©2011 maria nuria royo gascon all rights reserved
TRANSCRIPT
©2011
MARIA NURIA ROYO GASCON
ALL RIGHTS RESERVED
NEUROPLASTICITY OF SPINAL CORD NEURONS BASED ON PIEZOELECTRIC
STIMULATION AND ELECTROPHYSIOLOGICAL ANALYSIS AFTER STEM CELL-
DERIVED PROGENITOR TRANSPLANT
By
MARIA NURIA ROYO GASCON
A Dissertation submitted to the
Graduate School − New Brunswick
Rutgers, The State University of New Jersey
and
The Graduate School of Biomedical Sciences
University of Medicine and Dentistry of New Jersey
in partial fulfillment of the requirements
for the degree of
Doctor of Philosophy
Graduate Program in Biomedical Engineering
written under the direction of
Dr. William Craelius
and approved by
________________________________
________________________________
________________________________
________________________________
New Brunswick, New Jersey
October, 2011
ii
ABSTRACT OF THE DISSERTATION
Neuroplasticity of spinal cord neurons based on piezoelectric stimulation and electrophysiological
analysis after stem cell-derived progenitor transplant
By MARIA NURIA ROYO GASCON
Dissertation Director:
William Craelius
Repair strategies in the context of spinal cord injury cover a broad amount of fields. Different
approaches have been considered ranging from the chemical, mechanical, pharmacological,
material sciences, electrical and chemical engineering sphere. There is much interest in
combinational therapies since many approaches yield promising results yet none is beneficial
enough in functional terms. There is also a necessity of properly evaluate the improvement
these therapies pose from a functional point of view and do it with sufficient resolution to target
and invest in strategies with higher potential.
This thesis is born with the interest of contributing to the spinal cord injury field at those two
levels. On one side, I have developed an injury model and techniques to test the efficacy of a
therapy for spinal cord repair as compared to controls. On the other, I have proposed a
combinational therapy in the form of a scaffold that combines biomaterials and electrical fields to
stimulate regeneration
In the first part of this thesis, I have developed an animal model to test effectiveness of treatment
by analyzing the electromyography signal of the intercostal muscles. The respiratory system is a
good test bench, usually neglected in regeneration studies. I have used a mix of engineering
iii
approaches from signal processing to animal physiology analysis to provide the test with enough
resolution to identify improvement. Then, I have proved the efficacy of the model by using a stem
cell therapy.
In the second part of the thesis, I have tested piezoelectric polymers as a useful platform to
deliver electrical fields to neurons. I have shown the resulting increase in neuronal growth upon
exposure to alternating electrical fields, in concrete, in neuronal branching. These results
encourage the use of biocompatible piezoelectric polymers which are very versatile in nature, as a
source for combinational therapies. Future studies will translate this in vitro model into an in vivo
treatment which will be assessed with the strategy explained in the first part.
iv
Dedication
Lluís Montal, beyond time, distance and across borders of life, I hope you are proud.
This goes to you
Lluís Montal, més enllà del temps, la distància i les fronteres d’aquest món, espero que
n’estiguis orgullós. Aquesta va per tu.
v
ACKNOWLEDGMENTS
This PhD would have not been possible without the help of many people. I especially thank my
advisor, Dr. William Craelius. He got into this academic adventure with an electrical engineer
with a passion to cure Spinal Cord Injury. Bill, you are an impressive scientist but more
impressive person. Thank you for believing in me.
I also thank Dr. Jerry Scheinbeim, who opened the world of polymers to me with patience and
understanding and who always cared about my well-being beyond anything else.
I thank Dr. Bonnie Firestein, for offering her laboratory, ideas and friendship. Her passion for
science is contaminating. I am one of the lucky ones to be infected. I also thank all the people
from her lab, for the knowledge and discussion on tissue culture. I never annoyed them enough!!
You would not have this thesis in your hands if not for Dr. Troy Shinbrot. He stirred the fun for
discovery and gave me good advice: do what you enjoy in life because time will pass no matter
what.
I thank Dr. Dave Shreiber who always challenged me to improve and excel.
I thank Dr. Hans Keirstead, a driven scientist and a person of freedom, who inspired me in so
many ways. I also thank everybody in his lab for teaching all there is to know about animal work,
with amusement and style.
I thank the people at California Stem Cell, Inc, especially Dr. Monica Siegenthaler, for the cells,
vi
the expertise and the discussion on the stem cells studies. It was challenging and exciting!
I thank all my friends who have worked in the Rurehablab through the years, for being all such a
supportive team. Especially, and no words will be enough, for my dear friend Dr. Mike Wininger.
He is a brilliant scientist, a real mentor, and a true friend from whom I’ve learnt in so many
aspects of my career. He has been a guiding light. This message he will read next to an open
Matlab window. Mike, I owe you big!
I thank all my friends in Barcelona, New Jersey and California or anywhere life has tricked them
to go now. If I come out of this stronger and amazingly crazy is because of you. Especially have
suffered this thesis and need special mention the following:
Alberto and Rocío, my adoptive parents in the US. Mercedes, my home away from home. Nachi,
my Argentinian twin sister. Carles, who taught me that if I don’t believe in me, the battle is
already lost. Lavanya Peddada who has walked this long path with me. This PhD would make
sense if just for getting her in my life.
I thank all the people with Spinal Cord Injury and Spinal Muscular Atrophy, because they are the
example and inspiration for us working in the benches. We will make it happen!
My warmest gratitude goes to my family, in concrete my dear grandma. Last but not least, I thank
my parents and siblings. If I could make it so far and away, through all the happy and the rough
times, is because they are always with me. If I can live the life I’ve chosen, is because they have
my back. Mom, dad, Cèlia, Jaume and Roger, this and anything I will ever accomplish belongs to
you. You make me strong.
vii
Finalment, agraeixo als meus pares i germans el seu recolzament durant tots aquests anys. Si me
n’he sortit, tan lluny de casa, en els moments feliços i en els difícils. és perquè sempre sou amb
mi.. Gràcies a vosatres, tinc la vida que he escollit. Mama, papa, Cèlia, Jaume i Roger, aquest
doctorat i qualsevol cosa que aconsegueixi en aquest món, us pertany. Sou vosaltres que em feu
forta.
viii
TABLE OF CONTENTS
ABSTRACT....………………………………………………………………………………...…………….ii
DEDICATION…………………………..………………………………………………………………….iv
ACKNOWLEDGMENTS………………...………………………………………………………………...v
TABLE OF CONTENTS…………………………………………………………………………………viii
LIST OF TABLES…………………………………………………………………………………………xii
LIST OF ILLUSTRATIONS…………………………………………………………………………….xiii
CHAPTER 1 INTRODUCTION .................................................................................................................. 1
1.1. HYPOTHESIS ......................................................................................................................................... 3
1.2. CONTRIBUTIONS TO THE FIELD ............................................................................................................. 3
CHAPTER 2 ASSESSMENT OF TREATMENTS IN SPINAL CORD INJURY .................................. 5
2.1. GOAL OF THIS CHAPTER ........................................................................................................................ 5
2.2. ETIOLOGY OF SPINAL CORD INJURY ..................................................................................................... 5
2.3. CONSIDERATIONS ON PRIORITIES IN THE CONTEXT OF SPINAL CORD INJURY ......................................... 6
2.4. HISTOLOGY, MOLECULAR BIOLOGY AND GENETIC TESTS ...................................................................... 7
2.5. BEHAVIORAL TESTS .............................................................................................................................. 8
2.6. FUNCTIONAL TESTS ............................................................................................................................ 10
2.7. SIGNIFICANCE OF ELECTROPHYSIOLOGICAL ANALYSIS OF TREATMENT .............................................. 12
CHAPTER 3 ELECTROMYOGRAPHY ................................................................................................. 13
3.1. GOAL OF THIS CHAPTER ...................................................................................................................... 13
3.2. ELECTROPHYSIOLOGY OF THE SKELETAL MUSCLE .............................................................................. 13
3.3. AUTOMATIC MUP ANALYSIS ............................................................................................................. 18
3.4. IP ANALYSIS (IPA) ............................................................................................................................. 19
3.5. ELECTROPHYSIOLOGICAL PARAMETERS AFTER NERVE INJURY ........................................................... 24
3.6. ELECTROPHYSIOLOGICAL DIFFERENCES BETWEEN RATS AND HUMANS .............................................. 26
ix
3.7. SUMMARY .......................................................................................................................................... 27
CHAPTER 4 EMG SOFTWARE DEVELOPMENT .............................................................................. 28
4.1. GOAL OF THIS CHAPTER ...................................................................................................................... 28
4.2. NEED FOR SOFTWARE MODIFICATIONS ............................................................................................... 28
4.3. ELECTROCARDIOGRAM REMOVAL ...................................................................................................... 30
4.4. AUTOMATIC ZERO-CROSSINGS TRACKING .......................................................................................... 32
4.5. TURNS/AMPLITUDE ANALYSIS (TAA) ................................................................................................. 32
4.6. ANALYSIS OF THE ELECTROMYOGRAPHIC BURST ................................................................................ 36
4.7. SUMMARY .......................................................................................................................................... 37
CHAPTER 5 RAT MODEL OF INJURY ................................................................................................ 39
5.1. GOAL OF THIS CHAPTER ...................................................................................................................... 39
5.2. PHYSIOLOGY OF THE RESPIRATORY SYSTEM ....................................................................................... 39
5.3. ANATOMY OF INTERCOSTAL MUSCLES ................................................................................................ 41
5.4. RESPIRATORY FUNCTION OF INTERCOSTAL MUSCLES ......................................................................... 42
5.5. INNERVATION OF INTERCOSTAL MUSCLES .......................................................................................... 44
5.6. INJURY LOCATION AND TYPE .............................................................................................................. 45
5.7. EXPERIMENTAL PROTOCOL ................................................................................................................ 46
5.8. SUMMARY .......................................................................................................................................... 48
CHAPTER 6 RESULTS ON A STEM CELL REPAIR THERAPY ...................................................... 50
6.1. GOAL OF THIS CHAPTER ...................................................................................................................... 50
6.2. BACKGROUND .................................................................................................................................... 51
6.3. METHODS ........................................................................................................................................... 52
6.4. ELECTROPHYSIOLOGICAL RESULTS .................................................................................................... 56
6.5. BEHAVIORAL RESULTS ....................................................................................................................... 58
6.6. HISTOLOGICAL RESULTS ..................................................................................................................... 58
6.7. DISCUSSION ........................................................................................................................................ 64
6.8. SUMMARY .......................................................................................................................................... 65
x
CHAPTER 7 EFFECTS OF ELECTRICAL AND MECHANICAL STIMULI IN NEURONS ......... 66
7.1. GOAL OF THIS CHAPTER ...................................................................................................................... 66
7.2. STRATEGIES FOR SPINAL CORD INJURY .............................................................................................. 67
7.3. CHEMICAL GROWTH FACTORS ............................................................................................................ 68
7.4. STRUCTURAL SUPPORT ....................................................................................................................... 68
7.5. STIFFNESS ........................................................................................................................................... 70
7.6. MECHANICAL STIMULI ........................................................................................................................ 70
7.7. ELECTRICAL STIMULI ......................................................................................................................... 75
7.8. ELECTRICALLY ACTIVE POLYMERS ..................................................................................................... 80
7.9. SIGNIFICANCE ..................................................................................................................................... 82
CHAPTER 8 PIEZOELECTRICITY ....................................................................................................... 83
