after the duration of the experiment [6]. Longitudinal assessment in the same animal is
practically impossible with the conventional techniques. MRI has many advantages over the
conventional techniques that make it effective to study the experimental models of SCI. In addition
to being non-invasive and non-destructive, it also provides 3D volumetric information about the
morphology of the injured cord in a longitudinal manner such that each animal serves as its own
control [7]. Although in vivo MRI evaluation is difficult in rodent models, various studies have
shown the benefit of using MRI to study various histopathological findings such as edema,
hemorrhage, cyst localization and presence of biomaterials [7-10]. Some studies have utilized
Diffusion-weighted tensor imaging (DWI & DTI) to study the axonal structure, white matter sparing
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after injury and demyelination process [11-13]. It is evident that MRI can provide critical
information after spinal cord injury in a reliable, noninvasive, non-destructive and longitudinal
manner. However, there are only few studies that utilize the MRI to learn the environments around
the implanted biomaterial post injection [14, 15]. One of the potential limitations is that water
accounts for as high as 60-70% of most biomaterials used in the SCI applications. Therefore, MRI
contrast agents such as Gadolinium-based contrast agents are suggested to be incorporated into
the biomaterial for better visualization. However, gadolinium-based contrast agents are relatively
large molecules that can easily affect the gelation procedure, porous structure, stiffness modulus
and other chemically important sites of the biomaterial [16]. Considering the disadvantages of the
contrast agents’ incorporation in Gtn-HPA matrix, I began investigation into developing an MRI-
based protocol to visualize the implanted Gtn-HPA matrix, to be able to differentiate the Gtn-HPA
matrix from the surrounding healthy tissue and to gain insights into the secondary damage due
to the induced SCI in our rodent model without the use of contrast agents. Specifically, in the
context of evaluating a biomaterial matrix such as Gtn-HPA, MRI can provide a powerful tool to
measure Gtn-HPA gel volume, lesion volume, extent of the injury, and study the changes in the
gel volume over the duration of the experiment.
We worked on three different MRI approaches that were promising to exclusively show
the implanted Gtn-HPA matrix in an MRI image. The first approach (Approach 1) employed T2
weighted imaging sequence to generate coronal images of the cord with defect area in the center
of the field of view. The second approach (Approach 2) aims to implement a novel Magnetic
Resonance Elastography (MRE) sequence to identify the Gtn-HPA gel based on the differences
in the mechanical properties of the injury area. The third approach (Approach 3) utilized a
systematic approach using T1-weighted Inversion recovery (T1IR) using various inversion times
(ITs) to generate separate images for the Gtn-HPA matrix and surrounding tissue at the same
location. All images captured were coronal slices with the injured site in the center of field of view.
In addition, evaluator was blinded to the treatment groups in the analysis for method 1 and 3.
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5.2. Overall goal and hypotheses of this chapter
The overall goal of this chapter was to develop a reliable MRI protocol to visualize the
implanted Gtn-HPA matrix in the spinal cord cavity and distinguish the Gtn-HPA matrix from other
surrounding constituents without the use of contrast agents. In this chapter, the focus was to
achieve the goal in an ex vivo setting, which would lay the foundation for developing and
optimizing an in vivo protocol to visualize the Gtn-HPA matrix in longitudinal studies.
The chapter-specific hypotheses are:
1) MRI signatures (T1 or T2) of the Gtn-HPA matrix are different from the surrounding healthy
tissue, edema and hemorrhage to generate contrast using MRI
2) MRI generated images would be of high resolution and will exhibit enough contrast between
the Gtn-HPA matrix and other constituents to localize the defect and extent of the lesion.
3) Parameters such as Gtn-HPA matrix volume and lesion volume would be calculated using
directed analysis of the images generated from the protocol
4) The protocol would be feasible to be implemented in in vivo assessment for future studies
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5.3. Approach 1: T2-weighted MRI
5.3.1. Introduction: Sagittal T2-weighted MRI is the most common MRI sequence ordered
by the physicians in the hospitals for spinal cord injury patients either in the acute or chronic
phase. T2 is one the key MRI signature that is utilized to generate contrast between different
constituents. Transverse relaxation time (T2) is the time constant that measures the time taken
for spinning protons to be completely out of phase perpendicular to the main magnetic field [17].
Essentially, T2 is a transverse magnetization decay time constant (Figure 5.1). In addition to the
key anatomical abnormalities, T2-weighted MRI images show bright signal for CSF, edema and
hemorrhage in the cord after the injury [18]. Given that Gtn-HPA matrix would have high water
content, we hypothesize that the matrix would give strong signal at the injury site in T2-weighted
MRI but would have different signal intensity for edema so that Gtn-HPA matrix can be
distinguished four weeks post injection.
5.3.2. Methods:
5.3.2.1. Surgery procedure and experimental Design: The surgical procedure was
analogous to the one described in detail in Chapter 6 for inducing a 2 mm left hemi-resection SCI,
except that in this procedure, a 1 mm left hemi-resection defect was created. Briefly, 12 Lewis
female rats were induced a left 1 mm hemi-resection SCI at T8 level following a laminectomy. 10
µl of 8 wt% Gtn-HPA matrix with EGF was injected into the cavity after achieving homeostasis
(Table 5.1). All the animal care guidelines post-operation were followed as per the
recommendations of Veterans Affairs Institute of Animal Care and Use Committee (VA IACUC)
under the protocol 335-J. They were administered antibiotics and analgesics as per the
requirements. Animals were sacrificed four weeks after implantation of the Gtn-HPA matrix by
transcardial perfusion of heparinized saline and 4% PFA both kept at 4°C.
Figure 5.1. (Left) T2 relaxation decay of the MRI signal in materials with a short and long T2 (Right) Signal intensities of various materials as observed in a T2 weighted MRI. Reference – mriquestions.com
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Experimental treatment Group Study size (n) Duration of experiment
Control – no injection N 6 4 weeks
8wt% Gtn-HPA with EGF GE8 6 4 weeks
Table 5.1 Experimental groups investigated in the animal study with female Lewis rats
5.3.2.2. Sample preparation and acquisition of MRI: The spinal column was harvested
after the sacrifice and fixed in 4% PFA for 3 days at 4°C. Then, excess musculature around the
spinal column was removed. In order to prepare for ex vivo scan, the samples were immersed in
Fomblin oil to limit tissue dehydration and to generate better contrast. T2-weighted imaging was
performed at the Small Animal Imaging Laboratory (SAIL) at Brigham and Women’s Hospital
using Bruker Biospec 70/30 7.0T magnet with 30 cm bore horizontal magnet. 15 coronal MRI
slices were acquired with long TR (3500 ms) and long TE (80 ms) to produce a T2-weighted
image. Slice thickness was 250 µm and the scan time was 29 min per sample. The matrix size
was selected to be 192 x 192 with FOV being 25.6 x 19.2 mm. The imaging parameters were set
to optimize the visualization of Gtn-HPA gel.
5.3.2.3. Image analysis: RAW images were reconstructed using the inbuilt Bruker
software and were exported as DICOM files, which were later analyzed using ImageJ (NIH,
Bethesda, MD) to calculate the lesion volume using ROI manager. Study size was determined
based on the power calculation to determine significant a 30% difference in a selected outcome
variable with a 15% standard deviation, and with α=0.05 and β=0.05. The statistical significance
between the experimental groups was determined by one-tailed unpaired t-test because the
implantation of biomaterial has shown to reduce the lesion volume. Statistical analysis was
performed with GraphPad Prism (8.0).
5.3.3. Results: The protocol using approach 1 was able to generate high resolution T2
weighted images of the fixed spinal cord four weeks post injection. The resolution of the images
was measured to be 75 µm, considered to be high for similar applications. The images were able
to discern anatomical structures such as bones of the spinal column, vertebral discs, surrounding
musculature, and gray and white matter in the healthy cord in both the experimental groups –
control [N] and Gtn-HPA with EGF [GE8] (Figure 5.2 & 5.3). Importantly, we were able to identify
the injury location based on the expected intense signal due to edema and possibly the Gtn-HPA
gel. However, the images did not distinguish the implanted Gtn-HPA gel in the defect area from
the surrounding edema or fibrous connective tissue. Moreover, the morphology of the injury site
looked the same in terms of signal intensities looked the same in both groups. The images were
not able to distinguish between the fluid-filled injury site in controls [N] and Gtn-HPA matrix filled
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Figure 5.3 Representative 8 MRI images for a rat from Gtn-HPA with EGF [GE8] group clearly showing the location of the injury and the extent of the secondary damage. However, this method was not able to exclusively visualize the Gtn-HPA gel in the injury site.
Figure 5.2 Representative 8 MRI images for a rat from Control [N] group clearly showing the location of the injury and the extent of the secondary damage.
Figure 5.4 Representative H&E images of the (left) control group and (right) Gtn-HPA with EGF group. Gtn-HPA matrix was present four weeks after implantation in the injury site Despite the presence of Gtn-HPA gel, the T2-weighted MRI images failed to show any differences between the injury sites of both groups. Scale bars = 300 µm, Images acquired using 10x objective.
Gtn-HPA
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injury sites in the GE8 group. Histology of the Gtn-HPA with EGF group [GE8] did show the
presence of the Gtn-HPA confirming the presence of the gel in the injury site four weeks post
implantation (Figure 5.4, right). Nevertheless, these T2-weighted images were able to show the
extent of the injury and secondary damage. Therefore, we measured the lesion volume of each
sample using 9 best slices using ROI manager in ImageJ. The lesion area was manually marked
in each of the 9 slices. Total lesion area was calculated by adding the lesion areas of all 9 slices.
The lesion volume was calculated by multiplying the total determined lesion area with the slice
thickness of 250 µm. This was done for all the animals in both the treatment groups. An example
of the marking of the lesion area is shown in the figure 5.5, left. Quantitative analysis showed that
implantation of the Gtn-HPA matrix reduced the lesion volume and extent of secondary damage
compared to the controls (one-tailed t-test, p = 0.057) as shown in figure 5.5, right. Although the
p-value is not entirely less than 0.05, it is very close suggesting that the difference could be of
significance with more animals and with better method to measure the lesion area.
Lesion Volume = (Total lesion area) x 250µm (slice thickness)
Figure 5.5 (left) Lesion area from each of 9 slices were measured using ImageJ by manually drawing the area. The lesion volume was calculated by multiplying the total lesion area with the slice thickness (right) Implantation of the Gtn-HPA matrix reduced the lesion volume and extent of secondary damage compared to the controls, one-tailed t-test, p = 0.057. Although p<0.05 is not true, it is suggestive of real difference in the lesion volumes between the two groups. The result should be validated with other methods for future studies.
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5.4. Approach 2: Magnetic resonance elastography (MRE)
5.4.1. Introduction: Magnetic resonance elastrography (MRE) is novel MRI technique to
compare the biomechanical properties of the soft tissues in a non-invasive and non-destructive
manner. Fundamentally, the working mechanism of MRE is a three-step process. First, shear
waves of a certain frequency are introduced in the sample and image acquisition is performed
simultaneously. Second, the MRI sequence is sensitized to detect the perturbations in the tissue
due to the shear waves. Third, the displacements measured by the MRI acquisition is then
converted into stiffness to generate elastogram of the region of interest in the tissue [19]. The
differentiating feature that generates these elastograms is the displacement affect that the shear
waves have on a soft tissue compared to on a hard tissue. Given that many pathologies involve
significant change in the stiffness of the tissues, MRE is emerging as a diagnostic method to
detect and characterize various disease states [20]. Physicians have used MRE to study chronic
liver diseases to detect liver fibrosis that significantly hardens the liver tissue. Singh et al reported
that MRE had high degree of accuracy in determining the different stages of liver fibrosis
compared to the gold standard of liver biopsy [21]. Considering the promise of MRE in staging
the liver disease for prognosis, MRE has been used in experimental and clinical setting to evaluate
its utility for assessing the degree of fibrosis in the spleen, kidney, breast and brain due to various
pathologic processes [19]. Interestingly, glioblastoma tumors have also been characterized by
MRE and were reported to have lower stiffness owing to softer necrotic tissues [22]. Therefore,
this approach looked very promising to visualize Gtn-HPA gel in the spinal cord tissue because
the stiffness of the Gtn-HPA would be different from the affected tissue and the healthy tissue.
The stiffness maps of the spinal cord could allow us to measure the gel volume and lesion volume.
5.4.2. Methods: Phantom work involving the 8wt% Gtn-HPA gel was performed at the Small
Animal Imaging Laboratory (SAIL) at Brigham and Women’s Hospital using Bruker Biospec 70/30
7.0T magnet with 30 cm bore horizontal magnet. 8 wt% Gtn-HPA gel block was fabricated as per
the dimensional requirement of MRE (15 mm x 15 mm x 5 mm). MRI magnet was customized
with special setup to introduce the shear waves into the Gtn-HPA sample during acquisition
(Figure 5.6). Images were reconstructed as per the method developed by Dr. Samuel Patz. Due
to technical issues such as breathing motion artifacts, CSF motion artifacts, and difficulty in
designing the apparatus for rats, no live animal MRE was performed.
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5.4.3. Results: After overcoming several technical difficulties with the dimensions of gel block
because of its viscoelastic nature, we were able to scan 8wt% Gtn-HPA gel block on its own. MRE
elastogram was generated post reconstruction as shown in the figure 5.7. The elastogram shows
minor heterogeneity in the stiffness values in the block due to fabrication of the Gtn-HPA in such
large volume (~ 2000 µl).
Figure 5.6 Set-up for 8wt% Gtn-HPA gel block designed for MRE. (Left) The shear wave generator was connected to the coil apparatus by a yellow cylindrical tube to deliver shear waves to (right) the coil apparatus (close-up view) physically attached to the gel sample
Figure 5.7 Reconstructed images of the 8wt% Gtn-HPA gel block of size 15 x 15 x 5 mm. (Left) Normal proton density map of the block (right) MRE generated elastogram of the block indicating some minor heterogeneity in the stiffness of the Gtn-HPA gel block. Typically, Gtn-HPA would not be injected in such large volumes and it is homogeneous for the smaller volumes.
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5.5. Approach 3: T1-weighted Inversion Recovery (T1IR)
5.5.1. Introduction: T1-weighted imaging is widely used in the clinical radiology practice to
detect soft tissue contrasts in the MRI to study the fine anatomical structures and pathologic
abnormalities of the brain. In addition, T1-weighted imaging is a preferred technique in cases
where gadolinium-based contrast agents are used. T1-weighted imaging is widely used in the
experimental research to measure the spinal cord volume affected due to an injury or pathological
process [23, 24]. T1-weighted MRI uses short TR and TE times to generate the image. However,
the image contrast of the T1-weighted images is poor and therefore, various modifications of T1-
weighted sequences are utilized by the clinicians and researchers. One of the most common
method is T1-weighted fluid-attenuated inversion recovery (FLAIR). FLAIR has very long TR and
TE to attenuate the signal from CSF. FLAIR provides a remarkable contrast between the edema,
normal parenchyma and normal tissues in the brain [24, 25]. Other T1-weighted methods for
various applications include T1 mapping for cardiovascular imaging, short tau inversion recovery
(STIR), T1-weighted inversion recovery (T1IR) and double inversion recovery (DIR). T1IR is
extremely useful for differentiating the elements that have different T1 times but not different
enough to generate enough contrast by T1-weighted imaging. Image contrast can be generated
by selecting an appropriate inversion time (IT) without compromising the signs of MR signal
intensities [26].
Inversion recovery sequences have shown to be better in determining spinal cord lesions
and discriminate between the edema and healthy tissue in 1980s [27, 28]. The fundamental
working mechanism of the inversion recovery involves selectively nulling the signal for specific
tissues such as fluid, CSF or fat. IR sequences introduce a 180° inversion RF pulse before any
MRI sequence after a pre-defined time known as inversion time (IT). This inversion RF pulse
inverts the direction of the longitudinal magnetization followed by continuation of the T1 recovery
(Figure 5.8). The optimization of the image involves carefully choosing an inversion time such that
the signal from the unwanted element such as CSF, fat or fluid can be nullified to generate an
image with good contrast for the tissues of interest [29]. Clinically, inversion recovery sequences
such as FLAIR are useful in differentiating between intracranial cystic lesions such as arachnoid
cyst and epidermoid cyst. Arachnoid cyst contains CSF meaning that it will be nullified by FLAIR
but epidermoid cyst containing cellular debris is not suppressed using the same sequences [30].
In the context of our work, T1 relaxation values for Gtn-HPA matrix and surrounding spinal
cord tissue was different prompting us to utilize T1-weighted imaging. However, conventional T1-
weighted imaging did not produce good contrast between the Gtn-HPA matrix and the
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surrounding tissue. T1-weighted inversion recovery (T1IR) worked the best to achieve contrast to
scan rat spinal cords ex vivo four weeks after Gtn-HPA matrix injection.
5.5.2. Preliminary phantom work - Relaxometry
5.5.2.1. Methods: A systematic approach was taken to measure the T1 and T2
signatures of the normal spinal cord tissue and Gtn-HPA matrix. 600 µl of 8wt% Gtn-HPA gel was
fabricated. A healthy spinal cord tissue was isolated from the same anatomical location as that of
the injured site in our rodent model. Since our goal was to develop an ex vivo protocol to image
fixed spinal column in our animal studies, both the samples were fixed in 4% PFA overnight at
4°C. Bruker 7T Biospin 70/30 magnet with 30 cm bore was used to scan both the samples. The
MRI scanning was performed at Small Animal Imaging Laboratory (SAIL) at Boston Children’s
Hospital, Boston, MA. Bruker Relaxometry protocol was employed to measure the T1 and T2
properties of the Gtn-HPA gel and healthy spinal cord. Specific T1 and T2 were measured by
drawing an ROI in white matter of the cord, gray matter of the cord and the Gtn-HPA gel.
Figure 5.8. Comparison of the MRI sequence for conventional spin-echo and inversion recovery sequence highlighting the 180° pulse preceded by 90° pulse resulting in the inversion of the signal. Specific Inversion time can be selected to nullify signal from CSF, fat or tissue of interest. Reference – mriquestions.com
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5.5.2.2. Results: The relaxometry protocol was able to measure the T1 and T2 values
for gray matter, white matter and Gtn-HPA gel using the MRI images generated from the scan
(Figure 5.9). T1 and T2 values for the white matter of the fixed cord were 963 and 32 ms
respectively. T1 and T2 values for the gray matter of the fixed cord were 920 and 39.8 ms
respectively. Lastly, T1 and T2 values for the fixed Gtn-HPA were 1840 and 36.5 ms respectively.
The phantom work gave us a satisfying explanation of the failure of T2-weighted MRE to
distinguish the Gtn-HPA from the spinal cord because the T2 values of the cord and Gtn-HPA
were found to be very similar. As far as T1 values are concerned, the values for gray and white
matter of the cord were very similar. However, the T1 value of the Gtn-HPA gel was double the
value the both elements of the spinal cord (Table 5.2). This result was very encouraging for us
because such difference in the T1 value could be exploited to distinguish between the Gtn-HPA
gel and the spinal cord tissue.
Table 5.3 T1 and T2 values of spinal cord tissue and Gtn-HPA gel
White matter Gray matter Gtn-HPA gel
T1 963 ms 920 ms 1840 ms
T2 32 ms 39.8 ms 36.5 ms
Table 5.2 T1 and T2 values of the white matter and gray matter of the cord and Gtn-HPA gel block. Although T2 values were similar, T1 value of the Gtn-HPA was twice that of the spinal cord.
Proton density image T1 Relaxometry T2 relaxometry
Figure 5.9 T1 and T2 relaxometry MRI images used to measure the T1 and T2 values of the 4% PFA fixed spinal cord (top) and Gtn-HPA gel (bottom).
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5.5.3. Preliminary phantom work – T1IR
5.5.3.1. Methods: Based on the findings from the relaxometry phantom work, we
implemented T1-weighted imaging with various TR values but were unsuccessful in developing a
reliable protocol that achieved our objective. Next, we attempted T1-weighted Inversion recovery
with different inversion times (IT). Specimens used were the same as prepared in the ‘Methods’
section 5.5.2.1. Both the spinal cord and the Gtn-HPA gel block were scanned in the same plane.
Imaging parameters were set as follows: TR = 3500 ms, TE = 8.5 ms, and eight different IT = 300,
400, 450, 500, 600, 700, 800 and 900 ms. Multiple Inversion times were studied to find the
inversion time at which spinal cord signal could be completely nullified to exclusively scan only
the Gtn-HPA gel. Likewise, we were interested in finding an inversion time at which Gtn-HPA
signal could be completely nullified to exclusively scan only the spinal cord tissue.
5.5.3.2. Results: T1IR sequence generated individual images in the same plane
containing the spinal cord and the Gtn-HPA gel for eight different inversion times. The goal was
to find inversion time for which the signal from the spinal cord tissue could be nullified to
exclusively observe the Gtn-HPA gel signal. We found that with IT = 450 ms, the signal from the
spinal cord was completely nullified despite it being present in the same place. However, there
was strong signal from the Gtn-HPA block. In contrast, with IT = 800 ms, the signal from the Gtn-
300 ms 400 ms 450 ms 500 ms
600 ms 700 ms 800 ms 900 ms
Figure 5.10 T1-weighted inversion recovery (T1IR) images of the spinal cord and the Gtn-HPA gel in the same plane with different inversion times labeled above the respective image. It is evident that with IT = 450 ms, we only observe the signal from the Gtn-HPA gel nullifying the spinal cord tissue while with IT =800 ms, only the signal from the spinal cord tissue is observed nullifying the Gtn-HPA gel.
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HPA gel was completely nullified while there was strong signal from the spinal cord tissue (Figure
5.10). Proton-density image was acquired on the same plane of scanning to state that both the
Gtn-HPA gel and the spinal cord was present in the plane during the scan with T1IR (Figure 5.11).
Figure 5.11. Proton-density image of the spinal cord and the Gtn-HPA gel during T1IR image acquisition confirming the presence of both samples in the same plane of acquisition
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5.5.4. Ex vivo spinal cord T1IR - Protocol 1.0
5.5.4.1. Methods – surgical procedure and MRI acquisition: Based on the very
encouraging findings from the preliminary phantom work using T1-weighted inversion recovery
with 450 ms IT to visualize the Gtn-HPA gel and 800 ms IT to visualize the spinal cord tissue, we
optimized our protocol for ex vivo scanning of the harvested spinal columns with implanted Gtn-
HPA gels four weeks post injection.
Surgery procedure and sample preparation: The surgical procedure was identical to the
one described in detail in Chapter 6 for inducing a 2 mm left hemi-resection SCI. The same
animals were scanned using this protocol after sacrifice. Briefly, Lewis female rats were induced
a left 2 mm hemi-resection SCI at T8 level following a laminectomy. 20 µl of 8 wt% Gtn-HPA
matrix with EGF was injected into the cavity after achieving homeostasis. All the animal care
guidelines were followed as per the recommendations of Veterans Affairs Institute of Animal Care
and Use Committee (VA IACUC) under the protocol 378-J. They were administered antibiotics
and analgesics as per the requirements. Animals were sacrificed four weeks after implantation of
the Gtn-HPA matrix by transcardial perfusion with heparinized saline and 4% PFA both kept at
4°C. The spinal column was harvested after the sacrifice and fixed in 4% PFA for 3 days at 4°C
followed by 3 days in PBS at 4°C. Then, excess musculature around the spinal column was
removed. In order to prepare for ex vivo scan, the samples were immersed in Galden oil to limit
tissue dehydration and to generate better contrast.
Imaging parameters: T1IR imaging was performed at Small Animal Imaging Laboratory
(SAIL) at Boston Children’s Hospital in Boston using Bruker Biospec 70/30 7.0T magnet with 30
cm bore horizontal magnet. 16 coronal MRI slices each were acquired with long TR (3500 ms)
and short TE (8.5 ms) for both inversion times, 450 ms and 800 ms. Slice thickness was 220 µm
and the scan time was 34 mins per inversion time per sample totaling 68 min per sample. The
matrix size was selected to be 192 x 192 with FOV being 20 x 20 mm. All MRI safety precautions
and standards were followed as per the requirements set by the SAIL at Children’s Hospital.