8.1. GOAL OF THIS CHAPTER ...................................................................................................................... 83
8.2. HISTORY ............................................................................................................................................. 83
8.3. PIEZOELECTRIC CLASSES .................................................................................................................... 84
8.4. CONSTITUTIVE EQUATIONS FOR A PIEZOELECTRIC MATERIAL ............................................................. 85
8.5. PIEZOELECTRIC POLYMERS ................................................................................................................. 88
8.6. SUMMARY .......................................................................................................................................... 92
CHAPTER 9 EXPERIMENTAL DESIGN AND CHARACTERIZATION OF PIEZOELECTRIC
MATERIALS ............................................................................................................................................... 93
9.1. GOAL OF THIS CHAPTER ...................................................................................................................... 93
9.2. FABRICATION OF PIEZOELECTRIC FILMS ............................................................................................. 94
9.3. CHARACTERIZATION OF MATERIALS ................................................................................................... 94
9.4. CELL CULTURE ................................................................................................................................... 97
9.5. SETUP AND STIMULATION PROTOCOL ................................................................................................ 97
9.6. IMMUNOCYTOCHEMISTRY ................................................................................................................ 101
9.7. CELL ANALYSIS AND IMAGING .......................................................................................................... 101
9.8. STATISTICAL ANALYSIS .................................................................................................................... 103
xi
9.9. SUMMARY ........................................................................................................................................ 103
CHAPTER 10 GROWTH OF CELLS ON PIEZOELECTRIC POLYMER FILMS ........................ 104
10.1. GOAL OF THIS CHAPTER .................................................................................................................. 104
10.2. EFFECTS OF PIEZOELECTRIC POLYVINYLIDENE FLUORIDE IN SPINAL CORD NEURONS ..................... 104
10.3. DISCUSSION .................................................................................................................................... 109
10.4. SUMMARY ...................................................................................................................................... 112
CHAPTER 11 CONCLUSION ................................................................................................................ 113
CHAPTER 12 BIBLIOGRAPHY ............................................................................................................ 115
xii
LIST OF TABLES
Table 1- MUP features ............................................................................................................. 16
Table 2- MUP parameters obtained by a Multi-MUP Analysis and criteria used by it. .......... 19
Table 3- Parameters obtained from analysis of the IP signal. ................................................. 24
Table 4-IPA parameters provided by EMGvet software. ......................................................... 30
Table 5- Comparison of criteria for IPA parameters between Keypoint and EMGvet, where d
is duration of segments and amp is amplitude of segments. ................................................. 38
Table 6- Animal groups, condition,outcome measuremes, sacrifice points and number of
subjects (n) .............................................................................................................................. 52
Table 7-Rheolograph measurements for PZ films fabricated as described in methods ......... 95
xiii
LIST OF ILLUSTRATIONS
Figure 1.Schematic shows 2 motor unit pools and the patterns of innervation of each
neuron in different colors. ...................................................................................................... 14
Figure 2. Schematic of SFAP potentials recorded at short distance (A) and far distance (B). 15
Figure 3. Schematic representation of motor unit potential (MUP) generation and recording
by needle electrode. ................................................................................................................ 16
Figure 4. Main features of a MUP. .......................................................................................... 17
Figure 5- EMG signal of the IIIrd intercostal muscle before (A) and after (B) being processed
by the cleansing algorythm. In (A) it was also included the digitalized signal, which is green
for only EKG, magenta for respiratory burst, and red for smoothed signal. .......................... 32
Figure 6-Turns are depicted as black asterisk. Maximum are depicted in red dots for
maximum and green for minimum.in a time series (vector) .................................................. 33
Figure 7- Histograms of the segment amplitudes (blue) are shown, overlayed with scatter
plots of the values of the duration versus the amplitude of the individual segments (red),
and threshold values for activity (black) for two different animals (A and B) 1 week after
injury. ....................................................................................................................................... 34
Figure 8- Histograms of the segment amplitudes (blue) are shown, overlayed with scatter
plots of the values of the duration versus the amplitude of the individual segments (red),
and threshold values for activity (black) for two different animals (A and B) 4 weeks after
injury. ....................................................................................................................................... 34
Figure 9- Histograms of the segment amplitudes (blue) are shown, overlayed with scatter
xiv
plots of the values of the duration versus the amplitude of the individual segments (red),
and threshold values for activity (black) for two different animals (A and B) 5 weeks after
injury ........................................................................................................................................ 35
Figure 10- Inspiratory burst with activity regions based on definition of EMGvet. Activity is
presented in red while the general inspiratory burst in blue. ................................................ 35
Figure 11- Inspiratory burst with activity regions based on definition of EMGvet. Activity is
presented in red while the general inspiratory burst in blue ................................................. 36
Figure 12- (A) raw and digitized EMG signals of the IIIrd intercostal muscle for several
inspirations before processing. Digitalized signal was also included, green for only EKG,
magenta for respiratory burst, and red for smoothed signal. (B) EMG during a single
inspiration after processing by the cleansing algorithm. ........................................................ 37
Figure 13- IP analysis of EMG signal from intercostal muscles 2 (a) and 3 (b) presented as
turns per second. Data expressed as mean and standard error. Student t-test with *p<0.05,
**p<0.005. ............................................................................................................................... 57
Figure 14-BBB locomotor scores for MotorGraft treated and vehicle control animals with a
T3 SCI. Testing was performed at 5 days post injury prior to MotorGraft transplantation for
pretreatment comparison (5D Post-injury/Pre-MG). Testing was preformed again at 1 week,
2 weeks, 20 days, and 27 days after MotorGraft transplantation (1 Wk Post-MG, 2 Wks Post-
MG, 20 D Post-MG, and 27 D Post-MG, respectively). The locomotor capability of
MotorGraft and vehicle control treated animals did not differ. Data are expressed as mean ±
standard error. ........................................................................................................................ 58
Figure 15-MotorGraft cells survive and begin to express ChAT. a, d) Human nuclei (green)
xv
were detected in the spinal cords of transplanted animals. b, c, e, f) Hoechst nuclear counter
stain (blue) (b and e) colocalizes with human nuclear staining (c and f). g, h, i) ChAT positive
staining (red) is localized with MotorGraft (human nuclei in green) and surrounding cells
(Hoechst in blue). Images taken at 200X magnification. ........................................................ 59
Figure 16-a) The average number of neurons in a quadrant of spinal cord cross sections from
vehicle control (ave= 29) and MotorGraft transplanted animals(ave=38). There were
significantly greater number of neurons in transplanted animals (ave=38). b) The average
number of motor neurons in a ventral horn of vehicle control (ave= 7) and MotorGraft
transplanted animals.(ave=12) rostral to the injury epicenter. c, d) Representative NeuN
staining (red) in transplanted (c) and vehicle control (d) spinal cord sections. e, f)
Representative ChAT staining (red) in transplanted (e) and vehicle control (f) spinal cord
sections. Images taken at 200X magnification ....................................................................... 61
Figure 17- Comparison of serotonergic fiber sprouting (Stained by 5-HT) in vehicle control
and MotorGraft transplanted groups. MotorGraft transplantation resulted in increased
integrated density of 5-HT fibers (red) 2mm rostral (+2) to the injury epicenter. Analysis is at
2mm and 1mm rostral (+2 and +1, respectively) and 2mm and 1mm caudal (-2 and -1,
respectively) to the injury epicenter. Images taken at 200X magnification. Data is expressed
as mean ± standard error. *p<0.05. ........................................................................................ 63
Figure 18- Nomenclature of axis ............................................................................................. 87
Figure 19-PVDF Molecule ........................................................................................................ 89
Figure 20- Axis nomenclature for the piezoelectric matrix ..................................................... 90
Figure 21- PLLA molecule. The asterisk indicates the chiral carbon atom. ............................. 91
xvi
Figure 22- DSC graphic showing the melting temperature of PVDF. ...................................... 95
Figure 23- FTIR spectra of non-polarized PVDF (PV, top) and piezoelectric PVDF (PZ, bottom).
The characteristic peaks of the α-phase and β-phase have been marked for convenience. . 96
Figure 24- Set-up of the well plate for seeding. ...................................................................... 97
Figure 25- VIBES: Polycarbonate base for stimulation of well plate cell cultures. ................. 98
Figure 26- Mechanical characterization of well-plate stimulation system: contour plot of
frequency (left) and amplitude (right) across the well plate. ................................................. 99
Figure 27: Mechanical characterization of well plate stimulation at 80Hz, 50Hz and 20Hz. 100
Figure 28- Centrifugal labeling scheme. Processes attached to the soma (green circle) have
an order of 1. At each branch point the order is increased by one. ..................................... 102
Figure 29- Spinal cord cultures immunostained with a MAP2 antibody after 5 DIV in the four
conditions: (A) US-PV, (B) S-PV, (C) US-PZ and (D) S-PZ. Scale bar=30 µm. .......................... 105
Figure 30- Comparison of branching features between (A) US-PV and S-PV and (B) US-PZ and
S-PZ. *** p<0.001, **** p<0.0001 (Unpaired t-test/Mann-Whitney test). Standard error
depicted. ................................................................................................................................ 106
Figure 31- Comparison of average number of processes per cell between (A) US-PV and S-PV
and (B) US-PZ and S-PZ. * p<0.05, *** p<0.001, **** p<0.0001 (Unpaired t-test/Mann-
Whitney test). Standard error depicted. ............................................................................... 107
Figure 32- Comparison of average number of processes per cell between (A) US-PV and S-PV
and (B) US-PZ and S-PZ. (Unpaired t-test/Mann-Whitney test). Standard error depicted. .. 108
Figure 33- Comparison of Sholls analysis of the total number of neurite intersections
xvii
between (A) US-PV and S-PV and (B) US-PZ and S-PZ. Bar indicates significance * p<0.05
(Unpaired t-test/Mann-Whitney test). Standard error depicted. ......................................... 108
Figure 34- Comparison of neuronal densities for PV substrates (A) and PZ substrates (B) **
p<0.01 (Unpaired t-test/Mann-Whitney test). Standard error depicted. ............................. 109
1
Chapter 1
Introduction
Spinal Cord Injury (SCI) is a complex problem, with acute and chronic scenarios. In chronic
injuries, pressure sores, thermoregulatory and respiratory problems are the most common
problems, being diseases of the respiratory system the primary cause of death after one year of
injury. In order to find the cure for SCI, contributions at different levels are necessary, ranging
from strategies for tissue regeneration, effective tools of assessment, good modeling of the injury,
which also includes targeting physiological systems with high priority. Biomedical engineering is
a relatively emerging field within the engineering disciplines and it is defined by the application
of the engineering principles to a biomedical problem. It entails mostly interdisciplinary work and
interfaces between many fields. It is in a context like spinal cord injury, were biomedical
engineering approaches are most useful. This thesis is a comprehensive study which addresses
and contributes to different parts of this problematic from several biomedical perspectives.
First, I address the two most problematic aspects in the context of spinal cord injury research:
injury delivery and the measurement of the tissues. The first goal of the thesis is to develop
effective tools to track functional improvement of regenerative therapies for spinal cord injury in
animals. That aim is addressed in two ways. One is by developing an injury model that effectively
mimics motor neuron denervation of the respiratory system and the other is by developing a
2
technique that studies function, which also is more sensitive than behavioral tests.
Histology is a useful method to show improvement at cellular level but cannot show restoration
of function, while behavioral tasks may not be appropriate for the system under study.
Functionality is the missing link in measurements of regenerative improvement. Regarding injury
site, high priority systems like the respiratory system have traditionally been neglected in
comparison with the locomotor system.
Electromyography is a common functional outcome but is mostly confined to qualitative studies
due to low resolution. Commercialy available softwares are partly accountable for the low
resolution of automatic electromyography (EMG) analysis observed in animals. Those softwares
are intended for humans and do not take into account particulars of animal electrophysiology. As
the first part of my work, I developed new software able to do automatic analysis of the EMG
signal of animals, which also identifies the most sensitive parameters. Custom-made software
also allows for implementation of signal processing techniques that might have been neglected
before and add to overall resolution. I have validated the technique and the model of injury with a
stem cell transplant.
In the second part of the thesis I propose as a regenerative strategy for the neuronal population, a
piezoelectric substrate that can deliver electrical stimulation. This substrate would be the
precursor of a piezoelectric platform for combinational therapies. I present here the in vitro
studies with those substrates. Futures in vivo studies will test the efficacy of those scaffolds with
the model of injury and the technique I described in the first part.
3
1.1. Hypothesis
My contribution to the field of spinal cord injury strategies can be summarized by the answer to
the following hypothesis.
1. A thoracic level bilateral contusion injury is a suitable model of denervation of the
intercostal muscles via damage of the central nervous system.
2. Interference Pattern Analysis is a sensitive tool to study denervation and reinnervation of
the intercostal muscles and can detect effectiveness of treatment for Spinal Cord Injury
and Spinal Muscular Atrophy Type I.
3. Piezoelectricity enhances branching in neurons with independence of the material and
cell type and vibration.
1.2. Contributions to the field
In the context of the first hypothesis, I developed an injury model to study effectiveness of a
transplant by tackling the respiratory system.
In the context of the second hypothesis the tasks I carry out are:
Create custom software to perform interference pattern analysis in animals
Use different signal processing techniques to improve resolution on analysis of the
electromyography signal
Determine which electrophysiological parameters have an appropriate resolution to track
reinnervation
Test the electrophysiology technique in a stem cell transplant and correlate results with
histology and behavior.
Finally, in the context of the third hypothesis, the different tasks performed are:
4
Develop methods for fabricating and characterizing biocompatible and biodegradable
piezoelectric polymer films as cellular growth substrates.
Develop platforms to stimulate and test PZ polymers in tissue culture applications.
Test effect of PZ stimulation and vibration on neurons
5
Chapter 2
Assessment of treatments in Spinal Cord Injury
2.1. Goal of this chapter
Spinal Cord Injury is a deleterious condition with strong physical, psychological and economical
costs for individuals and society. Proper assessment techniques are crucial in targeting therapies
with regenerative capabilities. In this chapter, I present the current assessment techniques and the
gap there is left in two fronts. One is assessment of functionality and the other is targeting the
respiratory system. I review some of the most common functional methods and I focus on the
reasons of deciding on electromyography.
2.2. Etiology of Spinal Cord Injury
Spinal Cord Injury (SCI) is damage of the spinal cord that results in loss of function, such as
mobility or feeling, or dysfunction of its normal activities. Usually is a result of a traumatic event
like car accidents, falls and gunshots, although tumors, neurodegenerative or demyelinative
diseases can be causes of spinal cord injury as well.
According to National Spinal Cord Injury Statistical Center (NSCISC) report of 2006, the
6
annual incidence of spinal cord injury in the U.S., not including those who died at the scene of the
accident, is approximately 11,000 new cases each year, adding to a total of around 200,000 cases
[1]. The lesion can damage myelinated fiber tracts, gray matter and sensorimotor neurons, as well
as cause loss of neurons.
The average age at injury is 38 years and the expectancy of life for that population is between
24.7 and 11.1 years, depending on the severity of the injury. Unlike the peripheral nervous system
(PNS), the central nervous system (CNS) does not regenerate spontaneously. Although there
might be a certain degree of recovery during the first months, most of the losses are permanent
after a while. This leaves a big population of relatively young people with difficult challenges to
face.
The consequences of a spinal cord injury vary widely depending on the level of the lesion, the
type of the injury and the time and type of intervention. The outcome is often a certain degree of
paralysis or sensory loss, complete or incomplete, of the parts of the body controlled by the
segments of the spinal cord below the injury site. The loss depends on the extension of the lesion
as well as the concrete pathways that were affected. Neuropathic pain is also a common
dysfunction on people affected by spinal cord injury. Other health issues for those affected with
SCI are related to the disuse and decrease of self- autonomy imposed by the injury, as pressure
sores and urinary infections. Not less important are the psychological burden put on the
individual and their families.
2.3. Considerations on priorities in the context of spinal cord injury
According to the Anderson study from 2004, the priorities for people with spinal cord injuries do
not match the efforts from the scientific and clinical communities [2]. Locomotor function does
7
not rank the highest in the concerns of those suffering with spinal cord injuries, and yet, around
75% of the studies on spinal cord injury between 2001 and 2007 are focused on that subject. A
further analysis on the focus of spinal cord injury studies in scientific journal shows an obvious
under-representation of the autonomic system as well as of the nature of pain. [3]
Altogether, it suggests that the world of the benches needs to approach the necessities of the
people they intend to help. It can be done directly through studies to extend our knowledge on the
way SCI affects those systems, by finding specific approaches to treat these conditions or
indirectly by developing injury models focused in them to evaluate the effectiveness of therapies.
2.4. Histology, molecular biology and genetic tests
The first and most direct answer to the effectiveness of a treatment are histological, molecular and
genetic tissue studies. In the absence of functional and behavioral recovery, analysis of the tissue
targets effective therapies that have not yet reached their full potential. In the presence of
functional recovery, those analyses are means to identify the mechanism, adjust it to the purpose
and so enhance the efficacy of the therapy.
Various tissue analyses can detect reduction of secondary damage in the spinal cord, directly by
studying the spinal tissue as seen in tissue sparing, secretion of growth factors, up or down
regulation of certain genes, nerve sprouting, nerve tracing and types of cells present in spinal cord
or indirectly by observing the effect that reduction has in the innervated muscle, as decrease in
muscular atrophy and alteration of muscular composition. Here follows a list of them and some of
the assays to obtain them:
a) Morphometry: Changes in size and shape of the tissue are useful to identify the epicenter
of the injury and correlate results with distance
8
b) Tissue Sparing: Staining against Neuronal Nuclei (NeuN) or cleaved caspase 3 allows
observation and quantification of neuronal survival. A concrete population can be
targeted using the appropriate antibody like choline acetyltransferase (ChAT) for motor
neurons
c) 5-HT Sprouting: Immunohistochemistry against the 5-hydroxytryptamine (5-HT)
receptors identify serotonergic projections allowing visualization of axonal sprouting in
ascending and descending pathways.
d) Nerve tracing: Labeling nerves with 5-bromodeoxyuridine (BrdU) and using horseradish
peroxidase (HRP) to observe innervation and interrupted pathways.
e) Growth Factor Secretion: Specific antibodies can be used against different growth factors
like Neurotrophin-3T (NT-3), Neurotrophin-4/5 (NT-4/5), Nerve Growth Factor (NGF)
or Vascular endothelial growth factor (VEGF)
f) Gene Expression: Polymerase Chain Reaction (PCR) analysis is done in extracted RNA
of homogenized tissue. Reverse transcription is performed with selected primers to
identify up-regulation or down-regulation of genes
g) Protein Expression: Enzyme-linked immunosorbent assay (ELISA) is used to quantify
expression levels of different proteins and determination of human protein levels.
h) Muscular fiber diameters and fiber types: Staining with hematoxylin and eosin (H&E) to
observe cellular nuclei (deep blue) and boundaries of myocytes (clear). Further
classification of fiber types can be done using Adenosine triphosphatase (ATPase) at
different PH levels.
2.5. Behavioral tests
To study the efficacy of treatment through recovery of function, there are several behavioral tests
available. Most of them are devoted to locomotor function, although some examine the sensory
9
system as well.
The Basso-Beattie-Bresnahan test (BBB) [4] was developed to assess improvement in hind-limb
function in rats after a spinal cord contusion injury at the thoracic level. Nowadays is the standard
method of assessment of locomotor function in rats not only for injuries beyond the thoracic level
but also for injuries different than contusion.
Other locomotor tests address the shortcomings or improve the resolution of the BBB test at
certain time-points in recovery. For instance, to account for inter-limb coordination and remove
the scorer intervention in the BBB test that sometimes is needed to stimulate rats to make a pass,
some alternative tests are the GridWalk from Barth et al. [5] and Kunkel et al [6]., the horizontal
ladder beam of Soblosky et al. [7], which counts missed steps, and the straight alley of Wong et
al.[8].
To obtain and analyze gait-related data during locomotion exists the CatWalk automatic gait
analysis [9], which uses thermally-impressed paw-prints to obtain parameters as stride length and
swing duration, pressure during locomotion and support of the base during the stride. To evaluate
forelimb function there are grip strength meters as described by et al. [10], for vertical exploration
there is the clear cylinder test of [11] et al and the food pellet reaching task of Metz and
Whishaw [12].
Return of sensation and also the presence of hyperalgesia or allodynia can be tested by applying
controlled mechanical stimuli like in the pin-prick test and the Von-Frey hair test [13], or by
observing the response to temperature applied by the hot plate test. Other sensory tests are the
visual cliff test, which evaluates the ability to see the drop-off edge of a horizontal surface, the
10
acoustic startled test which sets thresholds for audition as well as noxious auditory amplitude by
observing flinching in the animal.
2.6. Functional tests
In addition to histology and behavior, efficacy of therapies can be assessed by functional tests.
They are better quantifiable and have a higher resolution than behavioral tests, especially for low
levels of improvement. As an advantage over histological results, they are able to un-mask
physiological changes that do not translate into functional improvement.
Most functional tests have been developed around systems that lack a primarily voluntary input,
mainly because of the absence of behavioral tests for those systems. Not many standardized
techniques are offered to evaluate the function of the cardiovascular, respiratory, sexual and
gastrointestinal systems, the lower urinary tract and thermoregulation but it is necessary to point
out that there are fewer studies on sensation compared to locomotion, and even fewer studies deal
with recovery in the autonomic system. Hereafter, I present some functional tests. Reviewed from
[3].
In the cardiovascular context, we can study problems like hypotension, tachycardias and
arrhythmias by monitoring fluctuations of blood pressure and heart rates with time. The
measurement is done by implanting a cannula and connecting it to a pressure transducer.
Malfunction of the gastrointestinal (GI) after SCI, for instance in motility, can be studied with
spectrophotometry by dye recovery on the GI tract after oral marker ingestion. Electromyography
(EMG) is another tool to study external anal-sphincter (EAS) hyperreflexia. EMG can be used
also to asses ending of spinal shock which is characterized by recovery of autonomic function.
11
Manometry measures pressure changes inside the GI tract by inserting a catheter filled with fluid,
which is later attached to the recording probes. However, a more feasible way of continuously
measuring pressure changes in moving animals is by using strain gauges. On another line,
hydrogen breath tests measures the increase in hydrogen expiration which follows carbohydrate
administration and is used to track oro-cecal transit time.
Cystometric urodynamic analysis is the common method for assessing low urinary tract (LUT)
function and it can be performed by a catheter implanted into the bladder dome, which allows for
simultaneous EMG recording of the external urethral sphincter (EUS). Bladder volume is
proportional to severity of SCI and consequently, the volume of unire expressed at micturition is
a very useful output as well.