5.5.4.2. Results and discussion: After localizing the injury area in the center of field of
view and setting proper geometry using quick localizer scans, the T1IR images at inversion time
of 450 ms showed strong signal indicating the presence of the Gtn-HPA gel in the animals injected
with the Gtn-HPA matrix. The final resolution of the images was 110 µm. The signal from the
spinal cord tissue was completely nullified. Simultaneously, T1IR images at inversion time of 800
ms showed signal from the fixed spinal cord tissue but the area of the Gtn-HPA gel was completely
dark. Images in the same geometrical location from both inversion times were complementary to
each other meaning the area with bright signal with 450 ms IT was dark in images with inversion
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time of 800 ms (Figure 5.12). In the control groups where no Gtn-HPA gel was injected, the injury
site showed no signal at IT of 450 ms demonstrating that the protocol is sensitive only to the Gtn-
HPA matrix and not edema, hemorrhage or fibrous interstitial tissue. Gtn-HPA gel volume four
weeks post injury was calculated using these MRI images in ImageJ (results are reported in
Chapter 6). Moreover, Gtn-HPA signal was observed in one hemisphere of the spinal cord as
expected in this hemi-resection model. Future studies should employ the final protocol in
complete-resection models and assess whether the signal from the Gtn-HPA matrix is seen in
both hemispheres at the injury location.
Overall, Protocol 1.0 was able to exclusively show the presence of the Gtn-HPA matrix in
the injury site in one hemisphere of the cord using T1IR imaging sequence with IT of 450 ms.
Images with IT of 800 ms served as the evidence that indeed the signal from the Gtn-HPA was
nullified as predicted by the phantom work describing the signal intensities of the spinal cord and
Gtn-HPA matrix. However, one ambiguity arose from the CSF signal seen in the subarachnoid
spaces along the spinal cord. The signal intensity of the CSF was high in images with IT = 450
ms. Although the anatomical location of the CSF was different from the location of Gtn-HPA
matrix, our goal was to optimize the protocol to reduce ambiguity between the signal from the
Gtn-HPA matrix and CSF. Another improvement in the Protocol 1.0 that we aimed was to increase
the signal-to-noise ratio to produce better quality images.
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IT = 800 ms IT = 450 ms
Gtn-HPA gel only
[G8]
Gtn-HPA with EGF
[GE8]
Control
[N]
Figure 5.22 Representative images from T1IR imaging of (top & middle) two rats showing the presence of the Gtn-HPA matrix in half of the injury site at IT = 450 ms while nullifying the Gtn-HPA signal with IT = 800 ms. However, (bottom) control group rat showing no signal at the injury site at both inversion times indicating that the protocol is sensitive to detecting the presence of Gtn-HPA matrix four weeks post injection
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5.5.5. Ex vivo spinal cord T1IR - Protocol 2.0
5.5.5.1. Methods – surgical procedure and MRI acquisition: The goal was Protocol
2.0 was to improve upon the Protocol 1.0 by reducing ambiguity in the T1IR images between the
Gtn-HPA matrix and the CSF signal in the images with IT of 450 ms. The surgical procedure,
tissue preparation for MRI and the imaging parameters remain the same as described in the
‘Methods’ section of Protocol 1.0. Given that CSF has a very long T1, we theorized that longer
inversion times could nullify the CSF signal. Hence, we did T1IR imaging of the fixed spinal cords
at higher inversion times of 900, 950, 1000, 1100, 1200 and 1300 ms. In order to improve the
signal-to-noise ratio, number of averages to generate the image was doubled to theoretically
increase the signal-to-noise ratio by 40%. The scan time was increased significantly as a result
of doubling the number of averages. In the final Protocol 2.0, each sample was scanned with
three inversion times 450 ms, 800 ms and 1100 ms. The scan time of each inversion time was 68
mins as a result of doubling the averaging, increasing the total sample acquisition time to roughly
3 hours 30 mins.
5.5.5.2. Results and discussion: Out of all the inversion times investigated, 1100 ms
was chosen to scan all the future fixed spinal cords. Although T1IR images at 1100 ms did not
completely nullify the CSF signal, we were able to observe a distinctive feature between Gtn-HPA
and CSF. There was no signal from the CSF while the signal from Gtn-HPA was visible in all the
preliminary samples with Gtn-HPA implanted. Therefore, IT of 450 ms showed the presence of
Gtn-HPA and CSF, IT of 800 ms showed the presence of spinal cord tissue and IT of 1100 ms
showed the presence of spinal cord tissue and the gel but not CSF. Additional inversion time of
1100 ms cleared the ambiguity between the signal from the Gtn-HPA gel and CSF (Figure 5.13).
Moreover, doubling the number of averages during acquisition improved the quality of the images.
Protocol 2.0 provided reliable and definitive distinction of the Gtn-HPA gel from the surrounding
tissue and CSF. This protocol would serve as the foundation to optimize an in vivo protocol to
study the location of Gtn-HPA, volume of Gtn-HPA and the features of the surrounding tissue
near the injury site over the duration of the animal experiment. One of the most interesting
application of in vivo imaging would be to generate a degradation profile of Gtn-HPA matrix in
each rat by calculating the Gtn-HPA gel volume each week post injection. Future studies should
involve in vivo imaging of the rats from various SCI models to gain real-time insights into the
progression of the injury and the Gtn-HPA characteristics in a longitudinal manner.
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IT = 800 ms IT = 450 ms IT = 1100 ms
G
tn-H
PA
+ E
GF
[G
E8
]
Gtn
-HP
A +
EG
F [G
E1
2]
Gtn
-HP
A g
el o
nly
[G
8]
Figure 5.33 Representative Ex vivo T1IR images from groups in which Gtn-HPA was injected in a left 2 mm hemi-resection cavity with three different inversion times (ITs) – 800 ms, 450 ms and 1100 ms. IT of 450 ms showed presence of Gtn-HPA gel and CSF while IT of 800 ms showed the presence of spinal cord tissue with dark regions where CSF and Gtn-HPA is present. IT of 1100 ms produced images that reliably differentiates between the Gtn-HPA gel and CSF because CSF appears dark while Gtn-HPA in the injury site shows greater signal.
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5.6. Summary and discussion
The long-term goal of the work presented in this chapter was to develop tools and methods
to study the presence of implanted biomaterial (Gtn-HPA in our work) and the progression of the
injury in SCI animal models in a longitudinal manner. Conventionally, histological and
immunofluorescent techniques are used to study the general morphology and the specific cellular
composition with identifying the ECM. However, these conventional methods are invasive,
destructive for the sample and rely heavily on the expertise of the investigator. Technological
advances in the MRI techniques has made MRI an enticing technique to study various
pathological conditions. For example, the extent of remyelination after an intervention is studied
with the help of advanced MRI methods such as diffusion tensor imaging (DTI), Magnetization
transfer (MT) imaging and myelin water fraction imaging [31]. Few pre-clinical studies have also
developed parameters acquired in MRI to study and progression of the spinal cord injury in
patients with Multiple Sclerosis [32]. However, only few experimental SCI studies implanting
biomaterials at the location of the SCI employ MRI to study the progression of the disease, track
the presence of implanted biomaterial and correlate the histological findings with MRI [9, 10].
Zhang et al studied the effect of fibrin-based hydrogel in regenerating axons using tractography.
They reported that the hydrogel was able to promote the restoration of fibers post SCI using DTI
[33]. In the context of our work, our first goal was to develop an MRI-based protocol that reliably
discriminates between the Gtn-HPA matrix, surrounding spinal cord tissue and other pathologic
features. Three approaches were taken to achieve the goal – T2-weighted imaging, magnetic
resonance elastography and T1-weighted inversion recovery.
T2-weighted imaging was successful in discerning the healthy spinal cord tissue from that
of affected injury site (Figure 5.3). Intense bright signal marked the affected spinal cord tissue at
the injury site. It is known that high water content elements would show high signal intensity in a
T2-weighted image. Since the injury area is marked by the presence of fluid-filled cavities, necrotic
tissue and Gtn-HPA in the case of gel-injected groups, images indeed show high intensity signal
at the injury site. However, the radiologic signatures were the same for both control and gel-
injected groups suggesting that T2-weighted imaging was not capable of differentiating between
the Gtn-HPA matrix from the fluid-filled cavity and other pathologic elements at the injury site. The
presence of Gtn-HPA was confirmed with the histological processing of the same samples (Figure
5.4). Nonetheless, we were able to measure lesion volume from the T2-weighted MRI images.
This was a significant improvement over the conventional histopathology techniques, where we
would have to use all the sections of the sample to calculate the lesion volume. Using MRI, we
were able to obtain this critical information in a non-invasive and non-destructive method. The
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quantitative analysis of the lesion volume showed that the tissue affected by the spinal cord injury
is reduced when Gtn-HPA treatment is injected after inducing a SCI (Figure 5.6). This is consistent
with the results from pre-clinical studies that claim the neuroprotective effects due to the
implantation of biomaterial into the cavity post SCI [34, 35]. Automated algorithms to identify the
injured area using MRI images with reduced slice thickness would be an interesting approach that
future studies can take, where image processing would be the central theme of the project. Given
that the slice thickness was comparatively high (250 µm), the measured lesion area is assumed
to be the same throughout the 250 µm, which might not be a good assumption. Therefore, smaller
slice thickness images can be taken to obtain a more accurate measurement of the lesion volume.
Overall, T2-weighted images gave critical insights into the injury area but was not able to
distinguish the Gtn-HPA matrix from the surrounding tissue.
Another promising approach that we attempted was the novel magnetic resonance
elastography (MRE). It has been clinically used to detect tissues with abnormal stiffness and have
applications in liver fibrosis, amyloid plaques, and studying tumors. We wanted to generate an in
vivo elastogram to differentiate Gtn-HPA matrix, interstitial fibrous tissue and surrounding healthy
tissue by exploiting the differences between their stiffness. MRE can be followed every week to
generate weekly elastogram to study the change in the modeling of the tissue properties at the
injury site and to study the changes in the volume of the Gtn-HPA matrix over the course of the
experiment. Our primary goal was to calibrate the MRE with our Gtn-HPA matrix such that the
values can be used in the reconstruction to accurately identify the presence of Gtn-HPA matrix.
Initial phantom work generated an elastogram for the Gtn-HPA matrix block, but the
reconstruction values were less reliable owing to heterogeneity in the sample (Figure 5.7). The
heterogeneity is attributed to the volume of the block fabricated to meet the dimensional
requirements of the MRE. There were other technical difficulties as well due to which we were not
able to work on the in vivo implementation of MRE to study the injury site after SCI. These include
anticipated motion artifacts from breathing and cardiac movement, motion artifacts from moving
CSF near the cord and the size of rat spinal cord. Moreover, the setup of the MRE was 3D printed
for mouse imaging in the collaboration facility. However, MRE is a promising technique that can
be applied well to our application. A project leader expert and interested primarily in the imaging
aspect of this work could gather the resources and expertise required to bring MRE to fruition.
The third approach with T1-weighted inversion recovery was the breakthrough protocol by
which we achieved our goal of developing a reliable method to distinguish Gtn-HPA gel from the
surrounding spinal cord tissue. The systematic approach was beneficial to developing T1IR based
protocol. First, T1 and T2 values of the spinal cord and the Gtn-HPA was measured. Our T2
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values for the white and gray matter of the fixed spinal cords were measured to be 32 and 39.8
ms respectively (Table 5.2). The measured values agreed with previously T2 reported values for
fixed spinal cord tissues within assumed experimental error. Carvlin et al reported T2 values of
white and gray matter as 44 and 46 ms respectively for fixed rat spinal cords [36]. Our T1 values
for the white and gray matter of the fixed spinal cords were measured to be 963 and 920 ms
respectively. Carvlin et al reported T1 values of white and gray matter as 250 and 253 ms
respectively for fixed rat spinal cords using 1.5T magnet. According to the physics of MRI, T1
values increase as the field strength of the magnet increases. Doubling the magnet strength could
increase the T1 value by 25% [37]. Considering this phenomenon, the T1 values agree with the
modified reported values. Interestingly, the T2 values of the Gtn-HPA gel and the spinal cord were
very similar. It explained the reason behind not being able to discern the Gtn-HPA gel from the
surrounding tissue using T2-weighted imaging in approach 1. However, T1 value of the Gtn-HPA
gel was twice compared to the spinal cord tissues. This difference in T1 values proved to be the
turning point to achieve our objective. Phantom work using T1IR with inversion time (IT) of 450
ms completely nullified the spinal cord tissue signal while the Gtn-HPA signal intensity was high.
T1IR with IT of 800 ms did exactly the opposite. This systematic phantom work laid the foundation
for achieving our objective with ex vivo scanning of fixed spinal columns containing the defect
site. T1IR of fixed spinal columns showed Gtn-HPA exclusively with IT of 450 ms and spinal cord
tissue exclusively with IT of 800 ms (Figure 5.12). Moreover, the anatomical location of the Gtn-
HPA presence was also reassuring – the signal intensity was highest in the injury site only in one
half of the spinal cord as expected in a hemi-resection SCI rats in which Gtn-HPA was injected.
This protocol 1.0 was an important feat because it gave irrefutable evidence regarding the
presence of the Gtn-HPA gel. With these images, we were able to calculate the Gtn-HPA gel
volume present in the injury site four weeks post injection (Gel volume data are reported in
Chapter 6). However, T1IR images with IT of 450 ms also exhibited high signal intensity from the
CSF fluid in the subarachnoid space. Although the anatomical location of the Gtn-HPA gel and
CSF was different, our next objective was to reduce or remove ambiguity between the signal from
the Gtn-HPA gel and CSF. Protocol 2.0 included T1IR imaging with an additional IT of 1100 ms
that showed that the signal from CSF was still nullified while there was appreciable signal intensity
from the area depicting the presence of Gtn-HPA gel in the images with IT of 450 ms. This
additional set of images with IT of 1100 ms distinguished the Gtn-HPA from CSF both regarding
MRI signal intensity and anatomical location (Figure 5.13). Protocol 2.0 also produced clearer
images with double averaging increasing the totals can time to 3 hours 30 mins per sample.
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We think ex vivo T1IR imaging with three ITs 450 ms, 800 ms and 100 ms coupled with
T2-weighted imaging of the spinal columns would give the most comprehensive view of the
characteristics and metrics of the Gtn-HPA gel and the injury site in our hemi-resection models.
Future studies could focus on validating the metric measured by MRI images with the histological
findings to assess if MRI metrics can be reliable and accurate predictors of the assessment
parameters conventionally obtained from the histopathological findings. For example, a validation
study comparing the Gtn-HPA gel volume measured with histology and MRI can be performed.
Moreover, future study can also investigate the percent volume acquired by the Gtn-HPA gel in
the lesion volume and can compare similar metrics measured by histological quantitative analysis.
If MRI seems to be a reliable and reproducible method to measure critical parameters such as
gel volume and lesion volume, in vivo implementation of the Protocol 2.0 should be implemented
to track the Gtn-HPA gel and its volume over the duration of the experiment in the longitudinal
manner. MRI could play a valuable role in giving important insights into the progression of the
injury, degradation behavior of Gtn-HPA gel and potential regenerative response in experimental
SCI models. Correlation between MRI metrics and functional recovery should be studied to
assess if MRI metrics could predict the functional recovery in the rats after SCI. It would drastically
improve the clinical role of MRI in predicting functional recovery in response to implantable
injectable biomaterials such as Gtn-HPA in promoting repair and functional recovery in patients.
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33. Zhang, Z., et al., Effect of hierarchically aligned fibrin hydrogel in regeneration of spinal cord injury demonstrated by tractography: A pilot study. Sci Rep, 2017. 7: p. 40017.
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Chapter 6:
Investigating the effects of Gtn-HPA
matrix with EGF and SDF-1α in a T8 2
mm hemi-resection rat model of SCI
after four weeks
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6.1. Introduction and clinical motivation
Traumatic spinal cord injury (SCI) is a life changing condition that causes severe disability,
dire reduction in the quality of life and high mortality [1]. Although the rehabilitation efforts assist
in improving the lives of the patients, the root cause of the problem – degeneration of the neuronal
tissue – is not addressed by the current clinical management. Therefore, various experimental
SCI models have been developed to understand the complex pathophysiological progression
after the injury and investigate the effects of potential therapies that could assist in unravelling a
promising treatment of SCI. Plethora of research approaches have shown promise in promoting
tissue repair and functional recovery after the SCI using the SCI models [2]. Various strategies
for SCI tissue repair such as pharmacological agents, exogeneous cells and growth factors have
been evaluated in pre-clinical studies and clinical studies for spinal cord repair and regeneration.
For the acute intervention, various pharmacological agents such as sodium channel blocker, anti-
NOGO antibodies, Rho-ROCK inhibitors, minocycline and myelin signal inhibitors to promote
neuroprotection and regenerative response after the injury [3]. Various cells such as induced
pluripotent stem cells (iPSCs), neural stem cells (NSCs), olfactory ensheathing cells (OECs),
schwann cells, and bone-marrow derived mesenchymal stem cells (bMSCs) have been implanted
after inducing SCI to investigate potential functional recovery in animals and regeneration of the
spinal cord tissue [4]. However, implanting cells in the spinal cord can be challenging and costly.
In addition, non-autologous cells also increases the risk of rejection worsening the injured tissue
after SCI [5]. Therefore, another approach is to deliver growth factors either in suspension,
intrathecally or in situ using a biocompatible biomaterial. The goal is to chemotactically recruit the
endogenous stem cells and glial cells to the injury site before the impenetrable glial scar is formed.
Therefore, these interventions are made either in the subacute or the intermediate phase of injury
progression after the SCI [6, 7]. Various neurotrophic factors such as nerve growth factor (NGF),
brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF), neurotrophin-3 (NT-3)
and epidermal growth factor (EGF) have been tested in animals models and have shown
promising neuroprotection, tissue remodeling and functional recovery after SCI [8, 9].
There are several factors that directed the choice of the hemi-resection experimental
animal model – clinical motivation, the type of therapy being tested, and the anticipated effect of
the therapy. In our work, we were motivated to propose a therapy for small cavitary lesions in the
spinal cord. Incomplete SCI accounts for 68% spinal cord injuries [10]. These cavities do not
generally affect the entire spinal cord in the axial plane. Since our goal was not aimed at studying
a specific function or a spinal tract, we employed left hemi-resection rat model of SCI. Clinically,
this is comparable to Brown-Sequard syndrome in which the patients suffer from ipsilateral upper
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motor neuron deficiencies and contralateral loss of temperature and pain [11]. Hemi-resection
models are widely used and considered to be ideal for studying axonal regeneration as a result
of growth-promoting trophic molecules, biomaterials implantation or cellular implantation [12].
Hemi-resection produces a standardized defect of preferred size and confirms the complete break
in the neuronal circuitry at the location of the cord. In addition, since the resection is made only
one side of the spinal cord, the contralateral side can serve as a control. The most important
advantage in the context of our work is that hemi-resection model creates a reproducible defect
in which biomaterial of a same volume can be injected along with growth-promoting factors such
as EGF and SDF-1α. Building on the results of the evaluation of the Gtn-HPA gel in a complete
resection model reported in Chapter 4 and a reliable MRI-based method T1-weighted inversion
recovery (T1IR) to visualize Gtn-HPA gel in the injury site as reported in Chapter 5, we were
interested in investigating the effects of delivering EGF and SDF-1α using injectable Gtn-HPA gel
in an 2 mm left lateral hemi-resection rat model. In this chapter, learnings from the Chapter 4 and
5 are considered to optimize the Gtn-HPA matrix to develop a second generation Gtn-HPA gel
for injecting into the injury site. In addition to testing the first generation 8wt% Gtn-HPA matrix,
we have evaluated a second generation 12wt% Gtn-HPA matrix to reduce the degradation rate
of the Gtn-HPA gel. Moreover, we have improved on the surgical parameters that could affect the
gelation and containment of the Gtn-HPA gel in the cavity created by a hemi-resection SCI.
Specifically, we have devised a better technique to achieve hemostasis after inducing SCI so that
Gtn-HPA gel can be injected into a ‘clean’ cavity devoid of blood. In addition, we modified the
dural replacement after injection of the Gtn-HPA matrix to better contain the biomaterial in the
cavity site. There were few procedural improvements done to improve the quality of histological
assessment of the spinal cord section. First, fixed cords were immersed in subsequent 15% and
30% sucrose solution to remove water molecules to prevent the formation of crystals during flash-
freezing, which can significantly alter the cellular architecture. Second, cryosection temperature
was optimized to reduce the horizontal and vertical tears in the section. Third,
immunohistochemistry protocol was optimized for several biomarkers, especially regarding the
antigen retrieval protocols. We believe that these methods and the second generation Gtn-HPA
gel is better equipped to improve the reparative and regenerative response in the injury site post
SCI. The results of the studies are promising and consistent throughout various assessment
parameters. Although the animal model and the proposed therapy has few limitations, findings
from this chapter provides a fundamental proof of principle demonstrating the benefit that local
delivery of EGF delivered using Gtn-HPA matrix provides in promoting functional recovery and
reparative response post SCI.
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6.2. Overall goal and hypotheses of this chapter
The overall clinical goal of the project was to develop an injectable polymer incorporating
therapeutic molecules for the treatment of cavitary lesions that can result from SCI. The
supposition was that improved clinical outcome will result from enabling neural tissue to fill the
cavitary defect and to establish neural connections with the surrounding cord tissue. Moreover,
injection of the polymer into an acute lesion may mitigate the secondary damage.
The goal of this chapter was to evaluate a novel gelatin-based conjugate capable of
undergoing covalent cross-linking after being injected as a liquid. The gelatin hydrogel (Gtn-HPA)
will be tested alone and incorporating epidermal growth factor (EGF) and/or stromal cell-derived
factor - 1α (SDF-1α) in a T8 2 mm hemi-resection rat model to assess the tissue healing and
functional recovery four weeks post injection of the treatment.
Chapter-specific hypotheses:
We hypothesize that the gelatin-based gel (Gtn-HPA) incorporating chemoattracting
molecules, EGF and SDF-1α, leads to recruitment of endogenous neural stem cells into the gel-
filled lesion. Moreover, we hypothesize that the acute injection of the gel improves functional
recovery in the rats after four weeks. Specific hypotheses are:
1) Gtn-HPA is biocompatible and does not elicit major inflammatory response.
3) 8% and 12% Gtn-HPA gel are present in the injury site after four weeks and is permissive of
cellular migration (e. g. stiffness would not be very high to impede cellular migration)
4) EGF can recruit endogenous neural stem cells (NSCs) to the injury site while SDF-1α can
recruit endogenous glial cells to the gel-filled lesion.
5) Better functional recovery is observed as a result of Gtn-HPA injection with or without the EGF
and SDF-1α. The functional outcome is better compared to the complete resection model. There
is greater preservation of spared tissue in Gtn-HPA injected groups
6) SDF-1α enhances the response in all assessment metrics compared to EGF.
7) MRI aids visualization of Gtn-HPA matrix and quantification of the gel volume in the lesion
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6.3. Methods
6.3.1. Gtn-HPA gel fabrication: Raw material of gelatin-hydroxyphenylpropionic acid
conjugate was obtained from Dr. Motoichi Kurisawa at Agency of Science, Technology and
Research (A*STAR), which was prepared as per the protocol described previously [13]. Briefly,
Gtn–HPA conjugates were prepared by a general carbodiimide/active ester-mediated coupling
reaction in distilled water. 3.32 g of HPA was dissolved in 250 ml of mixture prepared with 3:2
ration of distilled water and N,N-dimethylformamide (DMF) respectively. To this, 3.20 g of N-
hydroxysuccinimide and 3.82 g of 1-ethyl-3-(3-dimethylaminopropyl)-carbo-diimide hydrochloride
were added. The reaction mixture was stirred at room temperature for 5 hours, and the pH of the
mixture was kept at 4.7. Then, 150 ml of 6.25 wt% Gtn aqueous solution was added to the reaction
mixture and continuously stirred overnight at room temperature at pH 4.7. Subsequently, the
solution was dialyzed against 100 mM sodium chloride solution for 2 days, 25% ethanol in distilled
water for 1 day and distilled water for 1 day, successively. The purified solution was lyophilized to
obtain the Gtn–HPA conjugate.
8 and 12 wt% Gtn-HPA gel was fabricated as per the procedure described in Chapter 3
with few modifications. Specifically, 12% w/v (to prepare 8% Gtn-HPA gel) Gtn-HPA and 18% w/v
(to prepare 18% Gtn-HPA gel) conjugate solution was prepared fresh under sterile conditions and
kept on ice. Recombinant rat EGF (Peprotech, Cat# 400-25) and recombinant rat SDF-1α
solutions (Peprotech, Cat# 400-32A) were aliquoted as per the recommended concentration of
1.0 mg/ml and stored at -20°C. Depending upon the experimental group, EGF and SDF-1α were
added to the freshly prepared 12% or 18% Gtn-HPA conjugated solution at room temperature.