Sexual function is studied in animals by telemetric monitoring of pressure within the corpus
cavernosum (CC) and the corpus spongiosum (CSP). EMG of the perineral muscles during sexual
intercourse is one of complementary tests. Sexual arousal in cats might be studied by laser
doppler flowmetry, which records vaginal blood flow.
The effect that SCI has in thermoregulation can be tracked by measuring core temperature,
cutaneous temperature and blood flow. The latter gives information on the sympathetic
vasomotor pathways, which can be studied by micro-neurography as well.
Finally, in the context of the respiratory system, four tests yield useful information to track
recovery: phrenic nerve conduction, pneumotachometry, diaphragmatic electromyogrpahy and
plethysmography. A pneumotachometer measures the airflow and a plethysmograph changes in
air volume. They are both useful resources to study respiration in adult humans but they have
limitations when measuring from children and animals.
12
2.7. Significance of electrophysiological analysis of treatment
Histology and behavioral testing, mostly locomotive, pose the edges of a spectrum where there
are not many in-between outcome features and often neglects somatic or autonomic responses.
The functional output is the missing stone, better allowing to fine-tune therapies and identifying
where they are failing.
Electromyographic measurements are one of the most useful resources for therapeutic assesment
in terms of function, especially for systems with strong autonomic input. However, they have not
been used extensively as a validation tool of potential treatments. That is partly due to the
inherent variation of the technique which renders resolution too low to asses improvement with
statistical significance between injured and non-injured groups. Resolution of the
electrophysiology signal can improve with a combined approach. On one side, the use of
electrophysiological parameters most robust to variability, on the other, the use of signal
processing techniques to prepare the signal and finally creating software that can automatically
track the changes in
13
Chapter 3
Electromyography
3.1. Goal of this chapter
This chapter presents the main concepts related to electromyography (EMG), the effect of injury
in the electrophysiological parameters and the specific electrical features of muscle activity in
rats. First, I introduced the concept of the Motor Unit Potential (MUP) and common vocabulary
related to intramuscular EMG. The chapter focuses in the different analysis comprised under the
label of Interference Pattern Analysis (IPA). Then, the effects that injury and reinnervation have
in the EMG signal are described. Finally, I have presented the specifics of the EMG signal of rats
as compared to humans, thus justifying the necessity of new software to automatically perform
IPA in animals. Chapter 4 will introduce EMGvet, the custom software I developed to perform
IPA in rats based in specific characteristics of their MUPs.
3.2. Electrophysiology of the skeletal muscle
Electromyography is a technique to measure the electrical activity in muscle fibers. A motor unit
is the basic structure of the skeletal system. It is constituted by a single motor neuron and all the
fibers it innervates.
14
The neuromuscular junction is the location where the motor neuron axon synapses with the
muscle. The muscle at that site is known as motor end plate and it has a very excitable membrane.
Depolarization of the motor end plate as a response to the acetylcholine neurotransmitter released
by the motor neuron is called end plate potential (EPP). When the EPP reaches threshold, an
action potential is triggered and each muscle fiber enervated by that motor neuron contracts
Spontaneous depolarizations of the end plate, which are caused by random release of
acetylcholine vesicles by the motor neuron, are called miniature en potentials (MEEP). They
hardly ever are able to trigger an action potential by themselves.
In the skeletal muscle of mammals, there are no inhibitory synapses. Thus, activation of the α
motor neuron results in the contraction of all the fibers it innervates, which are all of them of the
same type, whether fast-twitching or slow-twitching. The fibers innervated by a motor neuron are
usually scattered through the muscle. The set of motor units that innervates a concrete muscle is
known as the motor neuron pool for that muscle. Figure 1 is a diagram depicting that fact.
Figure 1.Schematic shows 2 motor unit pools and the patterns of innervation of each neuron in
different colors.
There are two main different types of electromyography: surface electromyography (sEMG) and
15
intramuscular electromyography, also known as needle EMG. Intramuscular recordings are taken
using a bipolar electrode or a concentric needle electrode. All recordings performed within the
purview of this thesis work are of the latter type. Intramuscular EMG avoids the problems of
attenuation, absorption and scattering associated with tissue. Because of those effects, the
electrical signal in sEMG does not permit a proper analysis on Motor Units.
The electrode measures all the electrical activity from the fibers within its detection range. A
single fiber action potential (SFAP) has a shape as the one depicted in Figure2. The farther the
electrode is from it, the lower in amplitude and longer in duration the SFAP is.
A B
Figure 2. Schematic of SFAP potentials recorded at short distance (A) and far distance (B).
Activation of a motor unit reflects contemporaneous activation of all the fibers in the motor unit
pool. Therefore, activation of a motor unit creates an electrical profile which depends on the
addition of each individual SFAP for that MUP and their relative distance to the recording site.
Such a profile is known as a motor unit potential (MUP) and the study of its features can be used
as a characterization as well as diagnostic tool of muscle activity.
16
Figure 3. Schematic representation of motor unit potential (MUP) generation and recording by
needle electrode.
Important features regarding MUP are listed in Table 1.
PARAMETER DESCRIPTION
Amplitude Maximum peak to-peak amplitude
Duration Time interval between the first deviation of the signal from baseline and
its last return
Area Area under the rectified waveform
Thickness Area/amplitude
Number of phases A phase is the portion of signal between deviation from baseline to its
return to baseline.
Rise time Slope of the main phase of the MUP
Number of turns A turn is a change in polarization of more than 100uV
Satellite potentials Linked low-amplitude waveform linked to the main MUP
Complexity. A MUP with more than 4 phases (polyphasic), 5 turns or satellite
potentials is a complex one
Jiggle Vertical instability of the MUP
Firing pattern Periodicity of MUPs
Recruitment Pattern Number of MUPS present for different levels of force
Table 1- MUP features
Some of these concepts have been depicted in Figure 4.
17
Figure 4. Main features of a MUP.
Electrical activity in the EMG signal comes not only from action potentials elicited by the same
motor neuron but also from action potentials elicited by different motor neurons. For low levels
of activity, it is easy to distinguish between different MUPs since they are not-synchronous and
their firing patterns are different. However, for high levels of muscle activity MUPs overlap
making it difficult to distinguish each one individually.
Some considerations on factors influencing MUP parameters are due at this point:
The amplitude of the electrode recording of the SFAP diminishes with distance to the power of 2.
Therefore, the MUP amplitude and shape is dominated by those fibers close to the electrode. On
the other hand, the fibers remote from the electrode will contribute more significantly to duration
of the MUP. In general, the higher the absolute number of muscle fibers in a motor unit results in
a longer MUP duration.
In addition to that it should be pointed out that higher levels of force are achieved by recruiting a
higher number of active motor units and increasing their individual firing frequencies. In normal
18
activity, low-threshold motor units are recruited first, and levels of contraction are increased by
increasing their firing frequency. That value reaches a plateau, but simultaneously, higher-
threshold motor units have started getting recruited to maintain an increase in contraction levels.
The frequency at which a motor neuron starts firing is called the onset frequency, and the
frequency at which the next motor unit is recruited is the recruitment frequency.
Analysis of all of these MUP features and deviation from their normalized values allow
identifying abnormal EMG signal caused by trauma or disease and diagnose a diverse range of
neuropathies and myopathies.
3.3. Automatic MUP Analysis
Analysis of MUP features in an automated manner was first presented by Stalberg in their Multi-
MUP software [14]. Multi-MUP analysis is a decomposition analysis of the EMG signal which
allows automatic identification of MUPs. The analysis is performed over epochs of 4.8s and
gathers 6 different MUPs per epoch. It uses a decomposition method based on template matching.
MUPs are signals meeting the criteria of starting with a negative peak which exceeds 30uV,
having a positive slope higher than 30uV/0.1ms and a separation between other potential MUPs
of more than 2.5ms. Each of those signals joins a pre-existing class if the difference is below a
certain value or starts a new class. At the end, the six classes with more hits are each averaged.
MUP parameters provided in a Multi-MUP analysis have been listed in Table 2, along with the
criteria used by the analysis [14]:
19
PARAMETER CRITERIA Zero threshold Zeroing below 20uV to erase noise fluctuations
Duration Time difference between the end and starting point of the MUP. Time
points are determined using a slope criterion of more than 30uV/0.1ms, a
negative peak exceeding 30uV and separation with adjacent MUPs of
more than 2.5ms, after zero-thresholding.
Amplitude Difference between maximum and minim peaks of the MUP
Rise time Time between the maximum negative and the maximum positive peak
Area Area of the MUP within duration time
Thickness Area/amplitude
Number of phases Each phase being a section of an MUP that falls between two baseline
crossings and reaches an absolute value of more than 20uV, after zero-
thresholding.
Table 2- MUP parameters obtained by a Multi-MUP Analysis and criteria used by it.
3.4. IP Analysis (IPA)
MUAP analysis is a useful analysis to understand the MUP activity and recruitment but can only
be performed at low levels of activity when the EMG signal is not very full and individual
MUAPs can be identified. After a certain amount of activity (in voluntary muscles that would be
above 10% of maximum voluntary contraction) the summation and overlap of MUAPs creates an
interference pattern, which introduces a lot of error in the automatic MUAP analysis. In such
cases, the interference Pattern Analysis (IPA) is a good alternative. The interference pattern
depends on the number of recruited MUs, size, duration, firing rates, time of recovery…etc.
The inspiratory burst associated with the electrical activity of the intercostal muscles firing during
the inspiration cycle can be analyzed by different techniques. The Turns/amplitude Analysis
(TAA) is the closest approximation to MUP analysis and yields certain parameters very
dependent on MUP features. (Reviewed from [15], [16]).
The whole burst can also be studied through integration, room mean square or envelope. All of
them provide gross features of burst amplitude, burst duration and breathing frequency.
20
Alternatively, information can be extracted from power spectral analysis. In the end, some of
these analyses are redundant and convey the same information through different paths.
3.1.4. Zero-crossings
The number of zero-crossings through the zero line can indicate degree of muscle activity. To
avoid background noise, it is established a threshold level so only crossings between those two
thresholds are counted. Usually it is established 25uV as the threshold.
3.2.4. Turns/amplitude analysis (TAA)
Willison developed a method to study the IPA based in measuring turns on the Interference
Pattern of the EMG [17]. A turn is a local maximum that changes direction by at least 100uV in
amplitude compared to the preceding and subsequent turns (other versions of the algorithm
establish a threshold of 50uV for the turns). A threshold of 30uV is set to cope with noise. The
number of turns is linearly related to number of zero-crossings, but has extra value as a diagnostic
parameter.
Three useful derivations of this concept are the number of turns per second (T/S) often referred
simply as turns, the mean amplitude of the turns (A/T), which is the average amplitude between
two turns and the segment, which is the time lapse between turns. These parameters, together
with the ratio between them (T/S: A/T) are useful diagnostic tool of disease.
In myopathies, T/S during a fixed time increases, and in neuropathies, T/S does not change while
A/T increases. The ratio T/S: A/T was increased in myopathies with respect to the normal case
and decreased in neuropathies. [18]
21
Usually, in interference pattern analysis, the parameters under consideration are the recruitment
of MUPs with increasing force and the amplitude, duration and complexity of the MUPs. Stalberg
et al developed three features, activity, upper centile amplitude and number of small segments
which are equivalent to those MUP features.