The final dosage of the EGF and SDF-1α was 6 µg/rat. To this mixture, 2 µl of Horseradish
peroxidase (HRP) (Wako Pure Chemical Industries, Japan) was added with final HRP
concentration of 0.1 U/ml. Then, 2 µl of H2O2 (Sigma Aldrich) was added to give final H2O2
concentration of 6.8 mM. Addition of H2O2 initiated the cross-linking and gelation process. The
solution was mixed thoroughly for homogenous gelation and immediately injected into the cavity
before the solution turned into a hydrogel with an appropriate gelation time.
6.3.2. Animal surgical procedure and Gtn-HPA injection: Adult female Lewis rats
(Charles River Laboratories, Willington, MA) weighing 201-225 grams were chosen as the rodent
species in this study based on the previous work in our laboratory. Animals were given at least
48 hours to acclimatize before the major survival surgery of inducing a SCI. Before the surgery,
animals were singly housed in a cage as per the housing requirements after the SCI to minimize
the pain. Survival surgery was performed as per the approval from Veterans Affairs Boston
Healthcare system Institutional Animal Care and Use Committee (IACUC) on the protocol #378-
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J. Animal surgeries were performed at the Animal Research Facility (ARF) located in the research
building of the Veterans Affairs Jamaica Plains, MA campus. Animal surgery was performed by
the skilled and experienced Dr. Hu-ping Hsu, Department of Orthopedic surgery, Brigham and
Women’s Hospital. The hemi-resection model is based on the work done by Cholas et al [14].
After recording their weight immediately before the surgery, Lewis rats were anesthetized using
2-4 % isoflurane in oxygen through a nose mask for induction followed by 1-3 % isoflurane for
maintenance of anesthesia, during the surgical procedure. On average, the procedure lasted for
30-45 mins and isoflurane was administered for the entire duration. After confirming that the rat
is unconscious, rats were placed in the prone position on a flat operating board and all the limbs
were gently fixed using rubber bands to stabilize the rat. The hair on the back of the rat were
shaved and the skin was thoroughly cleaned with betadine. A 4-cm skin incision was made on
the thoracic spine portion. The skin and musculature overlying the thoracic spine was incised
along the midline and retracted laterally from the vertebral column. A laminectomy was performed
removing the dorsal aspect of T7-T10 using small bone rongeurs and microscissors, exposing
approximately 10 mm of spinal cord. Bleeding from the muscle was controlled by Gelfoam®®
(Pfizer, Andover, MA). The dura was incised in the midline and the dorsal spinal artery was
coagulated with a bipolar cautery. A sterile 2 mm plastic template was placed in the center of the
exposed spinal cord and a lateral hemi-resection of the spinal cord was created by making two
lateral cuts (2 mm apart) using scalpel #11 using the 2 mm template as guide (Figure 6.1A). After
the lateral cut, a midline cut was made by scalpel between the two lateral cut using dorsal spinal
vein as the anatomical marker for the midline. A 2 mm gap was created by removing the tissue
between the two lateral cuts. Bleeding was controlled using Gelfoam® placed into the defect site.
The Gelfoam® was kept for a total of 15 minutes changing the Gelfoam® every 5 minutes to
achieve better hemostasis (Figure 6.1B). Once hemostasis was achieved, the Gelfoam® was
removed from the defect and Gtn-HPA gel that was freshly fabricated immediately before the
injection was injected to fill the cavity (Figure 6.1C). In control animals [N], no treatment was
injected into the defect. However, 8 wt% Gtn-HPA gel was injected in the experimental groups as
per the dosage mentioned above. In 8wt% Gtn-HPA gel only [G8] group, Gtn-HPA gel was
fabricated and injected without addition of any factors. However, appropriate growth factors were
added to the Gtn-HPA conjugate solution prior to the gelation for local delivery of EGF and SDF-
1α [GE8 and GES8]. 12 wt% Gtn-HPA was injected into the Gtn-HPA with EGF group [GE12] and
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Gtn-HPA with EGF with ‘tucked-in’ fascia group [GE12T]. In all the groups that involved injecting
the Gtn-HPA gel into the cavity, 20 µl of the 8 wt% or 12 wt% Gtn-HPA gel, with or without the
factors were injected into the cavity using a pipette such that gelation will occur rapidly within in a
Figure 6.1 Top – Schematic of the surgical procedure, Gtn-HPA injection and dural replacement placement (A) 2 mm left hemi-resection cavity created by laminectomy at T8 level shown with the 2 mm template. It is important to note that bleeding covers the entire cavity (B) Gelfoam is used as a hemostatic agent and is kept for a total of 15 minutes in the cavity to achieve hemostasis (C) Gtn-HPA is immediately injected into the cavity (D) Cavity is covered with collagen membrane as dural replacement followed by suturing the overlying musculature
A B
C D
A B
C
Gtn-HPA gel
Dural replacement (autologous fascia)
T8 Left lateral hemi-resection Acute Gtn-HPA injection
Inject 20 µl 2 mm
Histological plane of analysis
153
few minutes to confine the biomaterial to the lesion site. The injury was closed after visual
confirmation of complete gelation and filling of the 2 mm hemi-resection defect with the Gtn-HPA
gel. Following gel injection, a thin collagen membrane was placed over the top of the spinal cord
wound as dural replacement to maintain positioning of the scaffold and separate the wound site
from the overlying tissue (Figure 6.1D). Following treatment and placement of the collagen
membrane, the overlying musculature was closed using 4-0 vicryl sutures (Johnson and Johnson,
Sommerville, NJ) and the skin was closed with wound clips.
6.3.2.1. Improvement in the dural replacement technique: Based on our observations
regarding the presence of the Gtn-HPA gel in the injury cavity after four weeks in both complete
transection and hemi-resection model, we brainstormed various ways to better contain the Gtn-
HPA gel in the cavity. As a result, a modification was made in the placing of the dural replacement
membrane. Specifically, the collagen fascia was trimmed small enough only to cover the spinal
cord at the injury site (Figure 6.2A). In addition, the fascia membrane was ‘tucked-in’ around the
perimeter of the exposed cord (Figure 6.2B). The goal was to fully contain the gel in the cavity by
reducing the likelihood of the Gtn-HPA being dislodged from the cavity due to movement of the
rats after the surgery.
6.3.3. Experimental design: There were six experimental groups evaluated in this study
with the seventh group being the control group that received no injection treatment for the total
duration of four weeks (Table 6.1). The animals were randomly assigned to the groups and were
blinded to the surgeon during injection. I was blinded to the treatment group and animals were
identified using their serial number only. Various assessment parameters were employed to
evaluate the effect of the proposed therapy including the behavioral assessment,
histomorphometric analysis and immunohistochemical analysis. The time course of the
Figure 6.2 Modification in the dural replacement technique for group 12wt% Gtn-HPA with EGF (A) Collagen fascia dural replacement was trimmed small enough only to cover the spinal cord area (B) Schematic showing the ‘tucking-in’ of the fascia membrane around the surface of the spinal cord to better contain the Gtn-HPA in the cavity (C) Smaller fascia placed around the spinal cord cavity site
A B C
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experiment describing the SCI injury, injection of the treatment, behavioral testing, manual
bladder expression and sacrifice are shown in figure 6.3. BBB scoring and the quantification
analyses were performed blinded to the treatment group.
Experimental Treatment Group Duration Study size (n)
Control – No injection N 4 weeks 6
EGF in suspension E 4 weeks 5
8 % Gtn-HPA gel only G8 4 weeks 6
8 % Gtn-HPA + EGF GE8 4 weeks 6
8 % Gtn-HPA + EGF + SDF-1α GES8 4 weeks 6
12 % Gtn-HPA + EGF GE12 4 weeks 6
12 % Gtn-HPA + EGF - ‘tucked-in’ dura GE12T Day 0 1
12 % Gtn-HPA + EGF - ‘tucked-in’ dura GE12T Day 1 3
12 % Gtn-HPA + EGF - ‘tucked-in’ dura GE12T Day 7 3
12 % Gtn-HPA + EGF - ‘tucked-in’ dura GE12T 4 weeks 6
Table 6.1 Experimental design to evaluate Gtn-HPA along with EGF and SDF-1α in a T8 2-mm left hemi-resection SCI rodent model
6.3.4. Animal care post-surgery and bladder function evaluation: Post-surgery, animals
were carefully brought to the pre-OR room and placed in a heated cage with constant supply of
oxygen at 1 L/min to maintain body temperature and breathing. Rats were subcutaneously
injected with warm 3 ml of lactated Ringer’s solution to compensate for the blood loss during the
Day 0 7 14 21 28
SCI & Gtn-HPA
injection Sacrifice
Bladder expression
Behavioral
assessment
Figure 6.3 Time course of the experiments detailing inducing SCI, Gtn-HPA injection, skin wound clip removal, manual bladder expression, behavioral testing by open field locomotor test and sacrifice after four weeks. Post-sacrifice harvested spinal columns were scanned with MRI. After MRI, spinal cord samples were prepared for histological and immunofluorescent analysis.
Skin Clip removal
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surgery. They were subcutaneously administered Meloxicam (1 mg/kg) as an analgesic and
Cephazolin (35 mg/kg) as an antibiotic to manage pain and infections, respectively. Rats were
monitored for breathing every ten minutes for an hour after surgery, every half an hour subsequent
two hours and every hour for the last three hours. Continuous oxygen supply was maintained until
they were transported to their housing room in the animal research facility. 6 hours post-surgery,
their bladders were expressed manually and placed in the cage with wood-chip bedding with free
access to food and water. Dry food was kept on the flood of the cage in the initial days for easier
access. Rats were kept in a light/dark automated cycle in the housing room as per the research
facility guidelines. Due to the loss of bladder control, their bladders were manually emptied using
Crede’s technique twice a day throughout the duration of the experiment with utmost care to
minimize the pain (Figure 6.3). Bladder volume was recorded in three categories – small, medium
and large during manual expression. In addition, the first day any rat showed some sign of return
of bladder function was noted in the records. At the end of four weeks, frequency of animals in
each treatment group was categorized into three urine output volumes – small, medium and large.
Moreover, the number of animals in each group that showed at least partial return of the bladder
function was noted. The effect of treatment on the bladder function will be quantified using Chi-
square test.
Rats were subcutaneously administered analgesic Meloxicam (1 mg/kg) once every 22-
24 hours for four days post-surgery while antibiotic Cephazolin (35 mg/kg) was administered twice
in a day for a week post-surgery. They were also administered lactated Ringer’s solution as
necessary based on their hydration status. One week after the surgery, skin wound clips were
removed after confirming the suturing of the skin (Figure 6.3). Animals were anesthetized using
2-3% isoflurane for painless removal of the wound clips. They were weighed once a week and
were noted of any flinching behavior and other concerns. The record was meticulously kept in the
animal housing room as per the VA animal research facility requirements.
6.3.5. Behavioral assessment – BBB scale: In order to assess the functional recovery of
the animals after the surgery, open-field locomotor test was conducted once every week until
sacrifice (Figure 6.3). Animals walked freely on the table with absorbent chute. Their movement,
especially of the hindlimbs, was digitally video recorded for three minutes. The animals were
scored using the well-established 21-point Basso-Beattie-Bresnahan (BBB) locomotor rating
scale on live animals and recorded videos [15]. The scale ranged from 0 (complete paralysis) to
21 (normal gait) as listed in Table 6.2. The evaluator was blinded to the treatment group during
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scoring of the both hindlimbs. Separate BBB score was assigned to the left and the right hindlimb
each week.
BBB score Description
0 No observable hindlimb movement
1 Slight movement (<50% of the range of motion) of one or two of the three joints – hip, knee and ankle
2 Extensive movement (>50% of range of motion) of one joint and slight movement of another joint
3 Extensive movement of the two joints
4 Slight movement of all three hindlimb joints
5 Slight movement of the two joints and extensive movement of the third joint
6 Extensive movement of two joints and slight movement of the third joint
7 Extensive movement of all three hindlimb joints
8 Sweeping movement (rhythmic movement of all three hindlimb joints) without any weight bearing (elevation of the hindlimb during movement) by the hindlimbs
9 Plantar paw placement with some weight bearing in stationary phase or minor weight bearing with dorsal stepping of the paw
10 Occasional (<50% of the times) weight bearing with plantar placement of the paw without any hindlimb-forelimb (HL-FL) coordination
11 Occasional weight bearing with plantar placement of the paw and occasional FL-HL coordination
12 Frequent (>50% of the times) weight wearing with plantar placement of the paw with occasional FL-HL coordination
13 Frequent weight bearing with the plantar placement of the paw and frequent Hl-FL coordination
14 Consistent (~100%) weight-bearing with plantar steps, consistent FL-HL coordination with rotational movement of the paw during stepping
15 Consistent FL-HL coordination with consistent dragging of the toes and rotational movement of the paw during stepping
16 Consistent FL-HL coordination with frequent dragging of the toes and rotational movement of the paw during stepping
17 Consistent FL-HL coordination with occasional dragging of the toes and rotational movement of the paw during stepping
18 Consistent FL-HL coordination with no dragging of the toes with parallel paw position during liftoff and initial contact
19 Consistent FL-HL coordination, no dragging of the toes, parallel paw position during liftoff and initial contact and tail is down part or all the time
20 Consistent FL-HL coordination, no dragging of the toes, parallel paw position during liftoff and initial contact and tail is consistently up
21 Coordinated gait, consistent toe clearance, predominant paw position is parallel during movement and tail consistent up with full trunk stability
Table 4.2 BBB scale implemented to assess the functional recovery for the rats after the SCI with the BBB score and corresponding functional description of the hindlimb
Adapted from the Basso et al, J Neurotrauma 1995. 12(1): p. 1-21.
6.3.6. Animal sacrifice and harvesting of the spinal column: After four weeks, animals
were sacrificed by transcardial perfusion. Rats were administered a dose of 150 mg/kg of sodium
pentobarbital and anesthetized. The level of anesthesia was checked by pinching their tail.
Quickly, a thoracotomy was performed to expose the heart and the needle attached to the
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peristaltic pump was inserted into the left ventricle and into the ascending aorta. The pump was
turned on at the speed of 4 initially and then the right atrium was quickly cut to allow open system
for the flow of perfusion solution. The speed of the pump was increased to 7 after cutting the right
atrium. A total of 120 ml of heparinized saline (20 U/ml) was circulated through the animal,
followed by 120 ml of 4% paraformaldehyde (PFA) without introducing any bubbles into the line.
Both the solutions were kept on ice during the perfusion. After completing the sacrifice, the spinal
column along with the musculature was cut from the neck region to pelvic region. The entire spine
was placed in 50 ml 4% PFA at 4°C overnight. The following day, the spine was trimmed of the
overlying musculature while leaving the vertebral bone and fascia over the defect intact. The
trimmed spine was kept in 4% PFA for additional 2 days at 4°C. After three days, they were kept
in 60 ml of PBS for three days at 4°C.
6.3.7. MRI ex vivo scan of the spinal column: The excess musculature around the spinal
column was removed to prepare them for the MRI (Figure 6.4A). In order to prime the spinal
column for ex vivo scan, the samples were immersed in Galden oil to limit tissue dehydration and
to generate better contrast in a 15 ml Eppendorf tube. Galden oil is considered proton free and is
expected to generate no signal during MRI acquisition. Galden oil is not shown to affect the post-
processing methods including tissue embedding and histological processing [16]. T1-weighted
inversion recovery (T1IR) imaging was performed at Small Animal Imaging Laboratory (SAIL) at
Boston Children’s Hospital in Boston using Bruker Biospec 70/30 7.0T magnet with 30 cm bore
horizontal magnet. Once the sample was positioned in the coil, quick localization scans were
executed to assess the location of the injury. The images were used as a reference to define the
location and the geometry for T1IR scanning. Then, either protocol 1.0 or protocol 2.0 was
implemented depending upon the group as mentioned in the detailed imaging parameters below
for both protocols. All MRI safety precautions were followed as per the requirements of the SAIL
at Children’s Hospital, Boston, MA.
Imaging parameters Protocol 1.0: The MRI protocol 1.0 was implemented as described in Chapter
5 to scan N, G8, GE8 and GES8 groups as outlined in Table 6.2. Briefly, 16 coronal MRI slices
each were acquired with long TR (3500 ms) and short TE (8.5 ms) for both inversion times, 450
ms and 800 ms. Slice thickness was 220 µm and the scan time was 34 mins per inversion time
per sample totaling 68 min per sample. The matrix size was selected to be 256 x 256 with FOV
set to 20 x 20 mm.
Imaging parameters Protocol 2.0: The MRI protocol 2.0 was implemented as described in Chapter
5 to scan GE12 and GE12T groups as outlined in Table 6.2. Briefly, 16 coronal MRI slices each
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were acquired with long TR (3500 ms) and short TE (8.5 ms) for three inversion times, 450 ms,
800 ms and 1100 ms. In addition, the averaging during acquisition was doubled than that of
protocol 1.0. Slice thickness was 220 µm and the scan time was 68 mins per inversion time per
sample totaling 3 hours 30 mins per sample. The matrix size was set to be 256 x 256 with FOV
set to 20 x 20 mm.
6.3.8. Gtn-HPA gel volume determination using MRI images: As described in Chapter
5, T1-weighted inversion recovery with inversion time (IT) of 450 ms was able to specifically
visualize the Gtn-HPA present in the injury site four weeks post injection. T1IR images with IT of
800 ms and 1100 ms were used to make accurate judgement about the presence of the Gtn-HPA
gel. Therefore, all the MRI slices obtained by T1IR imaging with IT of 450 ms were used to
calculate the area signaling the presence of Gtn-HPA in ImageJ using ROI manager feature for
each rat. Given that the slice thickness was known to be 220 µm, Gtn-HPA gel volume was
calculated by multiplying the total Gtn-HPA area in all the slices with the slice thickness (220 µm).
The quantification of the Gtn-HPA area was conducted blindly with respect to the treatment group.
6.3.9. Cryoprotection, tissue embedding and cryosection: After ex vivo scans, spinal
columns were wiped to remove as much Galden oil as possible. Then, spinal columns were
immersed in PBS overnight at 4°C. Next day, the spinal cord was carefully isolated and harvested
from the spinal column using bone rongeurs, surgical scissors, and a scalpel (Figure 6.4B). Then,
spinal cords were carefully placed in 15% sucrose solution in a 6 well plate until the cord sank.
Figure 6.4 (A) Gross image of the spinal column prepared for MRI ex vivo scan (B) Harvesting the spinal cord from the spinal column (C) gross image of the spinal cord showing the defect site filled with Gtn-HPA gel (D) spinal cord kept in Tissue Tek OCT compund prior to flash-freezing in isopentane cooled in liquid nitrogen (E) Frozen block with spinal cord mounted on the object holder in the cryostat for coronal cryosectioning
A B
C D E
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Subsequently, the cords were kept in 30% sucrose solution in a 6 well plate until the cord sank.
Usually, it took a day for the cords to sink in 15% sucrose solution while it took roughly two days
for the cords to sink in 30% sucrose solution. After the cryoprotection method, the spinal cords
with the injury site were embedded in Tissue Tek OCT compound solution kept in a rectangular
tinfoil mold (Figure 6.4CD). The spinal cords were then flash-frozen in the OCT compound
solution using isopentane cooled in liquid nitrogen. The frozen block with proper labeling with the
animal information was kept on dry ice until they were stored at -80°C. The blocks were serially
cryosectioned using Leica Cryostat CM 3050 as shown in figure 6.4E (Leica Biosystems,
Wetzelar, Germany). 30 µm thick coronal sections of the spinal cord were cut and were mounted
on a pre-treated Superfrost Gold glass microscope slides (Fisher Scientific, Hampton, NH).
6.3.10. Histology and histomorphometric analysis: Tissue sections were warmed to room
temperature and then baked at 60 °C for 3 hours prior to histological staining to improve the
adhesion to the glass slides. The tissue sections roughly in the middle of the defect in coronal
plane were chosen to best represent the tissue morphology for each rat. Then, after they reached
room temperature, tissue sections were rehydrated in PBS for 45 mins to dissolve the OCT on
the slides. Tissue sections were stained with Hematoxylin and Eosin (H&E) method to visualize
the tissue morphology and the implanted gel at the injury site. After being rehydrated in PBS, the
tissue sections were permeabilized in cold methanol for 10 min followed by immersion in water
for 5 min, rinsed in PBS for 10 min and again immersed in water for 5 min. Then, they were stained
with hematoxylin Gill #2 solution for 4 min followed by thorough wash in running tap water for 5
min. Then, the tissue sections were decolorized with 3 quick drops in acid alcohol and then were
thoroughly washed in running tap water for 5 min. Furthermore, they were stained with eosin for
40 seconds. Tissue sections were dehydrated with 100% alcohol (2 x 3 min) and processed in
xylene (2 x 3 min). Coverslip was mounted on using cytoseal and then the tissue sections were
air dried in a chemical hood overnight.
The brightfield microscopy images were acquired using Olympus microscope. In addition,
Zeiss Axioscan.Z1 automated slide scanner was used to scan the entire tissue sections (Zeiss,
Germany). For each animal, gel area and cellular migration area immediately next to the gel
islands were calculated using ROI manager in ImageJ (NIH, Bethesda, MD). Two analysis
methods were used to validate the findings. The first method (Method 1 – total area method)
measured the total Gtn-HPA gel area in all the gel islands and total cellular migration immediately
around all the gel islands in the injury cavity were measured in ImageJ using ROI manager tool.
The second method (Method 2 – standardized ROI method) measured the Gtn-HPA gel area and
cellular migration immediately surrounding the gel in three random standardized 0.3 mm2 ROI at
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the interface using ImageJ in three randomly chosen images. Average gel area and migration
area from each animal was calculated using both the methods and compared among the groups.
All histological analyses were conducted randomly and blindly with respect to the treatment group.
6.3.11. Spared tissue area and injury area quantification: Immediately after left hemi-
resection, the right hemisphere of the cord was kept intact. However, secondary damage after
the injury could lead to sparing of various degrees of the tissue in the right hemisphere of the
spinal cord. Using the H&E images, cross-sectional area of the spared tissue and cross-sectional
area of total lesion was calculated using ROI manager in ImageJ. Percent spared tissue and
percent injury area was calculated from these values for each animal. Next, percent spared tissue
and average BBB scores were correlated. The quantification of the spared tissue for each rat was
conducted randomly and blindly with respect to the treatment group.
6.3.12. Immunofluorescent staining and analysis: Tissue sections were stained with a
biochemical marker to gain more specific details regarding the cellular population in the injury site
and immediately surrounding the gel islands. Tissue sections were warmed to room temperature
and then baked at 60 °C for 3 hours prior to immunofluorescent staining to improve the adhesion
to the glass slides. Primary antibodies, dilutions and their antigen retrieval were performed as
shown in Table 6.3. Then, after they reached room temperature, tissue sections were rehydrated
in PBS for 45 mins to dissolve the OCT on the slides. Subsequently, antigen-retrieval was
performed for the tissue sections in low pH citrate buffer (Vector laboratories, CA, USA) at 97 °C
for 20 min. Then, tissue sections were cooled to room temperature in the antigen retrieval solution
and then immersed in 0.3% Triton-X in PBS for 30 min. Next, they were washed with quick dips
in PBS and then permeabilized in cold methanol for 10 min followed by PBS wash for 5 min. Then,
the slides were cleaned and marked with an IHC marker pen around the section to contain the
staining solutions to the sections. The tissue sections were blocked with serum-free protein block
(Dako, Agilent, USA) for 1 hr. Then, they were washed with 0.05% Tween20 in PBS solution (5
min) and then in PBS (2 x 5 min). Then, they primary antibodies diluted in Dako antibody diluent
solution were added to the sections in a black staining box with ample moisture. The slides were
then kept overnight at 4 °C.
The next day, slides were washed with 0.05% Tween20 in PBS solution (5 min) and then
in PBS (2 x 10 min) followed by addition of secondary antibodies diluted in Dako antibody diluent.
The slides were incubated with secondary antibodies for 2 hours at room temperature in the black
staining box. Subsequently, they were washed with 0.05% Tween20 in PBS solution (5 min) and
then in PBS (2 x 10 min). After 3 quick dips in deionized water, coverslips were mounted with
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DAPI-containing Fluoro Gel-II mounting medium (Electron Microscopy Sciences, Hatfield, PA)
and stored at 4 °C.
Primary antibody Dilution Antigen retrieval
Secondary antibody
Cells/molecules identified
Rabbit poly clonal anti-GFAP (abcam #ab7260)
1:400 Citrate buffer pH 6 (97 °C at 20 min)
Dylight 488 Donkey anti-rabbit IgG
Astrocytes
Rabbit monoclonal anti-NeuN (abcam #ab177487)
1:400 Citrate buffer pH 6 (97 °C at 20 min)
Dylight 488 Donkey anti-rabbit IgG
Mature neurons
Rabbit polyclonal anti-Iba1 (Wako #019-19741)
1:400 Citrate buffer pH 6 (97 °C at 20 min)
Dylight 488 Donkey anti-rabbit IgG
Microglia/ macrophages
Mouse monoclonal anti-CD68 (BioRad # MCA341R)
1:200 Citrate buffer pH 6 (97 °C at 20 min)
Cy3 Red Donkey anti-mouse IgG
Macrophages
Mouse monoclonal anti-Nestin (Millipore #MAB353)
1:200 Citrate buffer pH 6 (97 °C at 20 min)
Cy3 Red Donkey anti-mouse IgG
Neural progenitor stem cells
Mouse monoclonal anti-Vimentin (Santa cruz #sc6260)
1:200 Citrate buffer pH 6 (97 °C at 20 min)
Cy3 Red Donkey anti-mouse IgG
Neural progenitor stem cells
Rabbit monoclonal Mushashi-1 (Abcam# ab52865)
1:100 Citrate buffer pH 6 (97 °C at 20 min)
Dylight 488 Donkey anti-rabbit IgG
Neural progenitor stem cells
Rabbit monoclonal anti-vWF (Abcam # ab6994)
1:500 High buffer pH 9 (97 °C at 20 min)
Dylight 488 Donkey anti-rabbit IgG
Endothelial cells
Rabbit polyclonal anti-EGF (Peprotech #500-P277)
1:500 Citrate buffer pH 6 (97 °C at 20 min)
Dylight 488 Donkey anti-rabbit IgG
Rat EGF
Mouse monoclonal anti-MMP2 (Millipore #MAB3308)
1:300 Citrate buffer pH 6 (97 °C at 20 min)
Cy3 Red Donkey anti-mouse IgG
Gelatinase MMP2
Table 6.3 Immunofluorescent staining of the cells and molecules with their staining protocol
Fluorescent images were acquired using epifluorescence microscope (Olympus BX60)
and confocal laser microscope (Nikon). The images were analyzed using ImageJ to quantify the
signal from the biomarker of interest. Three random images were selected and analyzed with a
standardized 0.3 mm2 ROI. Images were converted to 8-bit images followed by a standard
threshold optimized for each antibody. Then, the percent area covered by the fluorescent signal
in the standardized 0.3 mm2 ROI was measured using ImageJ. The average percent area was
calculated and used for analysis for comparison among the treatment groups.
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6.3.13. Statistical analysis: Before the experiment, our power calculation for sample size
determination is based on the desire to determine significant a 30% difference in a selected
outcome variable with a 15% standard deviation, and with α=0.05 and β=0.05. The statistical
significance among the experimental groups for histomorphometric and IHC staining results were
determined by one-way ANOVA analysis using Tukey Post Hoc tests. Non-parametric data such
as BBB scores were analyzed for statistical significance using the Krusal-Wallis Test followed by
Dunn post-hoc test, Fisher’s Exact Test or Chi squared test. SPSS 25 (IBM) and Graphpad Prism
8.0 (Graphpad) were used for the statistical analysis.
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6.4. Results
6.4.1. Surgical observations and Gtn-HPA injection: The animals tolerated the
surgery well and had no major complications immediately after the surgery. 2-3% isoflurane was
able to maintain the anesthesia level during the surgery. However, it was noted that compared to
using sodium pentobarbital as an anesthetic for surgical SCI, isoflurane showed more bleeding in
the rat and in the SCI cavity. The use of Gelfoam® for 15 minutes in three 5-min sequential
Gelfoam® insertions reduced the number of the animals that exhibited bleeding during the
injection of Gtn-HPA gel in the cavity. Achieving complete hemostasis was difficult in 10 out of 49
rats despite the use of Gelfoam® for 15 minutes. Once the hemostasis was established, 20 µl of
Gtn-HPA was injected into the cavity. Visually, both the 8% and 12% Gtn-HPA gel achieved
complete gelation in 2-3 minutes. 12% Gtn-HPA gel was more viscous due to the increased
gelation content and therefore care was taken to inject the liquid as early as possible into the
cavity. Complete gelation was confirmed in the remaining amount of the gel and was noted in the
surgical records.
Post-operation, animals gained consciousness in roughly half an hour after the surgery.
Immediately after gaining consciousness, rats seemed to be in shock and they frantically
observed the surroundings. These movements also included movement of the spine at the
surgical site. All the animals showed good markers of recovery in terms of weight gain and
rehydration within a week. No animals were suspected of any infections after surgery. In terms of
the function, they had lost the ability to empty their bladder due to loss of micturition reflex
indicated by their full bladders, which were manually expressed twice a day. Moreover, both their
hindlimbs were completely paralyzed after the induction of SCI. By four weeks, they had partial
return of the function in their hindlimb movement and bladder function. They were well
acclimatized with the open field locomotor test to assess functional recovery. In the next two
sections, bladder function and motor function were compared among the treatment groups to
investigate the effect of the intervention on improving the functional outcome after SCI.
6.4.2. Functional evaluation – bladder function: Neurogenic bladder dysfunction is a
significant outcome of the SCI that severely affects the standard of living of the SCI patients.
There are various treatment options available to the patient based on the comfort and
requirements based on the severity of the functional loss. It is one of the leading symptoms of
SCI that is managed clinically [17]. Therefore, it is of prime importance to study the effect of our
proposed therapy on the return of the urinary function post SCI. We recorded three different
parameters in the rats across all the treatment groups to evaluate the effect of the treatment on
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urinary function – 1) number of animals that regained partial urinary function, 2) number of days
to regain partial urinary function and 3) urine retention volume during manual emptying for four
weeks.
Table 6.4 shows the results of the first two parameters – number of animals regaining
urinary function after four weeks and days required to regain partial urinary function. Qualitatively,
injection of Gtn-HPA with or without the factors increased the number of rats that showed partial
regain of urinary function (Table 6.4). However, the Chi-square analysis did not show a statistically
significant association between the treatment and total number of animals that showed partial
regain of urinary function after four weeks. Similarly, out of the animals that regained function, the
trend indicated that the injection of Gtn-HPA gel with or without factors promoted early regaining
of urinary function by 6 days compared to the controls (Table 6.4). However, one-way ANOVA
did not show statistically significant reduction in the days to regain partial urinary function.
Treatment # animals regaining
partial urinary function
# days to regain partial
urinary function
Control / EGF in suspension 5/11 21.25
8% Gtn-HPA gel only 4/6 21.5
8% Gtn-HPA + GFs 10/12 19.5
12% Gtn-HPA gel + GFs 11/12 18.2 Table 6.4 Trend shown in the table indicates that there is a statistically significant association between the treatment groups and number of animals that regain partial bladder function (Chi-square test). However, no significance was observed in the time to recovery among groups. However, the trend indicates an early recovery of bladder function in treatment groups.
The third parameter studied was the urine retentionvolume each time the bladder was
manually emptied using Crede’s maneuver. This parameter is clinically valuable tool and is
comparable to post-void residual volume (PVR). PVR is a noninvasively measured clinical metric
that is studied by the clinicians to evaluate voiding dysfunction. PVR is defined to be the residual
urine volume in the bladder after a voluntary void [18]. Similar measurements are made in the
urological animal models to study the effect of the treatment on the urinary function. Typically,
cystometric analysis involves use of catheterization to measure the urine retention volume [19].
However, catheterization was not performed to give free movement to the rats and to reduce the
chances of urinary infections. We applied a qualitative technique to measure the urine volume.
Urine retention volume was categorized as small, medium and large at each manual bladder
expression twice a day. In the beginning weeks, most all animals showed large residual urine
volumes while it decreased to medium and small in several animals. Table 6.5 shows the
frequency contingency table of the final average urinary output volume in the last four days with
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the treatment groups. Qualitatively, controls had more animals with large volume bladders (4/6)
while the 12% Gtn-HPA with EGF groups had zero animals with large bladder volumes indicating
that there might be some dependence between the treatment groups and urine output volume. A
chi-square test of independence was performed to examine the relationship between the
treatment groups and urine volume output four weeks post injection. The relation between these
variables was significant (Χ2 = 13.068, p < 0.05) suggesting that injection of Gtn-HPA with EGF
promoted significantly better regaining of urinary function such that rats were voiding more volume
on their own four weeks after SCI.
Treatment / urine volume Small Medium Large
Control / EGF in suspension 2 4 5
8% Gtn-HPA gel only 1 2 3
8% Gtn-HPA gel + EGF 5 4 3
12% Gtn-HPA gel + EGF 7 5 0
Table 6.5 Contingency table showing the experimental groups and urine output volume after four weeks. Chi-square Analysis shows statistically significant association between the treatment groups and the urine output four weeks post SCI and injection (Chi-square analysis, Χ2 = 9.299, p < 0.05)
6.4.3. Functional evaluation – motor recovery (BBB scoring): In order to assess the
functional recovery after the SCI, animals underwent open field locomotor test in which they freely
walked on the long operation room table covered with absorbent chute. Prior to surgery, all
animals displayed normal gait. However, immediately after the hemi-resection injury, they
suffered complete paralysis for both the right and left hindlimb. BBB is a well-established 21-point
scale ranging from score of 0 (complete paralysis) to 21 (normal gait) of the hindlimbs. Each week
after the implantation, rats were scored using BBB scale in a randomized and blinded manner
with respect to the treatment group. There were three methods by which BBB scores were
analyzed – 1) BBB scores during four weeks of the experiment, 2) rate of improvement in the BBB
scores, and 3) number of animals showing either poor, good and better improvement.
During the course of the experiment, one week after inducing SCI and injecting the
treatment, all the groups showed some recovery in both hindlimbs. There was no difference
between the BBB scores among all the seven treatment groups. All the groups scored average
BBB score between 1 - 4 for both right and left hindlimb. Although no differences were noted
between the right and left hindlimb for the same group, left hindlimb scored less than right hindlimb
for the same group. By the end of four weeks, average BBB score for N and E groups was scored
between 4 – 6 while the average BBB score ranged from 9 to 11 for the Gtn-HPA treated groups
with factors (GE8, GES8, GE12 and GE12T) groups (Figure 6.5). Krusal-Wallis Test showed that
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BBB scores were significantly dependent on the type of treatment four weeks post implantation
of the treatment (p < 0.01 for both hindlimbs). There were no differences among the treatment
groups at 1, 2 or 3-weeks post implantation. Subsequent Dunn post-hoc tests comparing four-
week scores showed that the groups delivering EGF and SDF-1α namely GE8, GES8, GE12 and
GE12T significantly recovered better than the control and EGF in suspension group for both left
Figure 6.5 Animals showed functional recovery after the SCI in both the right and left hindlimbs (Increase in the BBB score indicates functional recovery) during the four weeks of evaluation. Groups GE8, GES8, GE12 and GE12 showed better functional recovery compared to the controls and EGF in suspension group for both left and right hindlimbs four weeks after the implantation of the therapy. *p < 0.05, Krusal-Wallis Test followed by Dunn post-hoc test
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and right hindlimbs. However, there were no differences between the G8, N and E group
suggesting that injection of Gtn-HPA alone does not assist in better functional recovery. In
addition, growth factor delivery in suspension is inefficient given that the growth factors would not
be present locally to confer any potential therapeutic effect and therefore requires delivery via a
biomaterial such as Gtn-HPA. Furthermore, we were interested in evaluating the effect of the left
hemi-section on the right and left hindlimb function. Since our model employed a left hemi-
resection, one hypothesis would be that left hindlimb would be severely affected compared to the
right hindlimb. However, 2-way ANOVA did not show the difference between the left and the right
hindlimb for each treatment group (p > 0.05) (Figure 6.6).
Next, in order to study the rate of improvement during the course of the experiment, each
rat was treated as its own control and rate of improvement was calculated for each rat by
subtracting the one-week score from four-week BBB score to examine the influence of the
treatment on the rate of improvement in the BBB score. After four weeks, quantitative analysis
showed that the rate of improvement was greater in the groups GE8, GES8, GE12 and GE12T
compared to N and E in the left hindlimb while rate of improvement was higher in the groups GE8,
GES8, GE12 and GE12T compared to E in the right hindlimb (Krusal Wallis Test, p < 0.05).
However, there were no differences in the rate of improvement between N, E and G8 groups
(Figure 6.7). These results indicate that administration of EGF and/or SDF-1α with Gtn-HPA gel
in the SCI cavity promotes faster improvement in the BBB scores compared to N and E.
Figure 6.6 There were no significant differences in the BBB scores for left and right hindlimb for the same treatment group four weeks after implantation. However, treatment groups with EGF and/or SDF-1α implanted with Gtn-HPA gel showed significantly better functional recovery compared to the control and EGF in suspension group. (Bars = Mean ± SEM, p < 0.05, Krusal-Wallis Test with Dunn post-hoc tests)
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Third assessment parameter assessed was the number of animals that showed poor,
good and better improvement in the BBB scores by treating each animal as its own control. The
change in the BBB scores between first and fourth week was calculated. Three categories of
improvement were defined – poor (∆BBB = 0-4), good (∆BBB = 4-8), and better (∆BBB = 8+)
improvement. The frequency of rats in these categories are tabulated as shown in Table 6.6. No
rats from the control and Gtn-HPA gel only group [G8] showed more than 8 points improvement
in the score for both left and right hindlimb. However, Groups with EGF and/or SDF-1α delivered
by Gtn-HPA had 12/24 and 9/24 showed more than 8 points improvement in the BBB score in the
right and left hindlimb respectively. A chi-square test of independence was performed to examine
the relationship between the treatment groups and improvement in BBB scores four weeks post
injection. The relation between these variables was significant for the left hindlimb (Χ2 = 18.02, p
< 0.05) suggesting that injection of Gtn-HPA with EGF and/or SDF-1α promoted greater
improvement in the function compared to the controls. However, relation was not significant for
the improvement in the right hindlimb function.
Figure 6.7 Evaluation of the rate of improvement in the BBB score between week-1 and week-4 in both the hindlimbs. Quantitative analysis showed that the rate of improvement was higher in the groups GE8, GES8, GE12 and GE12T compared to N and E in the left hindlimb while rate of improvement was higher in the groups GE8, GES8, GE12 and GE12T compared to E in the right hindlimb. (*p<0.05, **p<0.01, Bars = Mean ± SEM
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RIGHT (∆BBB) 0-4 4-8 8+
Control 1 5 0
8% Gtn-HPA gel only 1 5 0
8% Gtn-HPA + GFs 0 6 6
12% Gtn-HPA + GFs 0 6 6
LEFT (∆BBB) 0-4 4-8 8+
Control 4 2 0
8% Gtn-HPA gel only 3 3 0
8% Gtn-HPA + GFs 1 8 3
12% Gtn-HPA + GFs 0 6 6
Table 6.6 Tabulation of the improvement in the functional BBB scores from week 1 to week 4 was categorized in three levels – poor improvement (improvement of up to 4 points), moderate improvement (improvement by 4-8 points) and high improvement (improvement of more than 8 points). A chi-square test of independence showed that the relation between the treatment groups and extent of improvement was significant for the left hindlimb (Χ2 = 18.02, p < 0.05) suggesting that injection of Gtn-HPA with EGF and/or SDF-1α promoted greater improvement in the function compared to the controls. However, relation was not significant for the improvement in the right hindlimb function.
Spared tissue
Injury area
Figure 6.8 Gtn-HPA incorporating EGF and SDF-1α promoted greater preservation of the spared tissue and exhibited smaller injury area compared to control and EGF in suspension group. Scale bar = 2000 µm, bars = Mean ± SEM, One-way ANOVA, Tukey post-hoc test, * p<0.05, **p<0.01, ***p<0.001
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6.4.4. Spared tissue and injury area quantification: Spared tissue was quantified based
on the histological images and was defined to be the unaffected tissue in the injury site continuous
with the rostral and caudal healthy spinal cord tissue. In addition, injury area was quantified to
assess if the injection of Gtn-HPA affects the extent of spread of the injury four weeks after SCI.
We found that Gtn-HPA incorporating EGF or SDF-1α significantly had lower injury area and
higher spared tissue compared to the control and EGF in suspension group (Figure 6.8). Gtn-
HPA gel alone was not sufficient promote greater sparing of the tissue near injury site.
6.4.5. Spared tissue correlation with functional evaluation: One potential explanation of
the better functional recovery in the treatment groups is attributed to the extent of spared spinal
cord tissue present in the injury site. A correlation analysis was undertaken between the percent
spared tissue and two functional evaluation – average BBB score of the left and right hindlimb at
weeks and days to regain urinary function. Spared tissue in the injury site was quantified using
H&E images of each animal. A reasonable positive correlation was observed between the percent
spared tissue in the injury site and the average BBB functional score (Figure 6.9). Correlation
analysis in Graphpad was found to be as r (41) = 0.7689, r2 = 0.5913, p < 0.001 suggesting that
there is a strong positive correlation between the spared tissue and functional recovery.
Figure 6.9 Correlation analysis between the percent spared tissue and average BBB functional score at four weeks showed variables to be positively correlated significantly r (41) = 0.7689, p < 0.001. The colorized data points show that treatment groups arguably promoted neuroprotection to exhibit greater spared tissue and therefore a higher functional score after four weeks
R² = 0.5913
0
2
4
6
8
10
12
14
16
0 10 20 30 40 50
BB
B s
co
re (
4 w
eek
s)
% spared tissue
Correlation between histology and function
N
E
G8
GE8
GES8
GE12
GE12T
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Out of the animals that recovered partial return of bladder function, similar correlation
between the percent spared tissue and days to regain the partial urinary function was performed.
A correlation analysis in Graphpad was found to be r (30) = -0.1072, r2 = 0.01148, p > 0.05
suggesting that there was no correlation between the percent spared tissue and the time to regain
the bladder function (graph not shown).
6.4.6. Gtn-HPA gel volume measurement using MRI images: Ex vivo MRI images using
T1IR method were analyzed to measure the Gtn-HPA gel volume across all the treatment groups
in a randomized and blinded manner. All the measurements were normalized to the average
volume of the control group. The analysis showed that on average, the average Gtn-HPA volume
present in the injury site after four weeks was about 5.67 mm3 in all the treatment groups.
Quantification analysis showed that the Gtn-HPA gel volume was greater in the Gtn-HPA injected
groups compared to EGF in suspension (Figure 6.10). However, there were no differences
observed in the Gtn-HPA gel volume among the Gtn-HPA injected groups namely G8, GE8,
GES8, GE12 and GE12T suggesting that presence of EGF and SDF-1α had no effect on the Gtn-
HPA gel volume present in the injury site after four weeks (One-way ANOVA, p < 0.001).
Moreover, the results suggest that the T1IR technique is able to reliably distinguish the Gtn-HPA
gel in gel-containing groups from the non-gel containing groups namely E and N.
Figure 6.10 Gtn-HPA gel volume quantified using T1-weighted inversion recovery (T1IR) sequence normalized to the control volume. There were no differences among the Gtn-HPA gel injected groups indicating the presence of factors had no effect on the Gtn-HPA volume in the injury site after four weeks. However, the technique was sensitive in visualizing Gtn-HPA gel evident by negligible volume measured in the EGF in suspension and control groups. (Bars = Mean ± SEM, One-way ANOVA *p < 0.05, **p < 0.01, ***p<0.001)
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6.4.7. Gross histological outcomes: General morphology and specific features regarding
the Gtn-HPA gel were studied using the H&E stained sections of all the rats across all the
treatment groups. As described in the experimental design section of the chapter, there were two
versions of the Gtn-HPA gel – 8 wt% Gtn-HPA and 12 wt% Gtn-HPA gel - based on the gelatin
content were evaluated in a 2 mm hemi-resection SCI model. After four weeks, histological
images showed important characteristics of the injury site and the response to the Gtn-HPA gel.
6.4.7.1. Control group – no injection: In the control [N] group, the injury site was
covered with dense fibrous tissue with empty regions, which were presumably filled with a cystic
fluid prior to the tissue processing (Figure 6.11A). Moreover, ferritin deposits were seen in the
injury site indicative of blood clots (Figure 6.12A). The injury site and the unaffected spinal cord
tissue was visibly different in terms of the morphology. Moreover, average percent spared tissue
in the injury site was 17.5% in the control animals. In the case where the injury has not caused
any secondary damage, we would expect to see 50% spared tissue given the right hemisphere
was the spinal cord in the injury site was not resected.
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A
B
C
D
Gtn
-HP
A o
nly
[G8]
Co
ntr
ol
[N]
Gtn
-HP
A +
EG
F
[GE8
] G
tn-H
PA
+ E
GF
+ SD
F-1
α
[GES
8]
Figure 6.11. Representative H&E stained images of the four groups – (A) Control [N] (B) Gtn-HPA only [G8], (C) Gtn-HPA with EGF [GE8], and (D) Gtn-HPA with EGF and SDF-1α [GES8]. Scale bar = 2000 µm
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6.4.7.2. 8 wt% Gtn-HPA gel groups: Normal histological features of white and gray
matter were observed in the unaffected tissue proximal and distal to the injury site. In the 8% Gtn-
HPA only [G8] group, injury site was substantially filled with the Gtn-HPA gel as opposed to the
dense fibrous tissue observed in the control group images after four weeks (Figure 6.12B).
However, dense fibrous tissue was also observed in the areas where the Gtn-HPA gel was not
present. The typical porous fibrillar structure of the Gtn-HPA gel was observed with weakly eosin
stained Gtn-HPA matrix as reported previously [20] (Figure 6.12B-D). Moreover, usually Gtn-HPA
gel was observed in ‘gel islands’ throughout the injury site and not as a one contiguous structure.
Gtn-HPA gel was not physically continuous with the surrounding tissue. Implanted Gtn-HPA
matrix was mostly void of any cellular infiltration around the gel islands. However, some cells were
A: Control [N] B: 8% Gtn-HPA only [G8]
C: 8% Gtn-HPA + EGF [GE8] D: 8%Gtn-HPA+EGF+SDF-1α[GES8]
Figure 6.12 Micrographs showing important observations of the four groups (A) Control - dense fibrous tissue (B) Gtn-HPA only - morphology of Gtn-HPA', minimal cellular migration around the island, poor interface (C) & (D) presence of the Gtn-HPA gel ‘islands’, greater cellular migration around the gel islands, well-defined interface with the surround tissue and migrated cells. Scale bar = 200 µm
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seen at the boundary of the gel island (Figure 6.12B). Spared tissue was present in the G8
sections with average percent spared tissue being 30.0% of the injury site.
The morphology of the Gtn-HPA with EGF [GE8] and Gtn-HPA with EGF + SDF-1α [GES8]
sections was very similar (Figure 6.11C-D). Like the Gtn-HPA gel only group, histological sections
of these group showed the substantial presence of the Gtn-HPA gel in the injury site in form of
gel islands. However, contrary to the Gtn-HPA gel only group, there was a well-defined physically
continuous interface that was established with the surrounding tissue in the injury site as shown
by high magnification micrographs. In addition, qualitatively, many endogenous cells infiltrated
the injury site and migrated immediately surrounding the gel islands (Figure 6.12C-D). This was
evident by a blue band as a result of hematoxylin stained nuclei of the cells immediately around
the gel islands. The migratory pattern of the cells did not seem to be unidirectional – cells migrated
all around the gel islands. Remarkably, there were few sections in which cells were captured
through the gel islands in a somewhat linear arrangement, which could give insights into the
formation of the gel islands and degradation of the gel (Figure 6.12C). Spared tissue was present
in both the groups and average percent spared tissue was quantified to be 31.8% and 30.3% in
GE8 and GES8 respectively.