Activity is a quantification of the time with MUP activity, whether coming from individual or
superimposed MUPS. As the force of contraction increases, more MUPs are recruited and the IP
signal becomes fuller. Activity measures that feature and we can see how it increases linearly
with the number of MUAP discharges up to 80% of its theoretical value. Activity is the time per
second that IP signal is active. Actually, this parameter is used not only to assess the number of
motor units recruited but also the firing rate of the active units.
For the IP Analysis as developed by Nandedkar, those thresholds were derived from the biceps
brachii of healthy humans at 10%, 25%, 50% and 80% of maximum force. Histograms of the
segments amplitudes overlayed with scatter plots of the values of duration versus amplitudes
were used to set the mentioned thresholds by trial and error.
As a result, segments are said to be active if duration is less than 5ms for amplitudes greater than
2mV, or less than 3ms for amplitudes between 0.5mV and 2mV, or less than 0.5ms for
amplitudes less than 0.5mV
The number of small segments (NSS) measures the number of segments with amplitude of less
than 2mV from the segments defined as active. NSS gives information about the low-amplitude
high-frequency components of the IPA and it is a good indicator of polyphasicity. A high value of
NSS would indicate absence of large amplitude MUP even if the UCA index detects one large
22
amplitude of MUP. Therefore, it is important to take into account UCA along with NSS [18].
The Upper Centile Amplitude (UCA) defines the upper limit of the peak-to-peak amplitude of
the motor unit action potential contained in the epoch. UCA is measured by the largest amplitude
change between successive turns after excluding the 1% largest values. As the force of
contraction increases, so does the amplitude of the largest spikes in the IP.
The EMG Envelope Amplitude (ENAMP) reflects the amplitude of the largest MUAP in the IP.
That value is obtained by measuring the difference between the peak that has the fifth most
positve amplitude from the peak that has the fifth most negative amplitude from an epoch of
500ms. Ignoring up to the 4th strongest peak allows avoiding solitary peaks of large MUAPs when
the contraction is close to its recruitment threshold. [19]
UCA and the ENAMP have a similar meaning. Along with activity those parameters increase in
value with the force of contraction [19].
These parameters relate directly or indirectly to MUAP features. Similarly, they can be used as a
diagnosis tool. It has been seen that a neuropathy results in an increase in the UCA parameter,
while the NSS stays normal or decreases. On the other hand, a myopathy results in increased
NSS, while UCA stays normal or decreases.
3.3.4. Amplitude measurement of electromyographic burst
There are different ways of depicting the general features of the inspiratory burst. Each one has
its tradeoffs.
Methods like root mean square or integration of MUPs can sometimes yield an estimate muscle
23
force level, but they are not immune to artifacts. The linear envelope using low-pass filters (5-
10Hz cuttoff) of the full-rectified signal can detect onset of the burst, but suffers from the same
problem. A more robust estimate of muscle force can be provided by applying the moving
average of the signal. The results such obtained correlate with the levels of contraction of the
muscle [15].
Data regarding a mean average analysis are typically expressed as mean EMG amplitude,
duration of contraction, mean EMG-spiking activity (ESA) and durations of activity or
contractions.
3.4.4. Power Spectral Analysis
The general observation that in patients with neuropathies the power spectrum is displaced
toward higher frequencies while in patients with myopathy, it gets tracked toward lower
frequencies suggests a power spectral analysis of the EMG signal a potential diagnostic tool. The
total power of the burst has been studied as an indication of activity and polyphasia [15].
However, the diagnostic capabilities of the power spectrum are still under scrutiny Kopec and
Hausmamowa-Petrusewicz have stated PS as a better diagnostic tool for myopathies. When
compared to individual MUP, it was found that PS analysis can detect myopathies better than
neuropathies [20]. Similar conclusions were reached by Fuglsan-Frederiksen [21]. As of now, the
PS stays a complementary technique and it should be used for redundancy.
Usual outcomes of the power spectral analysis are the mean frequency, and power at frequencies
140Hz, 1400Hz, 2800 and 4200Hz.
24
3.5.4. Summary of IPA parameters
The most common parameters obtained in analysis of the IP signal are listed in Table 3.
They have been organized according to the type of analysis that provides them.
ANALYSIS PARAMETER DESCRIPTION
ZERO-
CROSSINGS
Zero-crossings per
second
Number of voltage crossings of the baseline per
second
TURNS-
AMPLITUDE
ANALYSIS
(TAA)
Turns per second
(T/S)
Number of changes in polarization of more than
100uV per second
Amplitude per Turn
(A/T)
Average amplitude between two turns
Ratio (T/S:A/T) Ratio between T/S and A/T
Activity Time per second that IP signal is active
Number small
segments (NSS)
Number of segments with amplitude of less than
2mV from the segments defined as active
Upper Centile
Amplitude (UCA)
Largest amplitude change between successive
turns after excluding the 1% largest values
EMG Envelope
Amplitude (ENAMP)
Difference between the 5th most positive peak
amplitude from the peak with the 5th most
negative amplitude from an epoch of 500ms
BURST
ANALYSIS( by
moving average,
root mean
square or
integration)
Amplitude Average amplitude of the electrical burst
Duration Time length above a certain % of amplitude of
burst as defined by criterion
Burst rate Frequency between burst
POWER
SPECTRUM
Total Power
Mean frequency
Power at a certain
frequency
Usually at 140Hz and 1400Hz.
Table 3- Parameters obtained from analysis of the IP signal.
There is usually a zero-thresholding of around 25uV before performing Zero-crossings and TA
analysis to avoid fluctuations due to noise.
3.5. Electrophysiological parameters after nerve injury
Analysis of the EMG signal may provide information about injury and disease of the
musculoskeletal system and the nervous system. Neuropathies like Spinal Muscular Atrophy or
25
traumatic events like contusion of the spinal cord affect the population of lower motor neurons
and produce a characteristic electrical profile after injury. Diseases like primary lateral sclerosis
which affect the upper motor neuron population have a much different electrical profile.
In this document, I will focus on how a discrete insult to the nervous system can affect on the
EMG signal. The insult will cause partial axonal/motor neuron loss; surviving axons will be
present. Sequellae following injury include the process of regeneration, which has ongoing effects
on the shape of MUAPs as well as firing rates, power spectral profile and the burst activity.
Hereafter I will summarize some of those effects.
Partial denervation is first characterized by silencing of the muscle. Spontaneous activity appears
in the denervated fibers between one to three weeks after injury. The time depends on the length
of remaining nerve in the muscle. Electrical instability of the muscle membrane typical in a fiber
which has lost its innervation is the most probable cause. Positive waves and fibrillation
potentials are the most common instances of spontaneous activity.
It is believed that between 4 to 12 weeks is necessary for enough reinnervation to occur to track
changes in the MUP. Early findings during reinnervation include an increase in fiber density,
amplitude of the MUP, duration of the MUP and polyophasia [22].
During the reinnervation process, denervated muscle fibers are re-innervared by adjacent motor
units by axonal sprouting. This leads to increases in the amplitude of the MUP as well as duration
increases as well, since there are more fibers innervated, and thus, more long distance SFAP
contributing to the overall MUP., causing an. Long distance fibers have an effect in duration that
is not irrelevant. Moreover, inmature synapses have slow conduction velocity which leads to
dispersion of the signal and results in polyphasic MUPs. Fiber density also increases.
26
As the reinnervating process advances, synapses mature. Hence, polyphasia disappears and
duration of the MUP decreases. Amplitude in the MUP tends to decrease slightly although it will
remain above the ones characteristics of non-injured muscle.
In terms of firing patterns, the onset frequency and the recruitment frequencies of a muscle with
significant axonal loss are increased as compared to the non-injured muscle. Therefore, for a
given level of force, there are fewer MUPs firing at higher rates than in the normal muscle.
The power spectrum of the EMG signal of patients with neuropathies is displaced toward higher
frequencies.
3.6. Electrophysiological differences between rats and humans
In previous sections, I presented automated softwares commercially available to perform MUAP
Analysis and IP Analysis. All of them used thresholds for automatic identification of MUPs that
were obtained from normalized data of human MUPs and trial and error during implementation of
the code.
MUPs for animals have different characteristics than those for humans. Studies in the rectus
abdominis of humans have reported MUP amplitudes of 373.8uV and duration of 9.9ms [23]. The
equivalent in rats shows values of 193.4 uV for amplitude and 5.2ms for duration [183]. Despite
accepting the difference in function that might arise from difference usage in those muscles
between rats and humans, MUPs of rats are smaller in amplitude. The scaling from humans to rats
is approximately 2:1 for amplitudes. Therefore, the thresholding applied before the MUP
Analysis needs to be adjusted for rats. In Chapter 5, it will be presented the method and the
27
results achieved for automatic detection of rat MUPs.
3.7. Summary
Muscle denervation by traumatic injury or disease, reinnervation and myelination are processes
that affect different factors of the EMG signal. Diagnosis of injury, injury type and the stage of
reinnervation can be identified in the injured muscle by study of MUP features and the
parameters of IPA. Some of these parameters are especially sensitive to these processes, which
allow them to be used as discriminatory tools to set apart controls from animals treated with
therapies that enhance or speed up recovery of function in the muscle
Commercial softwares for automatic MUP analysis are available to study the EMG signal via
IPA, but these softwares are intended for clinical studies and have been developed for humans.
MUPs features differ across species. For rats, MUPs have lower amplitudes than for humans.
Therefore, commercial available softwares for automatic MUP or IP analysis need to be adapted
for animals if the EMG signal is to be used as assessment tool of regenerative treatments.
28
Chapter 4
EMG Software Development
4.1. Goal of this chapter
This Chapter presents EMGvet, the software I developed to overcome the lack of a program for
automatic Interference Pattern Analysis (IPA) of the Electromyographic (EMG) signal in rats. I
first introduce the necessity for developing such software, then I give some of the technical
details about its implementation, especially in terms of the algorithm to remove the ECG signal
and finally, I present the parameters I obtained, which allow performing automatic analysis of the
Interference Pattern (IP) in rats. I also present the methods that lead to these new thresholding
parameters. In Chapter 6, EMGvet has been used to analyze the EMG signal to show the
regenerative capabilities of a stem cell therapy by functional improvement of the respiratory
intercostal muscles as compared to controls. As a conclusion, EMGvet is a useful tool for
researchers to study electromyography in animals.
4.2. Need for Software modifications
Raw measurements of the EMG signal were recorded using the Dantec Keypoint® portable
system (Natus Medical Incorporated, San Carlos, CA) with standard filter settings (5-10Hz).
Keypoint®allows quantitative EMG recordings, single fiber EMG and nerve conduction studies.
29
Both intra-needle EMG and surface EMG are available. Moreover, Keypoint® provides
proprietary software which performs such common analysis as Motor Unit Potential Analysis
(MUPA), Interference Pattern Analysis (IPA), insertional activity and spontaneous activity.
Keypoint software is intended as a diagnosis tool in clinics and medical centers, therefore, MUPA
Analysis and IPA analysis have been coded as described by [14]. The method of Stalberg is an
automated analysis to detect and extract MUPS features from human EMG signals.
The Keypoint software required adaptation to the EMGs of rats. MUPs of animals are quite
different from those of human as seen in section 4.6. Indeed, the analysis of our data showed that
Keypoint software missed many MUPs that were obvious to a trained observer. Lost MUPs
cannot be modified manually without adding too much input from the researcher which,
eventually, compromises the bias in the study. To improve resolution of rat EMGs therefore it
was necessary to write custom routines.