It is important to note that the Gtn-HPA gel was not observed in in the injury site in all the
gel-injected groups. Table 6.7 shows the study size and number of animals that showed the
presence of the gel in the injury site. Only 2/6 rats in the GES8 showed presence of the Gtn-HPA
gel and therefore was not accounted for histomorphometric analysis. There are several reasons
for the absence of the gel in certain animals including incomplete gelation due to bleeding during
injection, displacement of the Gtn-HPA gel from the cavity due to animal movement or due to poor
containment of the gel by the collagen fascia membrane and degradation of the gel by four weeks.
Experimental treatment Group Duration of
evaluation
Study
size (n)
# of animals showing
presence of the gel
Control – no injection N 4 weeks 6 N/A
8 wt% Gtn-HPA gel only G8 4 weeks 6 4
8 wt% Gtn-HPA gel with
EGF GE8 4 weeks 6 4
8 wt% Gtn-HPA gel with
EGF and SDF-1α GES8 4 weeks 6 2
Table 6.7. Table comparing the number of animals exhibiting the presence of the Gtn-HPA gel in the injury site after four weeks in H&E staining
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6.4.7.3. EGF in suspension group [E]: The morphology of the EGF in suspension
group was similar to the control group. Dense fibrous tissue with empty areas covered the injury
site (Figure 6.12, left). The empty areas could be attributed to the cystic fluid present in the injury
site. Moreover, ferritin deposits were seen in the injury site indicative of blood clots (Figure 6.13,
right). The injury site and the unaffected spinal cord tissue was visibly different in terms of the
morphology with white and gray matter identifiable in the unaffected tissue. Moreover, average
percent spared tissue in the injury site was 15.5% in the animals with EGF delivered in PBS
suspension.
6.4.7.4. 12wt% Gtn-HPA groups: As shown in Table 6.7, all the rats in the 8wt% Gtn-
HPA injected groups did not show the presence of the gel in the injury site after four weeks. Two
strategies were evaluated – increasing the gelatin content (8 to 12%) and improving the
placement of the dural replacement to better contain the gel in the cavity. Furthermore, early
timepoints of Day 0, Day 1 and Day 7 were evaluated after implanting the 12% Gtn-HPA gel with
EGF [GE12T] to ensure that the Gtn-HPA gel was implanted properly and was not dislodged. The
H&E image of rat sacrificed on the same day (Day 0) showed the Gtn-HPA gel as a contiguous
block present in the left hemi-section defect with some degree of blood from hemorrhage (Figure
6.14A). It is important to note that there is no physical interface between the Gtn-HPA gel and
the surrounding tissue. Similar morphological features were observed in the H&E image of the rat
sacrificed the next day (Day 1). Gtn-HPA is present as a contiguous block in the left hemi-section
defect area with some degree of blood from hemorrhage after one day (Figure 6.14B). Moreover,
there is no physical interface between the Gtn-HPA gel and the nearby tissue. For both Day 0
Figure 6.13 (left) Representative general morphology of the EGF in suspension group – minimal spared tissue and empty areas are visible in the injury site, scale bar = 1000 µm (right) a micrograph showing dense fibrous tissue with ferritin deposits, scale bar = 200 µm
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and Day 1 images, right hemisphere of the cord is intact. However, there are notable different
features in Day 7 histology compared to Day 0 and Day 1. There is a continuous physical interface
between the Gtn-HPA gel and the surrounding tissue in the injury site (Figure 6.14C). The Gtn-
HPA gel is present in form of gel ‘islands’ by one week. These observations suggest that the
C, Day 7
B, Day 1
A, Day 0
Figure 6.14 Representative images from the implantation of Gtn-HPA with EGF [GE12T] at Day 0, Day 1 and Day 7. (A) Day 0 section shows the presence of the Gtn-HPA gel as a contiguous block in the left hemisphere of the cord and intact right hemisphere tissue. Blood is also observed in the section (B) Day 1 section shows similar morphology as that of Day 0 with blood deposit. Gtn-HPA is seen as a contiguous block and no interface between the Gtn-HPA gel and the surrounding tissue is established (C) Day 7 morphology is strikingly different than Day 0 and Day 1. Gtn-HPA is present in form of gel islands and physically continuous interface is existent around gel islands. Scale bars = 1000 µm
*
*
* *
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better placement of the fascia membrane by ‘tucking-in’ the dural replacement membrane helps
containing the implanted Gtn-HPA gel in the hemi-resection cavity after one week.
Two experimental groups were evaluated implanting 12wt% Gtn-HPA gel in the hemi-
resection defect four weeks after injection – 12% Gtn-HPA gel with EGF [GE12] and 12% Gtn-
HPA gel with EGF with ‘tucked-in’ dura [GE12T]. The morphology of the both the groups were
A: GE12
B: GE12T
C: 12% Gtn-HPA with EGF [GE12] D: 12% Gtn-HPA with EGF + ‘tucked-in’ dura [GE12T]
Figure 6.15 Representative injury site showing the presence of Gtn-HPA with cellular migration around the gel islands in (A) GE12, Scale bar = 1000 µm and (B) GE12T group, scale bar = 2000 µm. High-magnification micrographs of (C) GE12 and (D) GE12T group showing features of the Gtn-HPA gel with cellular migration around the gel. Scale bar for C-D = 200 µm
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very similar given that the treatment composition was the same for both groups. However, the
procedure and size of the fascia membrane as dural replacement was different in GE12T group.
The Gtn-HPA gel was present in form of islands and there was immense cellular migration around
these gel islands in the injury site similar to the GE8 and GES8 groups (Figure 6.15A-D). This
suggests that Gtn-HPA with higher gelatin content was permissive of the cellular migration and
EGF is able to recruit endogenous cells to the gel islands. Immunohistochemical assessment
would aid in identifying these cells. Interestingly, all animals (6/6) in the GE12T group showed the
presence of the Gtn-HPA in the injury site, reaffirming that the ‘tucking-in’ procedure improved
the containment of the Gtn-HPA in the hemi-resection cavity. Moreover, there was a continuous
physical framework between the gel islands and the surrounding tissue in the injury site. Percent
spared tissue was quantified to be 37.2% and 34.4% in GE12 and GE12T group respectively.
6.4.8. Histomorphometric evaluation of the injury site: The Gtn-HPA gel area and
cellular migration area around the gel was measured by two method. The histological sections
evaluated to measure the areas represented the midplane coronal section of the cord. GES8
group was not included in the analysis because only 2 out of 6 animals showed presence of the
gel in the injury site four weeks post implantation of the Gtn-HPA gel.
The first method (Method 1 – total area method) measured the total Gtn-HPA gel area in
all the gel islands and total cellular migration immediately around all the gel islands in the entire
injury site in ImageJ using ROI manager tool. Presence of the growth factors did not affect the
area of the gel present in the injury site for both 8% and 12% Gtn-HPA gel formulations (One-
Way ANOVA, Tukey post-hoc test, p > 0.05) (Figure 6.16A). Local delivery of the EGF with the
Gtn-HPA matrix significantly increased the number of the migrated cells immediately surrounding
the gel islands compared to Gtn-HPA gel only group for both 8% and 12% Gtn-HPA gel
formulations except for GE12 group (One-Way ANOVA, Tukey post-hoc test, p < 0.05) (Figure
6.16B).
The second method (Method 2 – standardized ROI method) measured the Gtn-HPA gel
area and cellular migration immediately surrounding the gel in the standardized 0.3 mm2 ROI at
the interface using ImageJ in three randomly chosen images. Presence of the growth factors did
not affect the area of the gel present in the injury site for both 8% and 12% Gtn-HPA gel
formulations (One-Way ANOVA, Tukey post-hoc test, p > 0.05) (Figure 6.16C). Like the results
observed in Method 1 (total area measurement), local delivery of the EGF with the Gtn-HPA matrix
significantly increased the number of the migrated cells immediately surrounding the gel islands
compared to Gtn-HPA gel only group for both 8% and 12% Gtn-HPA gel formulations (One-Way
ANOVA, Tukey post-hoc test, p < 0.001) (Figure 6.16D). These results are consistent with the
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findings in the complete resection model for 8wt% Gtn-HPA gel with EGF and/or SDF-1α
experimental groups (Chapter 4, figure 4.7 & 4.8).
Figure 6.16 Histomorphometric analysis results using both Method 1 (Total area method) and Method 2 (standardized ROI method)
Method 1 - (A) Delivering growth factors with the Gtn-HPA gel did not affect the amount of the gel present while (B) EGF presence in the Gtn-HPA recruited significantly greater number of cells immediately surrounding the gel in the injury cavity four weeks post injection for both 8% and 12% Gtn-HPA gel formulations except GE12 group
Method 2 - (C) Delivering growth factors with the Gtn-HPA gel did not affect the extent of the gel present while (D) EGF delivered with the recruited significantly greater number of cells immediately surrounding the gel in the injury cavity four weeks post injection for both 8% and 12% Gtn-HPA gel formulations
*p<0.05, **p<0.01, *** p < 0.001, One-way ANOVA followed by Tukey post-hoc test; Bars = Mean ± SEM
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6.4.9. Immunohistochemistry evaluation of the injury site: H&E staining provided
general morphology of the injury site. Next step was to identify the cell population that migrated
into the injury site and surrounding the gel islands. Sections were stained with various primary
antibody markers, results from which are reported below.
6.4.9.1. GFAP – astrocytes: GFAP+ cells were present in the injury site and at the
interface between the unaffected tissue and the injury site (Figure 6.17). In all the groups, the
density of astrocytes was high near the interface between the unaffected tissue and the injury
site, while it decreased towards the proximal and distal direction of the unaffected tissue. In
contrast to interface, the astrocytes were sparsely present in the injury site. Moreover, astrocytes
were rarely present in the cystic area of the injury site (Figure 6.17). In terms of morphology,
GFAP+ cells that had migrated into the injury site had long thin cells with their astrocytic
processes. However, GFAP+ cells away from the injury site had normal protoplasmic morphology
(Figure 6.18A-C). In order to compare the number of astrocytes, GFAP+ cells were quantified at
two different locations – in the injury site and at the interface between the unaffected tissue and
Interface
Gtn-HPA + EGF
A
B
C
Figure 6.17 Representative GFAP stained sections of (A) Control [N] (B) EGF in suspension [E] (C) Gtn-HPA-injected group [GE12 here] showing the interface, injury site and presence of gel in the injury site, Scale bars = 1000 µm
182
the injury site (Figure 6.17BC). Quantitative analysis of the area covered by GFAP+ cells in the
injury site indicated no significant difference among the treatment groups. However, the
quantitative analysis of the area covered by GFAP+ cells at the border of the unaffected tissue
and the injury site showed significant decrease in the presence of astrocytes in the gel-injected
groups (i.e. G8, GE8, GES8, GE12 and GE12T) compared to the control [N] and EGF in
suspension [E] group as shown in figure 6.18D (One-Way ANOVA, Tukey post-hoc test, p < 0.05).
Furthermore, migrated cells immediately surrounding the gel islands did not stain positive for the
GFAP indicating that the cells recruited by EGF to the gel islands were not astrocytes (Figure
6.19).
A: Away from injury B: Interface C: Injury site
Figure 6.18 Representative micrographs showing the GFAP+ cells’ morphology (A) in the unaffected tissue away from the injury site, (B) at the interface of the injury site and unaffected tissue and (C) in the injury site. (D) Quantitative analysis showed significantly reduced number of astrocytes at the interface outlining the injury site in the gel-injected groups [G8, GE8, GES8, GE12 and GE12] than the control [N] and EGF in suspension group [E] (One-way ANOVA, Tukey post-hoc test, *p < 0.05). However, percent area covered by GFAP+ cells was not significant in the injury site. Scale bars in A-C = 200 µm and Bars = Mean ± SEM
D
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6.4.9.2. Iba-1 - resident and/or activated microglia: Iba-1+ cells were present
throughout the injury site across all the groups. They were sparsely present in the unaffected
tissue of the spinal cord in its typical morphology – small ramified cells with processes (Figure
6.20A). Interestingly, similar morphology of Iba1+ cells was observed in the spared tissue in the
right hemisphere of the cord (Figure 6.20C). Iba1+ cells in the injury site had different morphology
– these cells were enlarged spherical cells without any processes (Figure 6.20D). This enlarged
morphology suggests that these cells are activated microglia [21]. Interestingly, Iba1+ cells at the
border of the injury site showed both types of morphology of Iba1+ cells (Figure 6.20B). Moreover,
spared tissue closer to the injury site also had presence of enlarged Iba1+ cells. Such dichotomy
in the morphology of the Iba1+ cells in the unaffected tissue and the injury site was consistent
across all the groups including the controls. Quantitative analysis of the Iba1+ cells in the injury
site showed that the percent area occupied by Iba1+ activated microglia was similar across all
the groups – N, E, GE8, GES8, GE12, and GE12T (Figure 6.20E). This indicates that Gtn-HPA
was well tolerated and had similar microglia response to the controls. There was very sparse
presence of Iba1+ cells immediately around the Gtn-HPA corroborating the finding that Gtn-HPA
was well tolerated in the injury site.
Figure 6.19 The cells that migrated immediately surrounding the gel block stained negative for GFAP indicating that these cells are not astrocytes (left) GFAP stained section showing the presence of the gel marked by white arrow and the cellular migration around the gel block marked by red asterisks. (right) The same animal H&E showing the presence of the gel and cellular migration for comparison with the GFAP stained section. Scale bar = 1000 µm
* *
*
*
* *
* *
Gtn-HPA+EGF Gtn-HPA+EGF
184
Figure 6.20 Micrographs showing the presence and morphology of Iba1+ cells in the (A) unaffected tissue away from the injury site, (B) in the interface bordering the injury site, (C) spared tissue in the right hemisphere of the cord and (D) in the injury site, Scale bars = 200 µm (E) Quantitative analysis of Iba1+ cells showed that percent area covered by the microglia/macrophages in the injury site is similar across all the groups suggesting that implantation of Gtn-HPA was well tolerated . Bars represent Mean ± SEM
A: unaffected B: interface
C: spared tissue D: injury site
E
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6.4.9.3. CD68 – Macrophages/monocytes: CD68 is widely considered to be the
marker for activated microphages/monocytes in response to tissue damage. This immune
reaction is not well detected by Iba1 [22]. Therefore, sections were stained with CD68-Ed1
antibody to study the immune response of macrophages after implantation of the Gtn-HPA matrix.
CD68+ cells were predominantly present in the injury site in the typical spheroidal shape with
enlarged nucleus without any processes in all the groups – N, E, G8, GE8, GES8, GE12 and
GE12T (Figure 6.21A). In the unaffected tissue away from the injury, there was sparse presence
of CD68 while the density of CD68 decreased away from the injury site in both rostral and caudal
direction. Consistent among all the Gtn-HPA injected groups, there were only few CD68+ cells in
the numerous cells that migrated immediately surrounding the Gtn-HPA gel islands (Figure
6.21B). This indicates that the migrated cells immediately surrounding the Gtn-HPA gel are not
macrophages/monocytes. Moreover, the observations are consistent with the findings of Iba1
staining that the implantation of the Gtn-HPA in the hemi-resection injury site did not elicit severe
immune reaction. As shown in figure 6.22, quantitative analysis of the percent area covered by
CD68+ cells did not reveal any statistical difference among all the groups – N, E, GE8, GES8,
GE12 and GE12T confirming the consistent finding that Gtn-HPA is a biocompatible matrix that
does not elicit severe immune response and is well tolerated (One-way ANOVA, p > 0.05).
Figure 6.21 (A) Representative micrograph showing CD68+ macrophages (red) in the injury site (B) Micrograph showing that very few cells were CD68+ in the migrated cells immediately around the Gtn-HPA gel in GE12T group. Moreover, very few CD68+ cells do not show the typical spheroidal morphology of macrophages. (C) DAPI (blue) staining of the same region showing numerous cells migrated around the Gtn-HPA gel for comparison. White dotted line marks the interface between the Gtn-HPA gel and surrounding tissue and scale bars = 200 µm
Gtn-HPA
+ EGF Gtn-HPA
+ EGF
A B C
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6.4.9.4. NeuN – mature neurons: NeuN+ staining was observed in the neuronal cell
bodies in the viable parenchyma away from the injury site in all the groups. Strong NeuN signal
was observed in the nucleus of the cell body of the neuron while the cytoplasm showed granular
NeuN signal. The staining was positive in the gray matter band of the unaffected tissue as
expected. The density of NeuN+ cells in the viable parenchyma was greater than near the
interface between the injury site and unaffected tissue. No NeuN signal was noted in the injury
site or spared tissue. These observations were consistent across all the treatment groups – N, E,
G8, GE8, GES8, GE12 and GE12T. Figure 6.23 shows representational micrographs of the NeuN
staining at different locations in the cord for GE12 group. Given that NeuN was negative for the
entire injury site across all the groups, no quantification of the NeuN+ cells was performed to
compare among all the treatment groups. Moreover, migrated cells immediately surrounding the
Gtn-HPA were negative for NeuN for G8, GE8, GES8, GE12 and GE12T group indicating that
EGF and/or SDF-1α had no effect on the presence of mature neurons four weeks post injection
of the treatment. It would be of importance to compare these findings at longer evaluation time
after the Gtn-HPA matrix injection into the hemi-resection defect.
Figure 6.22 Quantitative analysis of the % area covered by CD68+ cells did not reveal any statistical difference among all the groups – N, E, G8, GE8, GES8, GE12 and GE12T supporting that Gtn-HPA is a biocompatible matrix and is well tolerated by the spinal cord tissue after four weeks post Gtn-HPA implantation Bars represent Mean ± SEM (One-way ANOVA, p > 0.05)
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6.4.9.5. MMP2 - Gtn-HPA gel degradation: In the evaluation of the Gtn-HPA with EGF
and/or SDF-1α in the complete resection SCI model, we did a preliminary investigation into the
mode of Gtn-HPA degradation. 5 sections from G8 group and 5 from GE8, GE12 and GE12T
groups were stained with MMP2. Consistent with the complete resection model results, strong
MMP2 signal was observed for both Gtn-HPA gel only group [G8] and Gtn-HPA with EGF groups
[GE8, GE12, and GE12] only at the border of the Gtn-HPA gel (Figure 6.24). Quantitative analysis
of the percent area of MMP2 at the border showed significantly higher MMP2 presence in the
Gtn-HPA with EGF groups (Figure 6.24) indicating that the high cellular density of the migrated
cells released more MMP2 responsible for the degradation of the Gtn-HPA gel in vivo.
A: unaffected B: near the interface
C: injury site D: migrated cells around Gtn-HPA
Figure 6.23 Representational micrographs from GE12 group showing the NeuN staining in (A) unaffected viable parenchyma away from the injury in the gray matter of the cord, (B) near the interface between the injury site and surrounding unaffected tissue, (C) in the region where Gtn-HPA is not present and (D) around the Gtn-HPA gel island. White dotted line shows the border between the Gtn-HPA gel and migrated cells surrounding the gel. Scale bars = 200 µm
Gtn-HPA + EGF
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A: Gtn-HPA gel only [G8]
B: Gtn-HPA gel + EGF [GE12]
C
Figure 6.24 Representative image of the Gtn-HPA gel islands with MMP2 (red) and DAPI (blue) in the injury site of (A) Gtn-HPA gel only [G8] group and in (B) representational image of Gtn-HPA + EGF [GE groups]. Quantitative analysis shows significantly greater MMP2 presence at the border of the Gtn-HPA islands in GE groups compared to the G8 group (2-tailed t-test, **p<0.01). Scale bars in A-B = 200 µm and Bars in C represent Mean ± SEM.
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6.4.9.6. EGF – Gtn-HPA incorporated EGF: In order to evaluate the presence of EGF in
the Gtn-HPA gels four weeks post implantation of Gtn-HPA matrices in the hemi-resection defect,
6 sections from Gtn-HPA gel only group [G8] and 6 sections from Gtn-HPA + EGF groups (i.e.
GE8, GE12, and GE12T) were stained with anti-EGF antibody. All 6 sections stained positive for
EGF that co-localized with Gtn-HPA gel islands for Gtn-HPA gel groups (G8, GE12 and GE12T).
However, Gtn-HPA gel in the Gtn-HPA gel only group [G8] stained negative for the EGF,
confirming the presence of the EGF in the gel delivered with EGF four weeks post-injection (Figure
6.25). Fisher’s exact test showed significant association between the outcome of EGF staining
between both groups – G and GE groups (Table 6.8, p < 0.05). Therefore, EGF staining could be
used as reliable technique to stain the Gtn-HPA gel in the EGF-delivered groups and EGF is
locally delivered by Gtn-HPA gel four weeks post implantation in the hemi-resection defect.
Treatment Group + EGF staining - EGF staining
Gtn-HPA gel only [G8] 0 6
Gtn-HPA gel + EGF [GE8, GE12 and GE12T] 6 0
Table 6.8 Two-tailed Fisher’s exact test reports statistically significant association (p < 0.05) between the treatment groups (rows) and EGF staining outcomes (columns) four weeks post Gtn-HPA injection in the defect site. The finding suggests that EGF is being locally delivered after four weeks in the injury site.
EGF DAPI EGF/DAPI
G
tn-H
PA
ge
l +
EG
F
G
tn-H
PA
ge
l o
nly
Gtn-HPA
Gtn-HPA + EGF
Figure 6.25 Representative EGF stained micrographs showing the strong signal of EGF (green) and DAPI (blue) in the Gtn-HPA gel + EGF [GE12T] group while Gtn-HPA gel stained negative in the Gtn-HPA gel only group [G8]. Scale bars = 200 µm
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6.4.9.7. Nestin- neural stem cells: Nestin is a type VI intermediate filament protein
found in neural stem cells (NSCs). The groups in which EGF and/or SDF-1α was locally delivered
by Gtn-HPA gel (i.e. GE8, GES8, GE12 and GE12T), histological analysis showed significantly
greater migration of cells around the Gtn-HPA gel islands in the injury site. In these four groups,
Nestin stained positive in the cells migrated surrounding the Gtn-HPA gel islands (Figure 6.26D-
G). In the injury site where Gtn-HPA is not present, few Nestin+ cells are scattered. For the Gtn-
HPA gel only group [G8], a small number of Nestin+ cells are present throughout the injury site
and around the Gtn-HPA islands. Qualitatively, however, the density of Nestin+ cells around the
Gtn-HPA islands seem to be much higher in the GE8, GES8, GE12 and GE12T groups than the
G8 group (Figure 6.26C). Control group [N] and EGF in suspension group [E] did not show any
noteworthy presence of Nestin+ cells in the injury site (Figure 6.26AB). In terms of the
morphology, Nestin staining showed elongated cells oriented perpendicular to the border of the
Gtn-HPA gel indicating the radial migratory pattern of the cells. A high magnification image of an
area of interest shows the radially aligned Nestin+ cells at the border of the Gtn-HPA islands
(Figure 6.26H). Quantitative analysis of the percent area covered by Nestin+ cells in the injury
site around the Gtn-HPA islands showed that the groups in which EGF and/or SDF-1α was locally
delivered by Gtn-HPA gel (i.e. GE8, GES8, GE12 and GE12T) showed significantly greater
presence of NSCs immediately surrounding the Gtn-HPA gel islands and into the gel compared
to the G8, E and N groups (One-Way ANOVA, Tukey post-hoc test, p < 0.05, figure 6.28). This
finding supports the hypothesis that EGF can recruit NSCs into the gel-filled lesion. However,
there was no statistical difference between GE8 and GES8 groups indicating that SDF-1α did not
enhance the recruitment of NSCs to the injury site. There was no statistical significance between
the Nestin+ cells between the control [N], EGF in suspension [E] and Gtn-HPA gel only [G8] group
indicating that the Gtn-HPA gel alone is not able to recruit NSCs to the injury site. The gelatin
content (8% vs 12%) had no effect on number of NSCs recruited to the injury site as there were
no differences between GE8 and GE12 groups (Figure 6.28).
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Figure 6.26 (A-G) Micrographs showing the Nestin+ (red) cells in the injury site in all the experimental groups. Cells that migrated around the Gtn-HPA islands (shown by white asterisk) in the groups in which EGF was administered (GE8, GES8, GE12 and GE12T) show positive Nestin signal indicating that EGF recruited neural r stem cells (NSCs) to the injury site. (H) A high-magnification image (20x) shows the radial migratory pattern of these cells that are aligned perpendicular to the border of the Gtn-HPA islands. Scale bars = 200 µm
A: N B: E
C: G8 D: GE8
E: GES8 F: GE12
G: GE12T H: GE12-20x
* *
* *
*
*
*
*
*
*
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In order to confirm the identity of the migrated cells into the Gtn-HPA gel in the EGF-
delivered groups (i.e. GE8, GES8, GE12 and GE12T) as NSCs, representative samples from
EGF-delivered groups were stained with Vimentin, GFAP and Musashi-1. Nestin/GFAP double
staining showed that Nestin+ cells around the Gtn-HPA islands in the stained negative for GFAP
indicating these are NSCs and not astrocytes (Figure 6.27). Vimentin, another confirmatory
marker for NSCs, was double-stained with GFAP. The images showed that the migrated cells
immediately around the Gtn-HPA islands were Vimentin+ cells and stained negative for GFAP
Figure 6.28 Quantitative analysis of the percent area covered by Nestin+ cells in the injury site around the Gtn-HPA islands showed that the groups in which EGF was locally delivered by Gtn-HPA gel (i.e. GE8, GES8, GE12 and GE12T) showed significantly greater presence of NSCs immediately surrounding the Gtn-HPA gel islands compared to the G8, E and N groups. This finding supports that Gtn-HPA alone is not able to recruit endogenous reparative cells while EGF is necessary to recruit endogenous NSCs to the injury site. (One-way ANOVA, **** p < 0.001, Bars represent Mean ± SEM)
GFAP Nestin GFAP/Nestin/DAPI
Figure 6.27 GFAP (green) and Nestin (red) signal did not co-localize in the cells surrounding the Gtn-HPA islands in the GE12T group (representational) suggesting that migrated cells are NSCs and not astrocytes. DAPI (blue) shows the presence of the cells around the Gtn-HPA island (marked by white asterisk). Scale bars = 200 µm
* * *
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confirming the presence of NSCs (Figure 6.29). Musashi-1 is yet another RNA-binding protein
biomarker to study the presence of the CNS progenitor cells including the neural stem cells [23].
Few representative sections stained with Mushashi-1 showed Musashi-1+ signal in the cells
surrounding the Gtn-HPA islands confirming the presence of NSCs (Figure 6.30). The number of
Musashi-1+ cells seemed to be less than Nestin and Vimentin possibly indicating that Musashi-1
stains for a subset of NSCs. In addition, Nestin was double-stained with EGF marker to visualize
the presence of NSCs in the groups that delivered EGF by Gtn-HPA. We observed positive
staining of EGF in the Gtn-HPA gel and Nestin in the cells immediately surrounding the Gtn-HPA
(Figure 6.31). These observations were significant in establishing the identity of the migrated cells
in the EGF-delivered Gtn-HPA groups as neural stem cells (NSCs).
GFAP Vimentin GFAP/Vimentin/DAPI
* * *
Figure 6.29 Migrated cells around the Gtn-HPA islands are positive for Vimentin. GFAP (green) and Vimentin (red) signal did not co-localize in the cells surrounding the Gtn-HPA islands in the GE12 group (representational) suggesting that migrated cells are NSCs and not astrocytes. DAPI (blue) shows the presence of the cells around the Gtn-HPA island (marked by white asterisk). Scale bars = 200 µm
Figure 6.30 Representative micrographs of Musashi-1 (green) and DAPI (blue) showing positive Musashi-1 signal in the cells surrounding the Gtn-HPA gel in GE12 group further supporting that the migrated cells are NSCs. White-dotted line shows the border of the Gtn-HPA gel. Scale bars = 200 µm
Musashi-1 DAPI Musashi-1/DAPI
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6.4.9.8. vWF – endothelial cells: vWF was difficult biomarker to stain the frozen spinal
cord sections. The recommended antigen retrieval buffer (High pH 9 buffer) at 97°C for 20 min
detached the spinal cord sections from the slide. Few sections that remained attached partially
showed positive vWF signal for the presence of endothelial cells distributed in the injury site
(Figure 6.32). CD31 and CD34 staining was negative for the preliminary sections that were tried.
EGF Nestin EGF/Nestin
Figure 6.31 Double-staining of EGF (green) and Nestin (red) showing Nestin+ cells immediately surrounding Gtn-HPA gel containing EGF four weeks post injection in the hemi-resection defect.
Figure 6.32 Representative micrographs showing vWF staining in the injury site indicating the presence of endothelial cells in their typical morphology. Scale bars = 200 µm
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6.5. Discussion
In this chapter, we implanted two different Gtn-HPA gel formulations with EGF and/or
SDF-1α into the 2-mm hemi-resection injury induced in a T8 2-mm left lateral hemi-resection
surgical excision SCI model with female Lewis rats. First formulation tested was an 8% Gtn-HPA
matrix delivering the growth factors locally in the injury site. The second formulation investigated
had increased gelatin content to 12% Gtn-HPA. We evaluated the effects of implanting the Gtn-
HPA with or without the growth factors in recruiting of the endogenous cells to the injury site, in
inducing a reparative response of the damaged tissue and in aiding improved functional recovery
compared to the control group that received no injection. We evaluated these responses four
weeks after inducing the SCI and injecting the Gtn-HPA gel with or without growth factors into the
injury site. Since traumatic or contusion type of SCI model do not completely sever the cord,
surgical models such as complete or partial resection are utilized to study the healing response
and neuronal regeneration in the field of tissue engineering [24]. Moreover, lateral hemi-resection
models are widely used and considered to be ideal for studying axonal regeneration [12]. Hemi-
resection produces a standardized defect of preferred size and confirms the complete break in
the neuronal circuitry at the location of the cord. In addition, since the resection is made only one
side of the spinal cord, the contralateral side can serve as a control. The most important
advantage in the context of our work is that hemi-resection model creates a reproducible defect
in which biomaterial of a same volume can be injected along with growth-promoting factors such
as EGF and SDF-1α. In addition, we get a visual confirmation of the injection of the Gtn-HPA gel
into the hemi-resection injury site. In addition, hemi-resection models of various lengths have
been implemented to examine the ‘bridging gap’ therapies such as scaffolds, guidance channels
and pre-fabricated devices [12, 25, 26]. We studied a total of seven groups namely – control [N],
EGF in suspension [E], 8wt% Gtn-HPA gel only [G8], 8wt% Gtn-HPA gel with EGF [GE8], 8wt%
Gtn-HPA gel with EGF and SDF-1α [GES8], 12wt% Gtn-HPA gel with EGF [GE12] and 12wt%
Gtn-HPA gel with EGF with dural replacement ‘tucked-in’ around the cord [GE12T]. There were
several assessment parameters we employed to test our hypotheses mentioned in the ‘Overall
goal and hypotheses’ section of this chapter.
6.5.1. Functional evaluation: SCI results in loss of function in many systems of the body
drastically affecting the quality of life of the patients. Therefore, improvement in the function is the
most critical aspect of pre-clinical and clinical studies that aim to treat SCI. We studied bladder
function and motor function of the rats for four weeks post injection of the Gtn-HPA gels.
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6.5.1.1. Bladder function – urine retention volume: Post-void residual volume (PVR)
is a clinical metric that is studied to gain insights into the bladder function for patients with SCI. It
is well-established that decrease in the PVR correlates with improvement in the bladder function
of the patient [27]. 3 parameters were studied to evaluate the recovery of bladder function after
inducing a lateral hemi-resection injury – urine retention volume four after four weeks, number of
animals that regained partial bladder function, and time it took to recover partial urinary function.
Urine retention volume (URV) was qualitatively recorded as small, medium or large at every
manual emptying of the bladder twice in a day during the entire duration of the experiment. For
rats that showed partial recovery, the URV decreased from large to medium or large to small
indicating that they recovered partial voiding ability. For example, 7/12 rats in the 12% Gtn-HPA
+ EGF injected groups (i.e. GE12 and GE12T) had had small URV compared to 0/6 animals in
the control group [N]. Similarly, 4/6 animals in the control group had large URV compared to 0/12
in the GE12 and GE12T groups. Chi-square analysis showed significant association between the
treatment groups and URV after four weeks suggesting that the implantation of Gtn-HPA with
factors led to greater voiding ability in the rats (Table 6.5). Chi-square analysis showed significant
association between the treatment groups and the number of animals recovering partial bladder
function. Although not statistically significant, the trend suggested that treatment groups rats
recovered partial voluntary voiding ability earlier than the than the control group. However, no
statistical significance was observed (Table 6.4). These findings suggest that the treatment
facilitated in improving the bladder function after SCI. The mechanism of the partial bladder
function recovery was either the extent of spared tissue (correlation not observed) or re-wiring of
the neural pathways [28]. Injection of Gtn-HPA with EGF seem to affect the amount of spared
tissue and thereby providing viable parenchyma for rewiring of the neural circuitry (micturition
pathway) to improve bladder function. Future studies should perform a focused study where urine
retention volume is quantitatively measured using catheterization of the bladder or other
established methods to study URV in pre-clinical models of SCI [19]. Longer survival evaluation
times would add to the understanding of the bladder recovery function as well.
6.5.1.2. Motor function – BBB scale: Beattie, Basso, and Bresnahan (BBB) scale is a
validated, standardized and well-accepted behavioral scale that is used to assess the functional
integrity of rats’ hindlimbs post SCIs, usually with the underlying assumption that the cardiac and
respiratory functions are largely unaffected. BBB involves animals walking in an open field with
their hindlimb movement carefully observed. Various voluntary motor functions such as joint
movements, weight bearing, fore/hind limb coordination, paw placement, and the directions of the
tail were judged based on the 21-point BBB scale in a randomized and blinded manner [15, 29].
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Three analyses were undertaken to gain insights into the motor recovery function post
implantation of the Gtn-HPA matrices with or without EGF and/or SDF-1α – BBB scores over four
weeks, improvement in the BBB scores from first week to fourth week and the number of animals
showing the extent of improvement - poor (0-4 points), good (4-8 points) and better improvement
(8+ points).
Immediately after the 2 mm hemi-resection injury, animals showed complete paralysis in
both left and the right hindlimb. This is consistent with the observations of the previous hemi-
resection studies [30]. Paralysis in both hindlimbs is probably due to the presence of corticospinal
tracts in both hemispheres of the cord. Corticospinal tract (CST) is predominantly responsible for
the voluntary motor function with corticobulbar tract. Lesion in the CST tract sever 90% fibers on
the ipsilateral side while severs 10% on the contralateral side causing deficits in both ipsilateral
and the contralateral hindlimb of the lesion [31]. Despite the hemi-resection injury, most animals
showed some level of gain in the functional score on the BBB scale over four weeks (Figure 6.5).
Modest recovery was observed in the initial week across treatment groups which is attributed to
recovery from the spinal shock and ‘re-wiring’ to facilitate neuronal signal transmission [32].
However, after four weeks, EGF-delivered with Gtn-HPA groups (i.e. GE8, GES8, GE12 and
GE12T) showed significantly better recovery in the voluntary motor ability compared to the control
(N) and EGF in suspension group (E) at four weeks (Figure 6.6). After four weeks, animals in the
N, E and G8 groups showed modest recovery improving the average BBB score to 4-6 points,
animals in G8 group improved to average of 7 points while EGF-delivered groups (i.e. GE8, GES8,
GE12 and GE12T) showed improvement to 8-11 points on the BBB scale in both left and right
hindlimbs. The extent of the recovery in absolute BBB scores observed four weeks post hemi-
resection SCI in our study is consistent with the results of the similar studies [33-35]. Moreover,
there were no differences between G8, N and E groups. This indicates that Gtn-HPA gel alone is
not able to promote better functional recovery but a growth factor such as EGF is necessary to
be delivered locally to the injury site.
In order to account for the differences in the baseline scores of the animals, each rat was
treated as its own control to compare the differences in the scores at 1 (acute) and 4 (chronic)
weeks post injury. It is important to consider that since the BBB scale is not linear meaning that
the improvement from a score of 0 to 4 is different than the improvement in the score from 4 to 8
even though both would show net improvement by four points. Interestingly, the number of
animals that improved by more than 8 points were greater in the 8% or 12% Gtn-HPA delivering
EGF groups compared to the control and 8% Gtn-HPA gel only groups. Chi-square analysis
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showed statistically significant association between the treatment group and extent of
improvement in the BBB scores by four weeks for the left hindlimb but not for the right hindlimb
(Table 6.6). Similarly, EGF-delivered Gtn-HPA groups showed greater rate of improvement in the
BBB scores in three weeks compared to the control and EGF in suspension group (Figure 6.7).
However, it is difficult to pinpoint the exact underlying mechanism due to which more animals
showed better improvement in the EGF-delivered Gtn-HPA gel injected groups compared to the
controls and EGF in suspension, given highly sophisticated nature of the neural tissue response
after SCI. However, few mechanisms are proposed in the field.
The field recognizes that the mechanisms of motor recovery in the rats with or without
specific interventions are poorly understood [36]. Various explanations of recovery include the
greater sparing of the axons, intrinsic changes in the circuitry termed as neuroplasticity, and
stimulation of the lower motor neurons not directly affected by the injury to the upper motor neuron
[37, 38]. Still, it is well known that animals that underwent lateral hemi-resection spinal cord
injuries could show notable improvement in the motor function recovery on the ipsilateral hindlimb
as observed in our study [39]. Most probable explanation is the neuroprotection of the tissue by
mitigating further secondary damage caused by the injury due to implanted Gtn-HPA matrices
with EGF. This reasoning could explain the greater net improvement in BBB scores in the Gtn-
HPA injected groups with EGF compared to the N and E reported in this study. Similar
phenomenon has been observed in various studies where the implantation of a biomaterial or
exogeneous cells has shown neuroprotective effects in preventing further degradation of the
unaffected spinal cord tissue and thereby showing better functional recovery in biomaterial or
exogenous cells treated groups [9, 35, 40, 41]. Several mechanisms of the recovery point
specifically to the extent of spared tissue present in the injury site after SCI. Spared tissue
(unaffected tissue) on the other hemisphere can contribute to re-organizing of the wiring to re-
establish the connection with spinal circuits such as central pattern generators (CPGs) nearby
the injury site [42, 43]. In addition, pre-existing spared cross-over CST neurons in the spared
contralateral side could account for the ability to use the limbs on the same side as the lesion as
reported in primates [44]. Another mechanism suggests that reorganization of the local spinal
circuitry from the spared tissue and input signal from sensory afferents could be sufficient to
generate movement of the limbs via CPGs [43]. Interestingly, our spared tissue evaluation
indicates statistically significant positive correlation between the percent spared tissue and
functional BBB score after four weeks (Figure 6.9) supporting the mentioned mechanisms
hypothesizing that greater spared tissue leads to greater functional recovery. EGF delivery with
the Gtn-HPA matrix probably promoted better preservation of the secondary damage and
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therefore, we believe that EGF delivery with 8% or 12% Gtn-HPA gel, contributing to the
recruitment of endogenous cells, re-organizes the tissue response in the injury site leading to
greater presence of spared tissue in the treatment groups leading to greater functional recovery.
SDF-1α, as seen in all the three metrics of the functional evaluation, did not confer any
added advantage in the functional recovery compared to EGF. Moreover, there was no difference
between the 8% and 12% Gtn-HPA groups in functional recovery declaring that increased gelatin
content did not interfere with motor recovery mechanisms. Additionally, there were no differences
between the control and EGF in suspension group indicating that EGF without Gtn-HPA is not
able to localize in the injury site for longer time to exhibit any beneficial effect in the functional
recovery. In comparison with the complete resection model evaluation presented in Chapter 4,
rats showed greater motor function recovery explained by the difference in the severity of the
lesion. No spared tissue was present in the complete transection rats accounting for very minimal
recovery compared to the recovery observed in the hemi-resection rats four weeks post
implantation of the 8% Gtn-HPA gel. Future studies should validate the functional evaluation
findings presented in this chapter with other behavioral tests and electrophysiological
measurements.
6.5.2. Visualizing Gtn-HPA using MRI: spinal columns from all seven hemi-resection
groups after animal sacrifice were imaged with T1-weighted inversion recovery (T1IR) with the
protocols developed in a systematic approach described in Chapter 5. T1IR technique was able
to reliably distinguish the Gtn-HPA from the surrounding tissue. The location of the Gtn-HPA was
at the injury site as expected mostly in the left hemisphere of the cord. The analysis, performed
in randomized and blinded manner, showed significantly greater presence of the Gtn-HPA in Gtn-
HPA injected groups than the control and EGF in suspension groups indicating that the technique
is sensitive to visualizing only Gtn-HPA in the injury site in the spinal cord. Moreover, average
Gtn-HPA gel volume across all the Gtn-HPA implanted groups was found to be ~ 5.67 mm3
suggesting that out of 20 mm3 volume of the Gtn-HPA injected, 75% of the Gtn-HPA gel had
degraded by four weeks (figure 6.9). However, there were no differences in the Gtn-HPA volume
in Gtn-HPA injected groups, either in 8% Gtn-HPA gel or 12% Gtn-HPA gel. This finding suggests
that the qualitative degradation rate of the 8% and 12% Gtn-HPA gel in vivo is similar. Although
the protocol reliably distinguished the Gtn-HPA gel from the surrounding tissue, better
optimization of the protocol would need to be done before it is replicated for longitudinal in vivo
visualization of the Gtn-HPA.
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6.5.3. Histological evaluation of the injury site: Histological evaluation was performed
four weeks after inducing SCI in coronal sections. Based on the morphology of the tissue from
Day 0 and Day 1, it was evident that the surgery had indeed created a hemi-resection defect on
the left hemisphere of the cord (Figure 6.14). Day 0 injury site showed intact contralateral tissue
with Gtn-HPA contiguous block filling in the cavity on the left hemisphere where hemi-section
injury was induced. There was separation between the Gtn-HPA gel and the surrounding tissue
because there is reported to be retraction of the cord after a surgical excision. Similar morphology
of the tissue and Gtn-HPA gel was observed on Day 1 as well. The separation between the Gtn-
HPA gel and surrounding tissue could be as a result of retraction of the cord and some acute
inflammatory degradation of the end of the cord. However, Gtn-HPA had already formed islands
by Day 7 with notable infiltration of the cells in the injury site. By day 7, some mechanical breakup
of the gel due to the animal movement and additional acute inflammatory degradation of the gel
could contribute to the residual islands of gel. Moreover, across all the Gtn-HPA injected groups,
Gtn-HPA was present in most animals four weeks post implantation in the injury site in form of
Gtn-HPA islands consistent with the findings in Chapter 4 (Figures 6.12 and 6.13). It is important
to note that our study shows for the first time 8wt% and 12wt% Gtn-HPA gel is present in the
injury site after four weeks. Presence of Gtn-HPA four weeks after implantation is a significant
improvement over the previous Collagen-genipin gel formulation. Most or all of the Collagen-
genipin gel had degraded completely by as early as two weeks in the similar 3 mm hemi-resection
SCI model [45]. It is essential for the biomaterial to persist for longer duration to facilitate the
infiltration of cells into the defect, better cell-substrate signaling, remodel the underlying ECM and
deliver signaling molecules over long period of time [46]. As far as unaffected viable parenchyma
is concerned, the histological sections displayed typical characteristics of central gray matter in
both hemispheres in both the caudal and rostral sides of the injury site. The length of the injury
site was more than 2 mm supporting the well-known phenomenon that the contralateral tissue in
the hemi-resection model suffers secondary damage further progressing the injury [47, 48].
Although no treatment was applied to the control group [N], the injury site was filled two
kinds of morphology – ‘empty spaces’ and dense collagen fibrous tissue (Figure 6.11A). Similar
morphological findings were observed in the EGF in suspension group (Figure 6.13). The empty
spaces probably represent the cystic cavitations, which are formation of fluid-filled cysts in
response to the injury. These cavities are usually filled with macrophages and the growth-
inhibiting molecules deposited in response to degradation of myelin [49]. These cavitations are
consistent in the chronology of the pathophysiological response because these cysts are
developed in the subacute and intermediate phase of the response – which coincides with the
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four-week evaluation of our study [50, 51]. We did observe minimal spared tissue in both N and
E groups (17.5 and 15.5% respectively) suggesting that typical pathophysiological process after
SCI would lead to greater secondary damage compared to replacing the cavity contents with
growth-promoting materials. The extent of spared tissue present is consistent with the findings in
similar hemi-resection models in rats [52]. In addition, ferritin deposits were observed in both the
control and EGF in suspension group indicating the remains of the blood clot that was formed due
to the bleeding either from cord parenchyma or from surrounding muscles after creating the
surgical injury. It is possible that the blood clot provided a temporary matrix to the infiltrating
inflammatory cells especially because the dura membrane was compromised due to the surgical
nature of the model. Both the morphologies – cysts and dense fibrous collagen tissue is inhibitory
to the cellular migration into the cavity site for any potential reparative response. Therefore, goal
of this study to replace the growth-inhibitory environment with that of a biomaterial that would be
permissive of the cellular infiltration without eliciting major inflammatory response. Therefore, our
proposed therapy involves injecting a Gtn-HPA matrix in the defect site with chemoattractants,
EGF and SDF-1α to recruit endogenous neural stem cells and glial cells to the injury site.
There were two types of Gtn-HPA gel formulations implanted in the cavity created by left
lateral 2-mm hemi-resection in the cord – 8wt% Gtn-HPA and 12% Gtn-HPA gel. The gross
histological findings were similar in both 8% and 12% Gtn-HPA gels. After four weeks, Gtn-HPA
gel covered the predominant area in the injury site in all the treatment groups (i.e. G8, GE8, GES8,
GE12 and GE12T). The presence of the Gtn-HPA matrix in most animals in all the treatment
groups was assuring that the Gtn-HPA could serve as a reliable scaffold to promote regenerative
response after SCI. However, the number of animals that showed the presence of Gtn-HPA varied
across the groups. Only 2/6 of the GES8 groups showed presence of the Gtn-HPA gel while 4/6
animals showed Gtn-HPA in G8 and GE8 groups. We believe there are few potential explanations
that account for the absence of the Gtn-HPA gel in animals that were injected with Gtn-HPA matrix
in this study. First, bleeding in the cavity site during the injection could cause incomplete gelation
or more porous Gtn-HPA gel leading to a less stiff Gtn-HPA gel than anticipated. This would result
in faster degradation of the gel in vivo. Second, the animals were quick to gain consciousness
after the surgery and did movements that could lead to dislodging of the Gtn-HPA gel from the
cavity because the dural replacement collagen membrane was placed on the cord and not sutured
to the cord. Third, some animals could show more severe inflammatory response due to which
the Gtn-HPA gel degraded quickly. These potential explanations were considered in designing a
second generation Gtn-HPA gel and in better containing the gel in the cavity site. We made two
major improvements in the second generation Gtn-HPA formulation – we increased the gelatin
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content from 8 to 12% to reduce the degradation rate of the Gtn-HPA gel [GE12 group] and
modified the placement of dural replacement by ‘tucking-in’ the membrane around the cord only
to prevent the Gtn-HPA gel from dislodging itself from the injury cavity [GE12T group]. ‘Tucking-
in’ the dural replacement around helped contain the Gtn-HPA in the injury site as all 6/6 animals
showed the Gtn-HPA present four weeks after implantation. Future studies in our laboratory would
employ this ‘tucking-in’ technique due to the improved results reported in this chapter.
Another interesting feature of the morphology of the Gtn-HPA gel was the shape of the
Gtn-HPA gel after four weeks in the Gtn-HPA implanted groups. It was predominantly present in
the injury site in form of multiple ‘islands’ of varying sizes (Figure 6.11B-D and Figure 6.15A) as
opposed to a continuous block (Figure 6.15B). These islands could mimic the small cavitary
lesions, which are common clinical manifestation of the SCI due to various disease progressions
[53]. We believe there could be several mechanisms that could leads to the formation of ‘gel
islands. First, heterogeneity in the modulus of the Gtn-HPA gel during gelation could contribute
to mechanical differences in the gel. These differences could result in varying rates of the gel
degradation leading to the break in the continuous matrix resulting in formation of gel islands over
time. Second, the number and the types of cells present around the Gtn-HPA during acute phase
of the pathophysiological response could affect the extent of cellular migration into the Gtn-HPA
matrix. For example, macrophages are shown to occupy the injury site as early as a week after
the SCI and release various inflammatory cytokines and ECM degrading molecules, especially
Matrix metalloproteinases (MMPs) [54, 55]. MMP2 is a zinc-based gelatinase and we observed
strong MMP2 signal at the border of the Gtn-HPA gel islands in this study (Figure 6.24). Therefore,
it is possible that activated macrophages released significant amount of MMP2 at the border of
the continuous Gtn-HPA block resulting in formation of gel islands during the four weeks. Third,
animal movement immediately after the surgery and within initial weeks could mechanically break
the Gtn-HPA into physically separate entities leading to the formation of gel islands. Histological
evaluation as early as 7 days post implantation also showed these islands form (Figure 6.14C).