For this thesis, I have developed EMGvet, a software for electromyographic analysis customized
across species. Typical electrophysiological parameters of IPA as activity, turns/second,
amplitude/turn, upper centile amplitude (UCA) and the number of small segments (NSS) have
also been translated to comprise the particularities of animals. Moreover, EMGvet performs
moving and ensemble averages on the EMG burst to obtain amplitude and duration, onset times
and frequency of respiration. It also performs power spectral analysis.
A compilation of all the specifics of the software is out of the scope of this document, but general
considerations on some basic issues are presented hereafter. The following sections present some
of the solutions I adopted in the development of EMGvet that differ from available commercial
30
EMG software or that were necessary tools to achieve them. The IPA parameters provided by
EMGvet are listed in Table 4.
ANALYSIS PARAMETER
ZERO-CROSSINGS Zero-crossings per second
TURNS-AMPLITUDE
ANALYSIS (TAA)
Turns per second (T/S)
Amplitude per Turn (A/T)
Ratio (T/S:A/T)
Activity
Number small segments (NSS)
EMG Envelope Amplitude (ENAMP)
BURST ANALYSIS( by moving
average)
Amplitude
Duration
Burst rate
POWER SPECTRUM Total Power
Mean frequency
Power at a 150,hz, 1500Hz, 3000Hz, and 6000Hz
Table 4-IPA parameters provided by EMGvet software.
4.3. Electrocardiogram removal
Intercostal recordings were taken from the right side of the ribcage to minimize the interference
of the electrocardiogram signal (ECG). Even so, ECG interference is still very strong and present
in the respiration bursts, altering the parameters under study. The first requirement to any analysis
on the EMG signal is the effective removal of the ECG signal.
The ECG signal shares with the respiration burst a similar power distribution in the frequency
domain. Bruce (p.281) [24] suggests a 2nd
Butterworth high-pass filter with a cutoff of 70Hz to
remove it. In our studies, the distortion of the respiration signal of interest thus filtered is too high
to be acceptable.
Similarities of both signals in the temporal domain makes difficult to set them apart using
temporal techniques. The ECG signal may have stronger, smaller or a similar amplitude that the
31
signal of interested. Therefore, there is no option to use that as a discriminative feature to set both
signals apart. It was not taken a reference signal of just the ECG signal to allow adaptive line
enhancement (ALE) filtering.
Independent component analysis (ICA) could not be performed since principal component
analysis (PCA) did not find two independent signals. Motor Unit Potentials and ECG have such a
similar shape that confounds most of the shape tracking approaches, including wavelet analysis
using the Ricker wavelet that other groups have used.
The final algorithm used in EMGvet is a template tracking algorithm. The ECG-template is
constructed from an average of ECG signals identified within the regions with absence of
inspiratory activity.
The ECG-removal-function starts with conditioning of the signal by filtering with a Butterworth
band-pass filter of 4th order, with cutoff frequencies at 30Hz and 500 Hz. Mean of the signal is
removed from the signal prior to rectification to reduce the noise. Then, moving average filtering
is applied to the rectified signal using a triangle window of 41ms.
A digital signal constituted by respiratory pulses (EMG and ECG interference) and ECG-only
pulses is obtained by static amplitude thresholding. Pulses are identified as ECG-only or
respiratory based on a static thresholding in duration. Both thresholds were obtained empirically.
A synthetic ECG is constructed taking five different templates of ECG-only pulses and averaging
them after aligning their peaks. The period of the ECG is identified and the algorithm can predict
the approximate position of the ECG within the respiratory burst. The exact position is identified
by auto-correlation, then, the template of ECG is removed from the inspiratory burst.
32
A B
Figure 5- EMG signal of the IIIrd intercostal muscle before (A) and after (B) being processed by
the cleansing algorythm. In (A) it was also included the digitalized signal, which is green for only
EKG, magenta for respiratory burst, and red for smoothed signal.
4.4. Automatic zero-crossings tracking
The number of zero-crossings through the zero line is used as indication of activity on the
muscle. To avoid background noise, it is established a threshold level so only crossings between
those two thresholds are counted. The usual value in clinical studies establishes 25uV as the
threshold. EMGvet uses a lower threshold of 15uV to adapt it to rat IPA.
4.5. Turns/amplitude analysis (TAA)
EMGvet uses 30uV of difference between turns. Figure shows an inspiratory burst and the
identified turns as asterisks.
33
Figure 6-Turns are depicted as black asterisk. Maximum are depicted in red dots for maximum
and green for minimum.in a time series (vector)
Thresholds that determine activity are very dependent on MUP features as well: As it was shown
in section 1.4, MUP features for animals, and rats in concrete, are different than in humans.
Therefore, in order to analize rat EMG signals using IP analysis, EMGvet changed the thresholds
obtained by Nandekahr for humans [25],[26], [19]. In order to do so, EMGvet uses the same
procedure of plotting histograms of segment amplitudes overlayed with the scatter plots of
amplitude of segments versus their duration. However, Nandekarh was able to gradually increase
EMG activity by asking their subjects to produce gradually greater amounts of force and set their
thresholds for different amounts of activity. Rats cannot be directed to do so. Instead, it was used
the signal of the intercostal muscles at different time points after injury to display different
amounts of activity and set thresholds in those different situations. Figure 7, Figure 8 and Figure
9 show the histograms for two animals along with the thresholds of the activity criteria.
As shown in Figure 7, after injury the MUPS are very small and there are not many of them and
activity is low.
34
A B
Figure 7- Histograms of the segment amplitudes (blue) are shown, overlayed with scatter plots
of the values of the duration versus the amplitude of the individual segments (red), and
threshold values for activity (black) for two different animals (A and B) 1 week after injury.
Figure 8 depicts the situation four weeks post injury. MUPS have larger amplitudes since
remaining MUPS are reinervating the denervated nearby muscles and more activity is observed.
A B
Figure 8- Histograms of the segment amplitudes (blue) are shown, overlayed with scatter plots
of the values of the duration versus the amplitude of the individual segments (red), and
threshold values for activity (black) for two different animals (A and B) 4 weeks after injury.
Five weeks post-injury, MUPS decrease slightly in amplitude and diminish in duration as new
fibers get remyelinated as it is shown in Figure 9.
35
A B
Figure 9- Histograms of the segment amplitudes (blue) are shown, overlayed with scatter plots
of the values of the duration versus the amplitude of the individual segments (red), and
threshold values for activity (black) for two different animals (A and B) 5 weeks after injury
As a result, segments in EMGvet are said to be active if duration is less than 10ms for amplitudes
greater than 100uV, or less than 7ms for amplitudes between 30uV and 100uV, or less than 3ms
for amplitudes less than 30uV. Based on these thresholding, the activity of a typical inspiratory
burst is presented in Figure 10.
Figure 10- Inspiratory burst with activity regions based on definition of EMGvet. Activity is
presented in red while the general inspiratory burst in blue.
In EMGvet, the number of small segments (NSS) measures the number of segments with
amplitude of less than 100uV from the segments defined as active,
Upper Centile Amplitude (UCA) and EMG Envelope Amplitude (ENAMP) have been defined
36
similarly than in traditional. Figure shows the effects that it has in a burst of inspiratory activity.
Figure 11- Inspiratory burst with activity regions based on definition of EMGvet. Activity is
presented in red while the general inspiratory burst in blue
UCA and the ENAMP have a similar meaning. Along with activity those parameters increase in
value with the force of contraction [27].
4.6. Analysis of the electromyographic burst
Duration and mean amplitude of the inspiratory burst, onset time and breathing rate are features
that can be studied by using integrating techniques of the EMG signal.
EMGvet adopts a moving average technique. The moving average is a good method to
determine levels of contraction. EMGvet performs the moving average on the EMG once been
cleaned from ECG artifact. Before doing the sliding-average, the signal is full-rectified and the
mean is extracted to reduce the noise. This concrete implementation uses a rectangular window of
41.6ms. There is a delay in the onset of burst by the same amount but it is acceptable.
Digitalization of the clean signal is done similarly to the ECG-removal function but using a
dynamic amplitude thresholding of 6% of the mean of the signal. Then, the ensemble average is
37
done on four inspiratory bursts to increase smoothing without adding delay.
Moreover, to diminish the fact that different insertions yield fairly different EMG amplitude
(depending on proximity of electrode to different motor unit pools in each insertion), it was
averaged the result of the three insertions. Before averaging, it was assessed the quality of the
signal, to prevent aberrant measures to be included. Finally, it is obtained the mean of the burst
ensemble and the duration of the burst ensemble.
A B
Figure 12- (A) raw and digitized EMG signals of the IIIrd intercostal muscle for several
inspirations before processing. Digitalized signal was also included, green for only EKG,
magenta for respiratory burst, and red for smoothed signal. (B) EMG during a single
inspiration after processing by the cleansing algorithm.
4.7. Summary
In order to discriminate between spontaneous recovery and effective treatment, this method
requires of appropriate software to perform Interference Pattern Analysis. Aptitude to the task is
defined by how accurately the automated software identifies IPA features. Commercial softwares
do not evaluate properly the IP of animals because they use thresholds for humans to track rat
MUPs. I developed EMGvet to address this necessity.
A summary of the new criteria set in EMGvet which allows automatically identify rat MUPs and
38
properly quantify the principal parameters of IPA is listed in Table 5. These values have been
compared to the commercial available software Keypoint.
PARAMETER Keypoint CRITERIA EMGvet CRITERIA
Zero thresholding 25uV 15uV
Zero-crossings 30uV 15uV
Turns 100uV 30uV
Activity d<5ms & amp> 2mV
d<3ms & 0.5mV<amp<2mV
d<0.5ms & amp<0.5mV
d<10ms & amp> 100uV
d<7ms & 30uV<amp<100uV
d<3ms amp<30uV
Number small segments d<3ms & 0.5mV<amp<2mV
d<0.5ms amp<0.5mV
d<7ms & 30uV<amp<100uV
d<3ms amp<30u
Moving Average
window
Information not available 41.6 ms
Moving Average
threshold for duration
Information not available 6% of total amplitude
Power at a certain
frequency
140Hz, 1400Hz, 2400Hz,
4800Hz
150Hz, 1500Hz, 3000Hz,
6000Hz
Table 5- Comparison of criteria for IPA parameters between Keypoint and EMGvet, where d is
duration of segments and amp is amplitude of segments.
Aptitude to the task requires also a system with enough resolution to set apart treated from control
groups, if there is indeed a functional improvement in the former group. Several signal processing
techniques are available to increase resolution in EMG analysis, mostly involving the removal of
interference and noise in the signal. EMGvet has been designed with stress in resolution and
different techniques like averaging across time (embedding) and recordings were used to achieve
that goal. Especial attention was paid to filtering and ECG removal.
As a summary, EMGvet is a necessary analysis for researchers studying the EMG signal of
animals. Animal studies using commercial available softwares might be erroneously tracking
MUPs and/or investing much time in manual correction. EMGvet will soon be provided as Open
Access software to the scientific community increasing the accuracy and efficiency in those
studies.
39
Chapter 5
Rat Model of Injury
5.1. Goal of this chapter
Within the context of developing a model of injury and a technique to track functional
improvement of regenerative therapies for spinal cord injury, I decided on the electromyographic
signal (EMG) of the intercostal muscles as the most suitable method. Chapter 3 and Chapter 4
focused in the technique. I explained the usefulness of the EMG as a measurement of functional
improvement and the EMGvet software which was developed to analyze it with accuracy. In
Chapter 2, I presented the advantages of targeting the respiratory system as a model of injury.