Another feature important to discuss is the ability of the Gtn-HPA to establish a continuous
physical interface with the surrounding tissue to re-establish the lost framework due to the injury.
We observed that Gtn-HPA alone group [G8] was not able to establish an interwoven physical
structure with the surrounding tissue (Figure 6.12B). However, GE8, GES8, GE12 and GE12
groups displayed a well-defined continuous interface with the surrounding tissue in the injury site
(Figure 6.12 CD, Figure 6.15CD). Similar observations regarding the interface in the injury site
were made in the complete section model in Chapter 4. They are in accordance with the findings
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in various studies reporting that matrices without any form of the molecular cues failed at
establishing a well-defined continuous framework with the surrounding tissue in tissue repair
approach [56, 57]. Therefore, signaling molecules such as EGF and SDF-1α are critical to include
in the injection of Gtn-HPA matrices.
One of the hypotheses of the study was that EGF and SDF-1α would promote the
recruitment of different endogenous cells types such as the neural stem cells and other glial cells
to the injury site. Qualitatively, greater number of cells had migrated to surround the Gtn-HPA gel
islands in all GE8, GES8, GE12 and GE12T groups (Figure 6.11 and Figure 6.13) compared to
the Gtn-HPA gel only [G8] group (Figure 6.10B and Figure 6.12B). Similar results were reported
in the complete resection model in Chapter 4 confirming the ability of growth factors EGF and
SDF-1α in chemotactically recruiting endogenous cells to the injury site. Moreover, this
phenomenon highlighted in our study is in accordance with the conclusion that both 8% and 12%
Gtn-HPA gel is permissive of the cellular migration both in vitro and in vivo brain ICH model as
reported based on previous work in our laboratory [20] . Increased stiffness, such as in 12% Gtn-
HPA gel, could hinder the cellular migration into the injury site [58]. However, cellular migration
observed in the 12% Gtn-HPA seems to indicate that the stiffness of the 12% Gtn-HPA gel is
within the range of stiffness of materials that cells could migrated through. It would be interesting
to see if there are quantitative differences in the gel area and migration area between the groups
with 8% and 12% Gtn-HPA gels. Quantitative analysis of the Gtn-HPA gel area and cellular
migration immediately surrounding the Gtn-HPA gel islands showed that EGF and SDF-1α were
able to recruit significantly greater number of cells to the Gtn-HPA gel islands compared to the
Gtn-HPA gel only group (Figure 6.16). However, cellular migration area was similar for 8% and
12% Gtn-HPA gel suggesting that gelatin content within the range of 8-12% has no effect on the
cellular migration. We validated this result with two different quantification methods measuring
two parameters – gel area and cellular migration area immediately surrounding the gel. The first
method accounted for the all the gel islands and cellular migration immediately around all of them
present throughout the injury site. The second method measured both the parameters in randomly
chosen images in the injury site. Statistical analyses of these parameters in both the methods
showed significantly greater recruitment of cells by EGF and SDF-1α compared to the control and
Gtn-HPA only group (Figure 6.16BD). Therefore, we deduce that Gtn-HPA gel alone would not
be able to recruit cells to the injury site and therefore, signaling molecules, i.e. EGF and SDF-1α,
are essential in recruiting endogenous cells to the injury site after SCI. This is a critical result
supporting our central hypothesis of the study. Of note, GES8 group did not show significantly
greater cellular migration compared to the GE8 groups indicating that presence of SDF-1α did not
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enhance the recruitment of cells to the injury site – similar to the observation in the complete
resection model discussed in Chapter 4. As far as the Gtn-HPA gel area is concerned, there were
no differences among the Gtn-HPA injected groups in both the methods (Figure 6.16AC)
suggesting that introduction of the growth factor has no effect on the area of the gel present in
the injury site. Moreover, if gel area is any measure of the Gtn-HPA volume in the injury site, MRI
measurement of Gtn-HPA volume also showed no differences in the Gtn-HPA gel volumes
between the Gtn-HPA injected groups (i.e. G8, GE8, GES8, GE12 and GE12T) as shown in figure
6.10. In addition, there were no differences in the Gtn-HPA volume in groups with varying gelatin
content in the Gtn-HPA - either in 8% Gtn-HPA gel or 12% Gtn-HPA gel. This finding suggests
that the qualitative degradation rate of the 8% and 12% Gtn-HPA gel in vivo is similar.
Subsequent to the promising histological observations and conclusions, our goal was to identify
the cells that migrated around the gel islands and in the injury site.
6.5.4. Immunohistochemistry evaluation of the injury site: Several biomarkers were
stained to identify the presence and the extent of various cell types and important molecules in
the context of this study. Specifically, GFAP (astrocytes), Iba1 (microglia/activated microglia),
CD68 (macrophages), NeuN (mature neurons), Nestin (neural progenitor stem cells), Vimentin
(neural progenitor stem cells), Musashi-1 (neural progenitor stem cells), vWF (endothelial cells),
EGF and MMP2 (Gtn-HPA gel degradation) were stained to obtain a comprehensive composition
of the injury site post Gtn-HPA implantation with or without EGF and/or SDF-1α.
Astrocytes have been shown to play a multi-functional role in the context of repair and
regeneration after SCI. The multifaceted astrocytic response contributes to neuroprotection of the
spinal cord while inhibiting axonal regeneration [59]. Astrocytes are typically categorized into three
types in the CNS – naïve astrocytes, reactive astrocytes and scar-forming astrocytes [60]. Naïve
astrocytes help in maintaining the neuronal parenchyma and the blood-brain barrier while the
reactive astrocytes assist in limiting the spread of inflammatory cells in response to injury
promoting remodeling of the ECM and repair of the tissue [61]. These reactive astrocytes convert
to scar-forming astrocytes based on the environmental cues. They form the scar by adhering to
each other and releasing molecules like CSPG to produce a penetrating glial scar that impedes
axonal regeneration after SCI [62, 63]. We found the presence of GFAP+ cells in the unaffected
tissue and in the injury site but predominantly at the interface between the unaffected tissue and
the injury site. Quantification of GFAP+ cells showed no statistical differences between the groups
in the injury site (Figure 6.18D). Moreover, the cellular migration immediately around the Gtn-HPA
gel islands stained negative for the GFAP indicating that those cells are probably not reactive
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astrocytes because GFAP is considered to be the hallmark biomarker for reactive astrocytes
(Figure 6.19) [64]. However, quantification of the GFAP+ cells at the interface between the
unaffected tissue and the injury site showed significant reduction in the GFAP+ cells or astrocytes
at the interface in the Gtn-HPA injected groups (i.e. G8, GE8, GES8, GE12 and GE12T) compared
to the control group and EGF in suspension group indicating that implantation of Gtn-HPA matrix,
with or without the growth factors, into the cavity downregulates the astrocytic response near the
injury site (Figure 6.18D) . This phenomenon probably either delays the process of glial scar
formation or reduces the extent of glial scar formation in the Gtn-HPA injected groups. Similar
findings of reduced GFAP immunodensity was reported by Alluin et al at the interface of the injury
site [65]. Interestingly, one hypothesis proposed by Macaya in his doctoral work stated that the
elongated astrocytic processes towards the center of the defect are growth-promoting while the
astrocytes bordering the injury site with dense processes are inhibitory [45]. Similar morphologies
were observed in our evaluation at the interface and in the injury site. Our results show that
although the treatment groups do not increase the presence of the growth promoting astrocytes
in the injury site, the treatment reduces the number of the inhibitory astrocytes at the border.
Similar morphology remodeling has been characterized in the previous study by Sun and Jakobs
supporting the hypothesis proposed by Macaya [66]. Results in our evaluation provide critical
evidence to suggest that the treatment reduces the glial astrogliosis leading to the beneficial repair
and migration into the injury site.
Post-injury, the resident microglia migrate to the injury site while the monocytes
differentiate into macrophages in the acute phase of the pathophysiological process after SCI.
Since they are phagocytic in nature, they contribute to the debris clearing in the injury site to
mitigate the secondary damage [21, 67]. Macrophages have been classified into two phenotypes
– M1 and M2. M1 is shown to be promoting the release of pro-inflammatory factors while M2
contributes in wound healing and repair. Interestingly, M1 have been shown to have detrimental
effect in the SCI but M2 macrophages have shown to promote regenerative response after SCI
[68]. Iba1 is a microglia/macrophage specific marker that was used to assess the inflammatory
response to the Gtn-HPA gel while CD68 was stained to assess the immune reaction to the injury.
It is reported that immune reaction to tissue damage by macrophages is not well detected by Iba1
necessitating the staining of CD68 [22].The morphology of the Iba1+ cells varied depending upon
the location on the spinal cord section (Figure 6.20A-D). Unaffected tissue had typical ramified
phenotype cells with its processes indicating that these are resident microglia. Injury site
predominantly had enlarged macrophages indicating that these were the activated microglia or
macrophages responding to the injury and contributing the clearing the debris from the myelin
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degradation and other necrotic tissue. Spared tissue and the interface between the unaffected
tissue and the injury site had combination of both phenotypes possibly indicating that the resident
microglia are being recruited to the injury site in response to the cytokines released in the injury
site. This observation is consistent with the mechanism of the activation of microglia after SCI
[69]. Quantitative analysis did not show statistical differences among the number of
microglia/macrophages present in the injury site across all the groups including the control (Figure
6.20E). This result was validated using the CD68 staining across all the seven groups.
Morphology of the CD68+ cells was spheroidal indicative of activated macrophages phenotype.
There were very few CD68+ signal in the migrated cells around the Gtn-HPA islands in GE8,
GES8 and GE12 and GE12T groups but the morphology of the cells was not spheroidal possibly
suggesting that these were not macrophages. Even if they are truly macrophages, their number
was negligible compared to the numerous migrated cells. Similar to Iba1, analysis revealed no
differences between the groups in the injury site suggesting that Gtn-HPA does not elicit a severe
immune response greater than that observed in the control group. These consistent findings with
Iba1 and CD68 indicates that injection of Gtn-HPA, with or without the factors, did not elicit a
severe inflammatory response providing an in vivo evidence that Gtn-HPA is a biocompatible
biomaterial. Gtn-HPA and its constituents were well tolerated by the rats in the study. This is an
important finding in vivo for the Gtn-HPA material injected for the first time in the SCI models.
NeuN is a very specific biomarker for nuclear protein expressed in mature neurons. It also
has been used to study the differentiation of stem cells into neurons [70, 71]. Representative
sections from all the treatment groups were stained with NeuN to assess whether any neuronal
lineage was promoted by the potential regenerative effects of the migrated cells in the injury site.
Across all the treatment groups, we found that there were no NeuN+ cells in the injury site while
we were able to visualize characteristic neuronal cell body stained with NeuN in the unaffected
tissue and at the interface of the unaffected tissue and the injury site. Qualitatively, the number of
NeuN+ cells present in the unaffected tissue were higher than at the interface (Figure 6.23). No
NeuN+ cells were found in the cells migrated immediately surrounding the Gtn-HPA islands
indicating that Nestin+ NSCs did not have the cues required for differentiation into mature
neurons. NeuN staining is consistent with the NeuN images from complete resection model in
Chapter 4. We did not anticipate the presence of NeuN+ cells in the injury four weeks after Gtn-
HPA implantation. However, future studies should quantitatively investigate any differences
among the groups for the presence of neuronal differentiation in the injury site and near the
interface for longer duration post Gtn-HPA matrix injection.
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Gtn-HPA is a gelatin-based biomaterial and has excellent tunable properties to control the
gelation time and stiffness by varying the concentration of its constituents [13]. In our study, Gtn-
HPA was used in an in vivo application for the SCI for the first time. Therefore, it was important
to comment on the potential mode of degradation of the Gtn-HPA in vivo. Although Gtn-HPA was
injected in the brain ICH model, it’s in vivo degradation behavior was not studied previously [20].
Matrix metalloproteinases (MMPs) were reported to be involved into the cellular migration of
neural progenitor cells [72]. Preliminary MMP2 staining in the complete resection models showed
strong signal specifically at the border of the Gtn-HPA gel island in the Gtn-HPA injected groups.
Similar qualitative observations were noted in hemi-resection model as well. Since the MMP2
signal was strong only at the border of the gel island, we hypothesize that MMP2 is responsible
for the degradation of Gtn-HPA gel to promote cellular infiltration into the Gtn-HPA matrix.
Remarkably, MMP2 is a gelatinase A enzyme and therefore could be the agent to degrade gelatin-
based Gtn-HPA biomaterial in vivo. Upon quantification, migrated cells around the Gtn-HPA
islands in EGF-delivered with Gtn-HPA groups (i.e. GE8, GES8, GE12 and GE12T) released
significantly greater MMP2 compared to the Gtn-HPA gel only group [G8] (Figure 6.24). This could
explain the reason behind observing Gtn-HPA gel only in 2/6 animals in GES8 group. One
potential hypothesis is that presence of both chemoattractants led to greater cellular migration,
thereby increasing the MMP2 release near the Gtn-HPA islands resulting in faster degradation of
the Gtn-HPA. In addition to MMP2, EGF was the other protein stained with few representative
sections to confirm the presence of EGF in the GE8, GES8, GE12 and GE12T groups. The
stained micrographs confirmed the presence of EGF exclusively in the Gtn-HPA gel island as
reported in preliminary images in Chapter 4 (Figure 6.25). However, the Gtn-HPA gel island in
the Gtn-HPA gel only group [G8] stained negative for the EGF serving as a negative control
(Figure 6.25). Fisher’s exact test confirmed the association between the treatment group and EGF
presence in the Gtn-HPA four weeks post implantation. All EGF-delivered groups positively
stained for EGF while all Gtn-HPA gel only groups stained negative for EGF. These results are
reassuring that Gtn-HPA matrix can deliver EGF locally to the injury site at least over four weeks
and agree with the in vitro release of EGF from Gtn-HPA reported in Chapter 3. Furthermore, it
could also provide a reliable method to locate the presence of the Gtn-HPA gel in groups where
EGF was delivered with the Gtn-HPA matrix. Future studies should be designed to assess the
bioactivity of the EGF in vivo. For example, EGF delivered by the Gtn-HPA gel could be pre-
labeled with a fluorophore prior to injection and could be longitudinally followed over four or more
weeks and investigate if the signal is limited to the Gtn-HPA gel location. This experiment could
offer interesting insight into the degradation mechanism and EGF release mechanism in vivo.
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Due to difficulties with vWF staining, we were not able to stain sections from all the groups.
However, few representative rats showed the presence of vWF+ endothelial cells in the injury site
indicating that Gtn-HPA presence could promote angiogenesis in the injury site (Figure 6.32).
The identity of the cells migrated immediately surrounding the gel islands was determined
after sections were stained with Nestin to evaluate the presence of neural stem cellsin the injury
site. Nestin is an intermediate type VI protein and is considered as a biomarker for undifferentiated
CNS cells prior to committing to a specific cell lineage, considered to be the neural stem cells
(NSCs) [73]. It is reported that NSCs are found in a niche at the poles of the spinal cord ependymal
layer near the central canal. Similar niche is observed in the SVZ region in the brain [74]. These
niches stain positive for Nestin and negative for GFAP confirming the presence of NSCs [75].
There is a panel of biomarkers that can be used to further confirm the cells to be NSCs and
include Vimentin, Mushashi-1, and Sox 2 (R&D systems, SC205). In our study, cells migrated
around gel islands stained positive for Nestin, Vimentin and Mushashi-1 for EGF-delivered Gtn-
HPA groups (i.e. GE8, GES8, GE12 and GE12T) (Figures 6.26, 6.29 and 6.30). Positive staining
for all three markers in the migrated cells around Gtn-HPA further confirmed the identity of these
cells to be NSCs. In addition, double staining of GFAP/Nestin showed that these cells are not
astrocytes and similar conclusion was drawn after double staining of Vimentin/GFAP (Figure 6.27
and 6.29). Furthermore, EGF/Nestin were double-stained to confirm that NSCs migrate in
response to the presence of the EGF in the Gtn-HPA islands (Figure 6.31). We did not encounter
any Nestin+ cells in the injury site for the control or EGF in suspension group. There were some
cells that stained Nestin positive surrounding the gel islands in the Gtn-HPA gel only [G8].
Quantitative analysis of the Nestin+ cells showed significantly greater number of NSCs that have
migrated to the gel islands in GE8, GES8, GE12 and GE12T groups compared to the N, E and
G8 groups (Figure 6.28). These results show that Gtn-HPA gel alone is not capable of recruiting
important cells such NSCs while EGF and SDF-1α were able to recruit the endogenous NSCs
from the spinal cord ependymal layer niche near the central canal. Furthermore, there was no
difference between the GE8 and GES8 group indicating that SDF-1α either does not play a role
in NSCs recruitment or does not enhance the recruitment of NSCs to the injury site – finding also
observed in complete resection model in Chapter 4. This is consistent with the proposed
hypothesis that NSCs are highly responsive to EGF [76]. Hemann et al reported that intrathecal
administration of EGF resulted in significantly greater ependymal cell proliferation in the central
canal immediately rostral and caudal to the lesion compared to controls and in greater white
matter sparing [77]. Similar behavior of the NSCs was observed in our study in response to EGF
four weeks post Gtn-HPA with EGF injection. The migration of NSCs could explain the functional
209
recovery seen in EGF-delivered Gtn-HPA groups for both 8% and 12% Gtn-HPA gels. EGF could
promote better preservation of the spared tissue and reorganization of the injury site leading to
significantly better functional recovery in the rats with hemi-resection injury. Future studies should
include longer duration of evaluation and it would be interesting to find out the identity of the
migrated cells and assess if they differentiate into neural or glial fate to promote regenerative
response and improve functional recovery after the SCI.
In summation, a 2 mm hemi-resection of the spinal cord tissue at T8 level created a
standardized cavity in which 8wt% and 12% Gtn-HPA was injected with EGF and/or SDF-1α. Gtn-
HPA gel persisted in the injury site four weeks post injection. ‘Tucking-in’ the dural replacement
around the spinal cord after Gtn-HPA gel helped better contain the gel in the cavity created by
the hemi-resection in these rats. In the groups that delivered EGF and/or SDF-1α, rats showed
significantly better functional recovery in both urinary bladder function and motor function. Both
8% and 12% Gtn-HPA gels were permissive of cellular migration, making Gtn-HPA a promising
biomaterial for future studies. Injection of the Gtn-HPA matrix decreased the presence of growth-
inhibitory astrocytes at the border of the injury site thereby reducing the extent of glial scar
formation. Gtn-HPA is well tolerated and did not elicit a severe inflammatory response as
suggested by both Iba1 and CD68 biomarkers. Injecting the 8wt% Gtn-HPA matrix with EGF alone
or in combination with SDF-1α promoted recruitment of endogenous neural stem cells into the gel
islands in the injury site confirmed by positive staining for Nestin, Vimentin and Musashi-1.
However, SDF-1α did not enhance the recruitment response. Future studies should be planned
to evaluate the 12% Gtn-HPA with EGF in a contusion model of SCI to evaluate the potential
benefit of EGF delivered via Gtn-HPA to promote reparative healing response and improved
functional recovery as reported in this 2-mm hemi-resection model. The encouraging results in
this study would lay the foundation for further testing in different species of rats and in other SCI
species models such as minipigs and rabbits for eventual clinical application to improve the life
style of SCI patients – the goal that SCI researchers share throughout the world.
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Chapter 7:
Conclusions
215
The work presented in this thesis dealt with developing and evaluating an injectable
gelatin-based matrix (Gtn-HPA) incorporating EGF and SDF-1α in promoting tissue repair and
functional recovery after spinal cord injury. Specifically, Gtn-HPA was formulated to be injected
into a 2-mm cavity created by complete and hemi-resection SCI models. Following are the
conclusions which can be drawn from the data generated in this thesis:
1. EGF can be directly incorporated into a Gtn-HPA gel for its sustained release. In a neutral
buffer in vitro, 30% of the growth factor remains in the gel after 4 weeks. In vivo,
immunohistochemistry confirms that, after 4 weeks, EGF is present in gel remaining in the SCI
defect. By extension, SDF-1α, which has a molecular weight (10.6 kDa) larger than EGF (6.2
kDa) would likely display comparable release kinetics. These results are important in ruling out
the necessity of implementing secondary nano- and micro-meter particles as release vehicles for
EGF, and SDF-1α. Moreover, Gtn-HPA gel was persistent in the injury site after 4 weeks – a
significant improvement from the previously designed collagen-genipin gel for SCI.
2. Injection of Gtn-HPA/EGF immediately after SCI (a 2 mm long hemi-resection defect) provides
a notable neuroprotective effect resulting in a statistically meaningful improvement in behavioral
outcome at 4 weeks. However, injection of Gtn-HPA/EGF immediately after SCI (a 2 mm complete
resection defect) did not result in a statistically improved functional outcome at 4 weeks, likely
due to the extreme nature of the injury.
3. Gtn-HPA gel incorporating EGF accommodates the migrating of endogenous NSCs, clearly
demonstrated at 4 weeks post-injury. The results show that the gel is necessary, but not sufficient,
to enable the gel-filled defect to be infiltrated by cells with the potential to regenerate the neural
circuitry. EGF delivered without Gtn-HPA did not result in migration of endogenous NSCs
indicating the importance of delivery via Gtn-HPA. These results were consistent in both 2 mm
complete resection and 2-mm hemi-resection SCI models. Moreover, SDF-1α did not confer any
additional benefit of migration of endogenous NSCs into the Gtn-HPA gel.
4. An MRI protocol can be implemented to enable the visualization of the Gtn-HPA gel and its
distinction from spinal cord tissues and CSF. Specifically, T1-weighted inversion recovery (T1IR)
sequence with inversion time of 450 ms resulted in visualization of the Gtn-HPA gel.
5. Injection of Gtn-HPA, alone or incorporating EGF, resulted in reduced presence of reactive
astrocytes at the border outlining the injury area indicating delayed or reduced formation of glial
scar. However, injection of Gtn-HPA, alone or incorporating EGF, does not affect the number of
reactive astrocytes present in the injury site.
216
6. Gtn-HPA was found to be biocompatible because it did not elicit any severe inflammatory
response based on macrophage marker immunohistochemistry. Moreover, Gtn-HPA was present
in the injury site in form of ‘islands.
7. We found no differences between the 8% and 12% Gtn-HPA across various histological and
functional outcomes indicating that the 12% Gtn-HPA is also permissive of the cellular migration
into the Gtn-HPA gel.
217
Chapter 8:
Limitations and Future directions
218
8.1. Limitations of the thesis: Despite the extensive assessment performed to characterize
and evaluate Gtn-HPA/EGF/SDF-1α both in vitro and in vivo for spinal cord injury repair, there
were several limitations of this research work.
In vitro characterization:
• Although preliminary mechanical testing work was done specifically with 8% and 12% Gtn-
HPA to be used in our formulation, the technical difficulties with the shape and
homogeneity of the Gtn-HPA did not allow for an extensive assessment of the mechanical
properties of the gel to more accurate comparison with the stiffness of the spinal cord.
Moreover, the experiments characterized the stiffness modulus of the pre-formed Gtn-
HPA gels, the gelation rate profile for our formulations was not conducted due to the
evaporation issues encountered during preliminary trials. A proper mold for the fabrication
should be prepared for comparative analysis of 8% and 12% Gtn-HPA gel.
• EGF release profile from the 8% and 12% Gtn-HPA was studied for four weeks. However,
similar study was not planned with SDF-1α because SDF-1α was not incorporated alone
in the in vivo group. Moreover, we were not able to perform immunohistochemical
evaluation of the Gtn-HPA gel remaining after the four-week study to confirm the presence
of EGF primarily because of technical considerations in sectioning Gtn-HPA. Based on
the experience with the Gtn-HPA in prior work in our laboratory, it was difficult to
cryoprotect the Gtn-HPA gel to avoid artifacts and crystal formation during fast-freezing of
the Gtn-HPA.
• All the in vitro assessments were performed under conditions not encountered by the Gtn-
HPA gel in vivo such as Gtn-HPA degrading enzymes, blood during gelation, mechanical
forces experienced in the cavity during the movement of the animal, and breakage of the
Gtn-HPA gel due to perturbations. These factors could affect the gelation rate, gelation
time, final stiffness and the homogeneity in the Gtn-HPA gel in vivo.