This chapter describes the model of injury itself. First, I provide information on the anatomical
and functional background of the intercostal muscles as the rationale for the particulars of this
model. Later, I describe the level of injury, muscle of choice, location of recordings and
experimental protocol.
5.2. Physiology of the respiratory system
The main respiratory muscles are the diaphragm, intercostal muscles and abdominals. The
respiratory dynamics are mediated primarily by diaphragm contraction, which expands the
ribcage, diminishing the pressure inside of the lungs with respect to atmospheric pressure. As a
40
consequence, it forces entrance of air through the trachea into the lungs. Inspiration is facilitated
by the synchronized action of the external and internal intercostal muscles. Expiration is caused
by relaxation of the diaphragm which causes elastic retraction of the lung and tissues around the
thoracic cavity. Forced expiration, as it will be explained hereafter, is caused by interosseus
internal intercostals and assisted by other muscles in the thoracic and abdominal area. Reviewed
from [3].
Phrenic motor neurons located in the cervical spinal cord innervate the diaphragm, respiratory
intercostal muscles are controlled from the thoracic spinal cord and abdominal muscles are
innervated from the thoracolumbar spinal cord. The ventro-lateral medulla, modulates the activity
of these and other accessory muscle which facilitate respiration
The airways receive parasympathetic and sympathetic innervation too. Parasympathetic
neurons in the nucleus ambiguous innervate the trachea and bronchii, reducing airway
diameter upon activation. Their activation is excitatory via muscarinic cholinergic receptors.
On the other hand, sympathetic neurons at the 4th thoracic level (in rats and humans) project
to the paraventral ganglia activate their b-adrenergic receptors and as a result reduce
bronchial diameter they control.
The airways also have afferent innervation mostly through vagal mechanoreceptors, whose
cell bodies are in the nodose ganglia and whose central axons project to the nucleus of the
solitary tract in the brainstem. That link synchronizes respiration with the necessities of blood
flow.
41
5.3. Anatomy of intercostal muscles
Reviewed from The intercostal muscles are two layers of muscles laying in between pairs of ribs
and assisting the diaphragm to alter the ribcage dimensions in respiration. External intercostal
muscles attach at the tubercles of the ribs next to the vertebrae and extend dorsally up to the
costodronchal junction. Internal intercostal muscles extend dorsally from the sternocostal junction
in the sternum.
The two layers of muscles only super-impose laterally. Between the costodronchal junction and
the sternum, the external intercostal layer is replaced by the anterior intercostal membrane. Thus,
only the intercartilaginous internal intercostals are present in the ventral side of the thoracic cage.
This ventral portion of the internal intercostal muscles is also referred as paraesternals. Similarly,
internal intercostals do not reach the most dorsal part of the thoracic cage, close to the vertebrae.
External intercostal muscles at that point are mirrored by the levator costal. The parasternal
intercostal muscles are covered by the transversus thoracis. A deeper layer of internal intercostals
are the innermost intercostals which move together with the internal intercostals.
The muscle fibers of the internal and external intercostal muscles are skewed and run in opposite
directions. While the internal intercostal bundles run from rostral to caudal and dorsal to ventral,
the external intercostal muscles run from caudal to rostral and dorsal to ventral. The fibers of the
transversus thoracis are also set perpendicular to the internal intercostals.
Mechanically, because the bundles are skewed, the distance from the lower and upper insertion of
the muscle into the rib to the center of rotation of the rib is different, which translates into a
different torque magnitude for each. For the external intercostal muscles, the net torque produces
the ribs to upper and the lungs to inflate. The torque acting on the internal intercostal muscles is
42
smaller in the lower rib than in the upper one and the net effect is to lower the ribs and deflate the
lung. This generalization will be further extended on the following sections. This is a
generalization, which will be further extended in section.
5.4. Respiratory function of intercostal muscles
The traditional view sets that inspiration is produced mainly by contraction of the diaphragm and
the external intercostals, which elevates the ribs and the sternum, thus, expanding the transverse
dimensions of the thoracic cavity. During expiration, the diaphragm would relax, which would
decrease the dimensions of the thoracic cavity, driving the air out of the lungs. In forced
expiration, the internal intercostal muscles would contract to force the ribs down and drive air out
of the lungs. This is an oversimplification of the respiration process. It has been seen that specific
locations of both external and internal intercostals might have a role in inspiration and expiration.
It is suggested that its specific role depends on their mechanical advantage, even more, that the
electrical activity of the muscle is set according to its mechanical advantage [28].
In a canine model it was observed that contraction of the parasternal intercostal muscles by
electrical stimulation lifts the ribs up and increases lung volume, asserting the inspiratory role of
internal intercostals of specific locations in the dog [29]. Moreover, the inspiratory role of the
canine internal intercostal muscles decreases laterally and caudally at some point reversing its
role into expiratory [30], [31]. Therefore, the parasternal intercostal muscles at the 1st and 2
nd
interspaces have the strongest inspiratory function. In terms of nomenclature, throughout this
document the interscostal space between the 1st and 2
nd rib will be called 1
st intercostal , between
ribs 2nd
and 3rd
2nd
intercostal and the same for the 12th intercostal spaces of the rat.
The same trend has been seen in anesthetized or decerebrated cats, like any young cat usually is
43
[32]. Equivalently, the inspiratory effect of external intercostals decreases caudally and towards
the costochondral junction, up to the point of getting reversed into an expiratory function in the
lower and more ventral parts of the ribcage. Thus, both the external and parasternal intercostal
muscles in the upper spaces are active during inspiration and, in the lower spaces external and
internal intercostals tend to have an expiratory role.
Again, the same trend has been observed in humans [33]. A higher electrical activity in the upper
intercostal muscles has been measured not only qualitatively. The phasic inspiratory duration as a
percentage of total respiration time and the average discharging frequency of motor neurons (in
six subjects both decrease with the intercostal space in a rostrocaudal manner. All measurements
were taken close to the sternum
Troyer et al analized the mechanical advantage of different portions of the ribcage using an
extension of the mechanical model of Hamberger [34], and correlated those results with the role
in respiration of the internal and external intercostal muscles. Indeed, the topographic distribution
of mechanical advantage correlates with the topographic distribution of the respiratory effect,
both inspiratory and expiratory.
The net moment exerted by the muscle depends on the angular position around the rib, which for
the external intercostal muscle it is the strongest dorsally and is reversed in the ventral region.
The transition from expiratory to inspiratory effect depending on the position can be seen also for
internal intercostals [28].
In the dog, it has been seen that bundle of the para-sternal intercostal close to the sternum, which
have the greatest inspiratory mechanical advantage due to the angle of the bundle, contract the
most during inspiration than lateral bundles.
44
Histochemical study of the properties of intercostal muscles in terms of fiber type shows that
intercostal muscles routinely recruited in inspiration have significantly higher proportion of slow-
oxidative muscle fibers in the cat [35]. In the hamster parasternal intercostal muscles, it has been
observed a decrease in the slow oxidative fiber percentage and increase in the fast twitch fiber
percentage from rostral to caudal, which is in the same direction as inspiratory function decreases
[36].
5.5. Innervation of intercostal muscles
Trunk muscles in general are supplied by each spinal nerve deriving from its respective somite. In
concrete, innervation of intercostal muscles is segmentally distributed in the spinal cord [37],[38].
However, in humans it was found that intercostal nerves have communicating branches. There are
some conflicting results about the extent of innervation within the spinal cord of the motor
neurons for a concrete intercostal muscle Reviewed from [39].
In cats, it has been observed that segmented parts of the serratus dorsalis are supplied by a single
or double spinal segments, while intercostal muscles in adult rats and cats are supplied by each
segmental level. In neonatal rats, retrograde labeling located the soma of motors neurons
innervating external intercostal muscles within one or two segments [40] while electrophysiology
showed a strongest response to the stimulation of just one segmental level.
Therefore, injury of a segmental level of the spinal cord mostly denervates the corresponding
intercostal space, although it could have some effect in the innervation of adjacent intercostal
spaces. When the effect in intercostal innervation of spinal cord injury in an animal model,
special attention should be given to the fact that spinal cord segments are slightly decalated
45
rostrally with respect to their vertebral bodies.
5.6. Injury location and type
Under the light of the previous information it was decided as a suitable injury model a bilateral
contusion injury at the 3rd
thoracic level (T3). A thoracic injury affects the respiratory system and
yet the stress and surviving ratios for the animals are higher than in cervical injuries.
Regarding the intercostals, injury at the T3 level mostly denervates the 3rd
thoracic intercostals,
although it has also some effect in the 2nd
and 4th intercostals and it affects indirectly all the
intercostals in downstream pathways as well. Nevertheless, not being a complete injury like
transection, spares part of the function of the diaphragm and rest of intercostals. A contusion
injury also translates better than other models of injury, like hemisection, to the real scenario of
spinal cord injury in humans. According to the last etiology studies, contusion are the most
common type of spinal cord injury [1].
Electrophysiology of the intercostal muscles has been used to assess the functional improvement
that a potential beneficial treatment evokes on denervated intercostal muscles. Both internal and
external intercostals are affected by this type of injury. The parasternal intercostals are the muscle
of choice as site of recording.
Para-sternal intercostals allow for a minimally invasive setup of electromyographic recordings. In
a supine position, they offer easy access. Exposure of the parasternal intercostal only requires a
minimal incision of the ventral skin and tearing apart of the major pectoralis fibers. Altogether, it
has a minimal effect in pain and locomotion of the rat. Besides, it avoids the dorsal area where
the laminectomies were performed which would decrease the healing rate of those injuries and
46
increase distress in the animals. Moreover, by placing the rat in a supine position, there is no
restriction on its breathing due to body weight.
While more rostral intercostal muscles have stronger electromyographical signals, lesions at
higher levels in the spinal cord impose stronger disabilities in the animals. There is a trade-off to
reach. Ventral branches of the 1st and 2
nd thoracic nerves are part of the brachial plexus
controlling the forelimbs [41] and should be avoided to minimize the impact on the locomotor
system. At the T4 level there is an anatomical landmark in the form of an artery, which results in
death of the animal when torn. For practical reasons and to ensure survival, it is better to avoid
laminectomies at T4 vertebral body.
As a results of all those considerations, it has been decided T3 as the site of injury. It is important
to note that the function of internal intercostal muscles at the 3rd
intercostal space is solely
inspiratory and increases ventrally. Therefore, recordings were taken 5mm away from the
sternum at the midpoint between ribs.
5.7. Experimental Protocol
Twenty Sprague-Dawley female rats (200-275g of 6-8 weeks of age) were anesthetized by
injection of Xylazine (10 mg/kg, i.p.; Western Medical Supply, Los Angeles, CA) and Ketamine
(100 mg/kg). Animal losses are typically 10%. Because the laminectomy procedure for a T3
injury can result in severe blood loss due to critical arteries and veins being present within the
surgical area, we hypothesized that there would be more than 15% animal loss. Animals were
tagged at the time of surgery by tail markings as well as attachment of a clip with the animal
number to the ear. The dorsal area was shaved and disinfected with serial provodone and 70%
ethanol scrubs. A midline incision exposed the spinal column at the level of T1–T5, and the
47
paravertebral muscles were dissected bilaterally to visualize the transverse apophyses.
Laminectomy was performed at T3 and animals were subjected to a bilateral contusion injury
using the Infinite Horizons Device (Precision Systems, Kentucky, IL) with a 220Kdyne weight.