• The bioactivity and encapsulation efficiency of EGF and SDF-1α was not assessed after
being encapsulated in the 8% and 12% Gtn-HPA. Although the in vivo results provide an
indirect mechanism to study the biological effects of EGF, a standardized in vitro assay
was not performed.
• While we found the presence of EGF in the Gtn-HPA gel in vivo after four weeks by
immunohistochemical staining, no comment could be made regarding the bioactivity of the
EGF in vivo.
219
In vivo evaluation in SCI model:
• Although standardized surgical excision models employed in this evaluation were ideal to
create a reproducible standardized defect, to visually confirm the injection of the Gtn-HPA
gel in the cavity, and to study regenerative responses in the cavity, contusion models are
more relevant clinical models studied for spinal cord injury. One of the disadvantages of
contusion models, however, is the variability of the defect, with respect to size and
location. Typically, contusion models do not breach the dura and therefore exhibit reduced
inflammatory response. Moreover, the defects produced by contusion would be fully
contained within the cord tissue constraining the gel which would be injected.
Nonetheless, standardized surgical excision models provide a proof of principle of the
biocompatibility of Gtn-HPA and the chemotactic effect of EGF on the migration of
endogenous neural progenitor stem cells.
• Isoflurane was used as an anesthetic during the surgery while sodium pentobarbital was
used in previous studies. Animals are quick to gain consciousness due to the use of
isoflurane disrupting the gelation of Gtn-HPA in vivo by their movements post-operation.
Moreover, we believe that isoflurane causes greater bleeding in the musculature during
the surgery affecting the in-situ gelation of Gtn-HPA.
• The injection of the gel was timed to be immediately after the creation of the surgical defect
in order to enable direct visualization of the gelation process and to provide assurance
that the gel filled the defect. One of the disadvantages of the procedure was that the
retraction of the cut ends of the spinal cord, while slight, pulled the cord away from the gel,
creating a microscopic separation. Another disadvantage was that the acute inflammatory
response may have led to degradation of the ends of the cord further contributing to a
separation between the gel and the cord ends.
• The unconstrained nature of the hemi-resection defect enabled even the compromised
movement of the animals to dislodge and mechanically breakup the gel. While “tucking
in” the autologous fascia, which served as replacement for the excised dura, may have
helped to constrain the gel, there was still fragmentation of the gel.
• Although Gelfoam aided in achieving complete hemostasis in the injury site before
injection of Gtn-HPA, few animals showed bleeding in the cavity site during injection. Other
approaches to control the bleeding should be considered for future studies.
• Fabrication step of the Gtn-HPA was not ideal given the subsequent addition of H2O2
after HRP. In an ideal case, a two-syringe model should be employed for simultaneous
delivery and mixing of HRP and H2O2.
220
• Since the Gtn-HPA was injected using a pipette directly into the cavity, the injection rate
of Gtn-HPA was not controlled due to the cumbersome nature of an automatic injector.
• We performed two types of functional evaluation – open field locomotor test and bladder
function. However, these functions do not address the other aspects after spinal cord
injury such as sensory function, pain, electrophysiological deficits. Although the scoring
for BBB scale was done in a blinded and randomized manner, a greater number of
evaluators should score the animals for hindlimb function.
• Our results present the findings from the acute injection of Gtn-HPA because the Gtn-HPA
gel was injected immediately after the injury – a scenario not replicable in the clinic.
• We evaluated the effects of injection of Gtn-HPA/EGF/SDF-1α for four weeks post
injection. Therefore, we are able to assess the beginning aspects of the tissue remodeling
and regeneration. In addition, although preliminary histological was performed on Day 0,
1 and 7 post injection, a comprehensive study with immunohistochemical staining was not
performed at early time points.
• Considering that Gtn-HPA does not embed well with paraffin-embedding procedure, we
used the fast-freezing method to prepare serial sections of the spinal cord. However, the
thickness of the sections was limited to 30 µm as opposed to 6 µm achieved with paraffin-
sectioning protocol. In addition, despite cryoprotection of the spinal cord samples with
sucrose solution, there were artifacts introduced in the sections due to crystal formation in
the tissue resulting in incomplete picture of the injury site after four weeks.
• Histomorphometry analysis was performed on one section per animal from the middle of
cord. Although that was representative of the injury site for the animal, volumetric analysis
could be performed in future studies to gain more insight into the injury site.
• Immunohistochemical analysis was restricted to one section per animal. New methods
such as CLARITY could give a volumetric analysis of the injury sites in terms of various
cells and biomarkers.
• MRI-based protocol was reliable in distinguishing Gtn-HPA from the surrounding tissue.
However, the protocol was not sensitive to detect edema, hemorrhage and other
pathophysiological features. Moreover, MRI examination was performed using fixed spinal
columns ex vivo. Future studies should employ longitudinal in vivo assessment of the
location and the volume of the Gtn-HPA over the duration of the experiment.
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8.2. Future directions: The research work presented in this thesis commends further
investigation of injectable Gtn-HPA incorporating EGF for tissue repair and regeneration after
spinal cord injury. The future directions are based on the findings and the limitations of this work.
In vitro characterization:
• More comprehensive characterization of the mechanical properties of the Gtn-HPA should
be done to evaluate the effect that injecting into cavity filled with blood or varying injection
rates has on the gelation profile of Gtn-HPA. Moreover, reliable protocol with proper
dimensions of the Gtn-HPA should be devised to study the stiffness of the Gtn-HPA gel
using compression testing and frequency sweep testing.
• 3D migration assays with two different formulations of Gtn-HPA - 8% and 12% - can be
performed with various cells such as neural progenitor stem cells, astrocytes, and
macrophages. In addition, the ability of Gtn-HPA/EGF in recruiting oligodendrocytes would
be interesting to evaluate in future experiments. In addition, the migration assay could
study the dose-dependent migration of these cells into the core.
• Detailed release profile kinetics studied with both EGF and SDF-1α can give critical
information about the behavior of the Gtn-HPA in sustained release of these factors. In
addition, embedding procedure for Gtn-HPA should be optimized so that
immunohistochemical assessment of the presence of EGF and SDF-1α can be performed
after four weeks.
• Other injectable biomaterials such as HA-Tyr and collagen should be evaluated both in
vitro and in vivo in comparison with Gtn-HPA. Moreover, other mitogenic factors such as
FGF-2, BDNF, PRP and NGF should be studied in combination with EGF if the
combination can enhance the response seen in these experiments.
In vivo evaluation:
• Contusion model of the spinal cord should be employed to validate that Gtn-HPA/EGF can
induce the migration of neural progenitor stem cells into the injury site. Since contusion is
a more relevant clinical model, it would be necessary to reproduce the data in higher
animal contusion models such as minipigs and monkeys from a translation point of view.
• Although the treatment groups showed promising results after 28 days, longer evaluation
more than four weeks (i.e. 8 weeks or longer) should be studied to gain insight into the
chronic effects of the proposed intervention.
222
• BBB scale is a well-established scale to measure functional recovery after spinal cord
injury. Different functional evaluation tests such as electrophysiological studies and
retrograde tracing should be incorporated in future to get a comprehensive view of
functional recovery. Quantitative analysis of urine retention volumes using catheterization
could also be considered.
• This study provided a comprehensive study of experimental groups to evaluate the effect
of Gtn-HPA/EGF/SDF-1α in a rodent spinal cord injury model. Few groups not evaluated
– SDF-1α in suspension, SDF-1α alone in the Gtn-HPA and 12% Gtn-HPA gel alone
should be evaluated in future studies for comparison.
• Since immediate injection of the treatment is not feasible in the clinicals setting, a modified
model to inject Gtn-HPA/EGF 7-14 days after inducing SCI should be considered.
• This work studied the serial coronals sections of the injury site in which axonal
regeneration is difficult to detect. Axial sections of the cord should be simultaneously
studied to get a comprehensive view of the injury site.
• MRI evaluation was able to distinguish Gtn-HPA in a reliable manner. Further optimization
of the ex vivo protocol may be performed to identify edema, hemorrhage and cavitation.
Furthermore, we believe that T1IR method in conjunction with T2-weighted image would
provide the most information about the injury site. In addition, future studies may compare
the histological or functional findings with the MRI metrics. Therefore, the protocol may be
optimized for in vivo imaging of the rats so that the degradation characteristics of the Gtn-
HPA gel can be studied in a longitudinal manner in future. It would be important to consider
the effect of cardiac and respiratory movements of the rat on the MRI images during
acquisition.
• In future, one may consider greater panel of biomarkers to study various cell types such
as oligodendrocytes and Schwann cells shown to promote regeneration in the defect area.
Moreover, ECM proteins in the injury site should be comprehensively studied using
histological and immunohistochemical methods.
• Finally, future formulation of the Gtn-HPA may plan to include cells delivered in the injury
site in a contusion or surgical excision models. These cells should be chosen such that
their recruitment would be difficult to achieve using chemotactic factors. This could ensure
repopulation of the spinal cord defect with all the major cell constituents present in the
healthy tissue.
223
Appendix:
1. Table outlining the total number of animals evaluated in thesis work:
Defect Experimental group Duration Study size (n)
3 mm left hemi-resection
8% Gel [G8] 2 weeks 2
8% Gel [G8] 4 weeks 2
8% Gel + SDF-1a [GS8] 2 weeks 1
8% Gel + SDF-1a [GS8] 4 weeks 2
8% Gel + SDF-1a in PCN [GS8P] 2 weeks 2
8% Gel + SDF-1a in PCN [GS8P] 4 weeks 2
1 mm left hemi-resection
Control [N] 4 weeks 6
8% Gel + EGF [GE8] 4 weeks 6
2 mm full-resection Control [N] 4 weeks 5
8% Gel [G8] 4 weeks 6
8% Gel + EGF [GE8] 4 weeks 3
8% Gel +EGF +SDF-1a [GES8] 4 weeks 5
2 mm left hemi-resection
Control [N] 4 weeks 6
EGF in PBS suspension [E] 4 weeks 5
8% Gel [G8] 4 weeks 6
8% Gel + EGF [GE8] 4 weeks 6
8% Gel +EGF + SDF-1a [GES8] 4 weeks 6
12% Gel + EGF [GE12] Day 0 1
12% Gel + EGF [GE12] Day 1 3
12% Gel + EGF [GE12] Day 7 3
12% Gel + EGF [GE12] 4 weeks 6
12% Gel + EGF - tucked-in dura [GE12T]
4 weeks 6
Total 90
224
2. Supplies to ensure before starting animal study
ARF (Diane/Sylvia):
- Oxygen cylinders
- Isoflurane
- Nembutal
- Heparin
- Lactated ringers
- Meloxicam
- Baytril/Cephazolin
- Heating pads & empty cages
- 4 face masks
- Surgical gloves
- Weighing scale
- Syringes (3 ml, 1 ml)
- Suture clips
- Scalpels
- Clip removers
- Razor for shaving rat’s hairs
Dr. Hsu
- Number clips for ears
- Gelfoam
Surgery and preparation:
- EGF (aliquots)
- SDF-1α (aliquots)
- PBS
- Gtn-HPA
- calibrated pipettes, tips
- Filters
- HRP (aliquots)
- H2O2 (aliquots)
225
3. Rat Spinal Cord Surgery Preparation and Post-Operative Care Checklist
Pre-surgical Set Up- Day++ before:
Confirm availability for the dates with Dr. Hsu
Order animals through BVARI (email [email protected] and cc Dr. Spector, Diane Ghera from ARF and Nickey Ortega for approval), procedures must wait 48 hrs after arrival
Set up room adjacent to O.R. a. Get 4 (short) empty plastic rat cages from the 3rd floor. b. Place cages on heat pad. c. Place a small cloth (blanket) in each cage. d. Set up oxygen and tubing with face masks for 4 rats.
Autoclave 1.5 ml tubes, water (if needed for reconstitution)
Place box with syringes and gloves into procedure/animal care room.
Make stock solutions of Meloxicam and Cephazolin. Keep refrigerated. a. Meloxicam (1mg/kg): 5 mg/ml bottle, 0.04 ml for 200 g rat, 0.05 ml for 250 g rat b. Cephazolin (35mg/kg): Add 9 ml saline to 1 g cephazolin, 0.07 ml (for 200 g rat, 0.09
for 250 g rat) Pre-surgical Morning of:
Turn on heating pads, make sure water level is OK, put on bag of lactate ringers
Take care of any animals already operated on (Bladder, Meloxicam & Baytril)
Have EGF and SDF-1a (as needed) ready on ice – do all the calculations before)
Fabricate gel and hold on ice Pre-surgical Immediate:
Weigh and record weight of rat
Take the rat to Dr. Hsu and set the isoflurane at 4% isoflurane. Once the rat is unconscious, keep isoflurane level at 2.5% to maintain anesthesia)
Intra-operatively:
Once the rat is under anesthesia (2.5%), shave the back, especially the surgery region
Get ready with the gel, PBS, any growth factors for the injection
Confirm with Dr. Hsu that it is 2 mm left hemi-resection defect
Inject 20 ul (2 mm left hemi-resection defect) of the gel into the injury site – calculations
As Dr. Hsu is finishing up, weight the rat and bring the second rat in. Post-Operative Care:
1. Take rat from the O.R. and place in heated cage in adjacent room. 2. Inject rat with 3 ml of warm Lactated Ringer’s Solution 3. Inject rat (1 mg/kg) Meloxicam - 5 mg/ml bottle, 0.04 ml for 200 g rat, 0.05 ml for 250 g rat 4. Inject rat (35 mg/kg) with 0.07 ml (for 200 g rat, 0.09 for 250 g rat) Cefazolin 5. For the first hour after surgery, monitor rat every 10 minutes for breathing 6. Thereafter, monitor rats every 45 minutes
Once the rat regains consciousness, express the bladder and place in cage with wood-chip bedding, a long nozzle water bottle, and 2-3 pieces of dry food on the bottom for easy access to the rat. Fill out postoperative card!!!
7. Return in the evening to express bladder and administer Cephazolin as per dosage guidelines (Ensure light/dark timings in the room with Diane Ghera or Sylvia Smith)
Daily Care thereafter:
1. 2x Daily: a. Bladder expression, b. Cephazolin – twice daily for 1 week 2. 1x Daily: Meloxicam - once every 22-24 hours for 4 days 3. 1x Weekly: a. Weigh b. videotape movement
Keep track of everything on the VABHS record sheet for each rat
226
4. Functional evaluation – Open field locomotor test and 1-week clip removal protocol
BBB:
- Bring the animals (maximum 4 at a time) on the second floor wrapped in the gray cloth
cover in my box located in the rat room on 3rd floor.
- In the pre-OR room, have 2 green chux tightly on the table using tape.
- Weigh the animal and record in the VABHS record sheet
- Gently take the rat and place her in the middle of the table and record video for 3 min
- Put rat back in the cage and repeat for others
- Take notes of the hindlimbs movement with BBB scale
- Randomize the videos for randomized and blind rating
Clip removal (after 1 week – reserve time with Diane Ghera / Sylvia Smith)
Items needed: Clip remover tool, tweezers, Vetbond (if needed)
- After BBB, bring the cart with rats in the surgery room 216A.
- Turn on the oxygen at 1.5-2 L/min
- As you are ready to bring in the rat, turn the isoflourane to 4%
- With the rat glove in the right hand, bring the rat and put her into the nose cone. At 4%, it
will roughly take 1-2 minute for the rat to anesthetize
- Pinch the tail to make sure rat is anesthetized. Once anesthetized, turn the isoflourane
to 2%
- Using the clip remover tool, take out all the clips. Sometimes, due to blood and/or other
material, it will take some force to take out the clips.
- If you notice any open area due to clip removal, use vetbond to close it.
- Once all the clips are removed, turn off isoflurane and put the rat back in the cage.
- Repeat the procedure for each rat.
- Once all are done, turn off the oxygen and isoflurane. MAKE SURE both are turned off
- Clean up and throw away the clips
- Bring the rats back to the rat room on 3rd floor wrapped with gray cloth cover and put
them back in their original place.
- Make sure the rats are awake in the cage!
227
5. Animal sacrifice - transcardial perfusion of female Lewis rats
Items needed:
• Medium dissecting scissors
• Small dissecting scissors, scalpel
• 18 gage needles
• 4% paraformaldehyde solution (PFA)
• PBS
• Heparin solution
• Ice bucket
• Perfusion pump
• Specimen containers – 50 ml Eppendorf tube
• Red biohazard bags
• 1 ml syringes with 25 gage/ 5/8 inch long needle (for administering IP Nembutal)
• Nembutal
• 100ml measuring cylinder
• Metal pan with grill
• Two glass beakers
• Rat glove
• Box of gloves
• Paper towels, paraffin
Prepare:
-120 ml of heparin solution at 20 units/ml PBS, put on ice (Add 2.4 ml Heparin in 120 ml PBS
located on the shelf opposite the chemical hood – labeled with my name)
-100 ml paraformaldehyde on ice
-50 ml Eppendorf tube with 4% PFA – label it with animal number and the surgery date
-Once both are prepared in glass beakers, put them tilted at an angle in the ice bucket
-The ‘in’ tube should be in the heparinized saline beaker: use tape to fix the tube in the solution
to avoid air bubbles.
-The ‘out’ tube should be fixed with 18 Gage needle (pink cover) wrapped tightly with paraffin to
ensure no leakage
Procedure:
1. Flush air from pump tubing with heparinized saline solution (remove any air bubbles) 2. Anesthetize rat with 150 mg/kg pentobarbital (Nembutal) IP injection (200 g → 0.6 ml,
201-225 g → 0.65 ml, 225g → 0.675 ml, 225-250 g → 0.7 ml, 250 g → 0.75 ml) 3. Using medium dissecting scissors cut skin across abdomen 4. Using medium dissecting scissors cut skin along midline of chest 5. Using medium dissecting scissors cut sternum along midline of chest 6. Make lateral cut from bottom of sternum to the left side of thorax 7. Expose the heart
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8. Insert needle into left ventricle and please always be on the lookout for the needle in the ventricle. Due to animal movement the needle could come out of the ventricle so hold the needle inside during the perfusion
9. Turn on pump at medium setting and speed 3 10. Quickly cut right atrium using small dissecting scissors 11. Increase pump speed to 7 12. Just as the heparin solution is about to finish, turn off the pump and switch to PFA
solution (be careful not to introduce any air bubbles into the line), then restart the pump at speed 7.
13. Once the rat is completely perfused with PFA, discard the needle in the sharps container.
14. Flip the rat body and dissect out the skin layer to expose the collagen fascia cover. Any sized scalpel would work well for this.
15. Make cuts along the ribs on the sides. Top cut should be around the neck and the bottom cut should be around the ‘white’ fascia region to ensure the defect site is part of the resected spinal column
16. Put the column in 4% PFA in 50 ml Eppendorf tube and store it in 4C cold room on the first floor.
17. Discard the rat carcass in the red biohazard bag. Please discard all the blood, saline and PFA in the container under the chemical hood. For thick blood, use paper towels to clean the metal pan and the grill. Those paper towels can go into the red biohazard bag.
18. Clean all the tools with plenty of water in the sink. Air dry the metal pan, grill and the white board while the tolls (scissors, etc) can be cleaned and dried with paper towels.
19. Put the pump back into its box! 20. Please discard the red biohazard bag in the animal facility carcass freezer room #R2-
205 (room is near 2nd floor entrance in animal facility) 21. Keep the dirty cage in the wash room on the 3rd floor animal facility. Please make sure to
keep the VABHS white sheet and the animal cards with you (the red and the pink card).
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6. Fast freezing spinal cords and embedding procedure
Supplies to ensure:
• Medium and small dissecting scissor, Bone ronguers
• Scalpel/sharp blade
• Dry ice
• Aluminum foil & mold
• OCT Tissue-Tek compound
• Liquid nitrogen
• Isopentane (2-methylbutane)
• Metal bowl and tongs
• PBS
• Surgical gloves & markers
Spinal cord harvest from spinal column:
• Get liquid nitrogen, dry ice, isopentane
• Make Aluminum foil molds for tissue embedding
• Carefully take out extra tissue (muscle and bone) from the spinal column
• Using small dissecting scissors, slowly and carefully isolate the cord
• Make cuts at 45 angle and then proceed carefully not to puncture it/damage it
• Do not let the tissue dry out so dip in PBS as needed
• Once the cord is isolated, rinse it in PBS and then put it in the Al foil mold (marked with the sample #) with OCT compound
o Ensure that the cord is covered by OCT on all surfaces o Ensure that the cord is stable in the mold – cord at the bottom with floor of the
mold o Ensure there are no air bubbles o Ensure the cord is nicely placed in the middle of the mold
Tissue Embedding using isopentane and liquid nitrogen
• Fill isopentane in the metal bowl
• Fill Styrofoam box with liquid nitrogen (take all necessary precautions and wear protective gear)
• Lower the isopentane metal bowl into the liquid nitrogen
• Let it cool till the bottom of the bowl is opaque (white)
• Remove the bowl using tongs and then submerge the mold with the cord in isopentane for fast freezing
o If the isopentane turns completely clear, it is not cool enough. Cool it in liquid nitrogen again
o Lower one mold at a time in isopentane. Do not try to embed 2 spinal cord tissues in isopentane
• Let the OCT freeze in isopentane completely. You should see outside to inside freezing. Freezing of the entire block should take less than a minute
• Place the frozen block in dry ice for some time to ensure all the vapors either from isopentane or liquid nitrogen are gone
• Cover the block with Al foil marked with sample #
• Store the tissue blocks at -80 C (cryosection to produce serial 25-30 µm thick sections with CT ~ -17-19 C and OT = -20 C)
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7. Typical immunohistochemistry staining protocol
Antibody Company Dilution Antigen retrieval
2 abs Dilution
Iba-1 (Rb) Wako 1:400 pH6 buffer 488 Rb 1:300
CD68 (Ms) Biorad 1:200 pH6 buffer Cy3 Red 1:300 Procedure:
1. Warm the tissue sections at 550C in slide warmer for 3 hours.
2. Allow the slides to cool at room temperature (RT) for 15 minutes.
3. Immersed the slides in PBS at RT for 30-40 min.
4. Antigen retrieval step - citrate buffer pH 9 (200 ml diH2O + 1.875 ml unmasking solution) or high
pH 9 buffer
a. Preheat the buffer containing the staining rack in pressure cooker at 1000C
b. Set up the temperature to the required level well in advance and check in between for
proper increase in temperature. Immerse the slides in preheated buffer in staining rack
and heat in the pressure cooker at the required temperature for 20 minutes.
c. After heat retrieval step, the slides are allowed to cool in the buffer itself for 15 minutes
at RT.
5. Immerse the slides in 0.3% TritonX-100 in PBS for 30 minutes.
6. Immerse the slides in PBS for quick dips and then for 5 minutes.
7. Fix the slides in ice cold methanol (precooled at -200C) for 10 minutes.
8. Then transfer the slides to PBS and allow to stand for 5 minutes.
9. The slides are taken out from PBS and the sides are blotted with care being careful not to wipe
off section. Mark a closed circle around tissue section with an IHC marking pen.
10. Protein block step
Block the slides with serum-free protein block for 1 hour.
11. Wash the slides as follows:
a. 0.05% Tween20 in PBS (5 min)
b. PBS (5 min)
c. PBS (5 min)
12. Dilute the primary antibodies in DAKO antibody diluent to the working dilution and add to the
slides. Slides are incubated at 40C overnight.
13. Wash the slides as follows:
a. 0.05% Tween20 in PBS (5 min)
b. PBS (10 min)
c. PBS (10 min)
14. Incubate with secondary antibodies for 2 hours (at room temperature). (Secondary antibody
constituted using DAKO antibody diluent.
15. Wash the slides as follows:
a. 0.05% Tween20 in PBS (5 min)
b. PBS (10 min)
c. PBS (10 min)
16. After 3 quick dips in diH2O, blot excess liquid and mount in DAPI containing fluorescent mounting
media (DAKO) with cover slip.
17. Store at 4 C
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8. Quantification of Immunohistochemical biomarkers
1) Import original IHC images into ImageJ/Fiji.
2) Assign appropriate scale.
3) Convert the image to 8-bit image
4) Uniform threshold was applied to all the images for each biomarker (threshold for each
biomarker was determined using at least 15 samples images and compared with original IHC
images.
5) Post-threshold, a standardized ROI of 0.3 mm2 was created.
6) Percent area of the signal in the standardized ROI was measured.