After injury, animals are kept in a heating pad for 12 hours and receive a solution of 5ml ringers,
0.02 ml Baytril (2.5 mg/kg/d; s.c.; Bayer, Shawnee Mission, KS), and 0.4ml of dilute
buprenorphine (0.025 mg/kg/d, s.c.; Western Medical Supply, Los Angeles, CA) subcutaneously
during 2 days post injury. Animals are bladder expressed twice a day and monitored for health
problems for a minimum of 3 days or as needed.
Five days after injury animals are divided into two groups of ten subjects comparable in terms of
injury behavioral outcome according to the Basso, Beattie and Bresnahan Locomotor Rating
Scale procedure, which was described in Chapter 2. One week post-injury, one of these groups is
exposed to the treatment whose effectiveness wants to be assessed while the other group is treated
to become their appropriate controls, whether that implies injecting a vehicle solution, implanting
an inert bioscaffold, etc...
EMG recordings from the 2nd
and 3rd
intercostal spaces were taken before injury to act as
baselines, one week post-injury before transplant, four weeks post-injury (three weeks after
transplant) and five weeks post-injury ( four weeks after transplant). Special care was taken while
recording to minimize the effect of noise and interference. Proper grounding and a Faraday cage
effectively removed most of the noise and the 60Hz interference.
Animals are placed in a supine position within the Faraday cage with the limbs immobilized to
avoid disturbance during the procedure. The animals need to be anesthetized but Xylazine should
be avoided in this protocol because it is a muscle relaxant. Ketamine is a sedative and it is not
48
acceptable for use as the primary anesthetic method in surgeries that require incision. Isofluorane
is a general anesthetic which potentiates the effect of muscle relaxants but its use at 2% was
found to have a slight muscle relaxant effect itself. The final approach was a combination of 60
mg/kg Ketamine and isoflurane at a lower dose of 1.0%.
Electrophysiological recordings are taken with a 30 gauge concentric needle with a standard
recoding surface (0.07mm2), placed 5 mm away from the sternum, since the electrical activity
decreases dorsally. At the same time, recordings were taken at the midpoint in between ribs and
approximately 3mm inside the muscle, ensuring by observation of the electromyographic signal
that the muscle was active during inspiration and that the electrode had not gone too far and
reached the transversus thoracis instead.
Recordings were taken from the right intercostal to minimize the effect of the electrocardiogram
(ECG) signal. A reference electrode was placed near the left side to minimize that presence
although signal filtering has been applied by software to eliminate the remainders
Three electrode insertions were performed approximately at the same spot, per animal per
session, and at least 20 seconds of recordings were taken from each one of them. Although these
recordings are minimally invasive, animals receive an injection of 1 ml of the analgesic solution
(5ml ringers, 0.02 ml Baytril and 0.4ml of dilute buprenorphine).
5.8. Summary
One of the goals of this thesis is the development of an injury model that targets denervation of
the respiratory system and assess efficacy of regenerating therapies with a functional test, being
49
EMG the specific functional technique here applied. In this Chapter, I have proposed the contused
spinal cord at the thoracic level 3 as a good model of injury to mimic denervation of the accessory
muscles of respiration due to injury or disease. I have also presented the recovery of the
intercostal muscle functionally as a good indicative of neuronal regeneration. At the same time,
not affecting the phrenic nerve or producing much locomotion or homeostasis deficits like
cervical injuries entail, the survival ratios of the animals and all the practical considerations
relating maintenance and recurrent EMG measurements are facilitated.
50
Chapter 6
Results on a stem cell repair therapy
6.1. Goal of this chapter
In previous chapters, I presented my work on developing a rat-injury model and
electromyography (EMG) program to test efficacy of cures in conditions affecting the motor
neuron population. In this chapter, I validate the model and the technique by assessing the
efficacy of a stem cell therapy for the treatment of Spinal Muscular Atrophy (SMA) type I and
identify the parameters with enough resolution to use as a discriminatory tool between groups.
The MotorGraft (Motor Neuron Progenitors) transplantation into the injured spinal cord of rats
produced accelerated regeneration, as I documented using electrophysiological methods.
Histological and behavioral tests are also presented to correlate with the functional outcomes of
EMG. The results confirm that EMG is a useful tool to identify beneficial therapies where
behavioral tests lack resolution or target the wrong system and yet there is functional efficacy. I
performed the work here presented in the Hans Keirstead laboratory at University of California-
Irvine in collaboration with California Stem Cell, Inc.
This study represents one of the first successful uses of stem cells in restoring respiratory neurons
51
and the work here presented is part of the application to the Investigational New Drug (IND)
program of the Food and Drug Administration (FDA), as a preliminary requirement to enter
Phase 1 clinical trials for SMA Type I. If the application is cleared, it will become the second
clinical trial in the stem cell therapy field.
6.2. Background
Spinal Muscular Atrophy (SMA) type I is a genetic disease characterized by degeneration of
certain pools of motor neurons, including the anterior horn motor neurons of the spinal cord.
Symptoms presented by children with SMA type I are hypotonia and a progressive weakness of
voluntary muscles. The diagnosis of SMA Type I is usually made before 3 months of age. A
common scenario with life-threatening implications is the presence of abnormal respiration
patterns and respiratory insufficiency due to weakness of the chest muscles, including the
intercostal muscles.
Transplanted stem cells can influence the injured environment by acting as survival factors,
guidance molecules, or cues for proliferation and differentiation of endogenous stem and
progenitor cells [42] [43, 44] [45, 46]. Motorgraft© is a population of high purity motor neuron
progenitors derived from hESCs by California Stem Cell, Inc. which has been shown to produce
growth factors in vitro [47]. In this study, Motorgraft©, has been transplanted into rat thoracic
spinal cord according to the model presented in chapter 3 with the aim of sparing the motor
neurons that control the intercostal muscles. Two action mechanisms are considered: secretion of
growth factors to delay loss of motor neurons and replacement of dying motor neurons.
52
6.3. Methods
6.1.3. Spinal cord injury:
Injury was performed according to the experimental protocol described in Chapter 3.
6.2.3. Basso, Beattie, and Bresnahan (BBB) Locomotor Rating Scale Procedure:
Five days following spinal cord injuries, all animals were tested according to the BBB test in
order to place them in groups with similar injury profiles. The groups have been listed in Table 6.
Following transplantation, BBB was performed on animals from groups I and II once a week for
the duration of the study. Rats were scored independently by two observers in a blinded fashion.
Groups were compared at the end of the study using a mixed factorial ANOVA with differences
(p>0.05) used for assessment of statistical significance.
Group Condition Outcome Measures Sacrifice
points
n
I MotorGraft
Transplant
BBB, EMG, Tissue sparing analysis, 5HT
sprouting, growth factor
immunohistochemistry
1 month post
transplant 18
II Vehicle
Control
BBB, EMG, Tissue sparing analysis, 5HT
sprouting, growth factor
immunohistochemistry
1 month post
transplant 18
III MotorGraft
Transplant RNA extraction for PCR
14 days post
transplant 5
IV Vehicle
Control RNA extraction for PCR
14 days post
transplant 5
V MotorGraft
Transplant Protein extraction for ELISA
1 month post
transplant 10
VI Vehicle
Control Protein extraction for ELISA
1 month post
transplant 10
Total 66
Table 6- Animal groups, condition,outcome measuremes, sacrifice points and number of
subjects (n)
The result from groups III and IV for gene expression and groups V and VI for growth factor
expression analysis have not been included in this report.
53
6.3.3. Immunosuppression and Cell Transplantation:
MotorGraft was manufactured by California Stem Cell, Inc., using protocols similar to those
described in [47] and implanted 1 week post injury. Twenty four hours prior to transplantation,
animals began daily immunosuppression with Cyclosporin A (20 mg/kg/d, s.c.; Bedford
Laboratories, Bedford, OH). Animals also received antibiotic treatment of Baytril (2.5 mg/kg/d;
s.c.; Bayer, Shawnee Mission, KS) beginning one day prior to transplantation, and continued as
necessary thereafter.
Animals were anesthetized as above and the laminectomy site re-exposed. A total of 100,000
MotorGraft cells or Cell Transplant Solution (CTS) (vehicle control) was injected in a total of
four sites, 2 bilateral injections rostral and caudal to the injury site (0.5mm lateral, 1.2mm deep)
to target the ventral horn. Each injection was a total volume of 1 µL, at a dose of 25,000 cells/
µL.
6.4.3. Electromyography measurements:
Electromyography was performed in groups I and II according to the Experimental Protocol
described in Chapter 3.
6.5.3. Tissue Extraction:
Spinal cord injury epicenter segments of animals in groups III and IV were fresh extracted 14
days post transplantation. The spinal cord injury epicenter segments of animals in groups III and
IV were also fresh extracted 1 month post transplantation to extract protein for analysis with
ELISA. Animals in groups I and II were sacrificed by trans-cardiac perfusion with 4%
paraformaldehyde. The entire spinal cord was extracted and cryopreserved for sectioning. The
54
spinal cord was cut into 1 mm transverse segments and embedded into OCT compound. Sections
of the embedded spinal cord were then cut with a cryostat into 20µm sections and placed onto
slides. Ten slides from each animal were then used for 5-HT staining and anti-human nuclei
staining, NeuN and anti-human nuclei staining, and ChAT and anti-human nuclei staining.
6.6.3. MotorGraft Cell Survival:
Animals in groups I and II were used for MotorGraft cell survival analysis. Animals in group II
served as a negative control for the presence of human cells. Transverse sections of the spinal
cord were analyzed for presence of human cells using an antibody against human nuclei (1:200;
Abcam, Cambridge, MA). MotorGraft cells, as determined by human nuclei staining, were
expected to be located near the injection site.
6.7.3. Immunohistochemistry (IHC):
The spinal cords from animals in groups I and II were used for analysis of MotorGraft
engraftment, neuronal sparing, motor neuron sparing and serotonergic sprouting. Rat spinal cords
were cryosectioned into 20 m sections, and placed onto slides. Five slides per animal, spaced
200 m apart, from transplanted and non-transplanted groups were used for each analysis.
IHC staining was performed on slides using mouse anti-NeuN (neuronal nuclear protein, 1:200;
Millipore, Billerica, MA) primary antibody and rabbit anti-Ku80 (human nuclear protein, 1:200;
Abcam, Cambridge, MA) primary antibody for neuronal sparing analysis. For motor neuron
sparing analysis, IHC staining was performed on slides using mouse anti-ChAT (choline
acetyltransferase, 1:200; Millipore, Billerica, MA) primary antibody and rabbit anti-Ku80 (human
nuclear protein, 1:200; Abcam, Cambridge, MA) primary antibody. For serotonergic sprouting
analysis, IHC staining was performed on slides using mouse anti-5HT (serotonin, 1:200; Abcam,
55
Cambridge, MA) primary antibody and rabbit anti-Ku80 (human nuclear protein, 1:200; Abcam,
Cambridge, MA) primary antibody. Hoechst was used as a nuclear counter-stain. Following IHC
staining, the tissue was imaged using an Olympus AX-80 microscope. Images were taken at 200x
magnification of the dorsal and ventral horns using the MicroFire software. The transverse
section of the spinal cord was broken down into four quadrants; left and right dorsal horns, and
left and right ventral horns. These images represented more than 30% of the total grey matter.
Analysis of human nuclear staining was performed on all stained tissue sections. The presence of
human nuclear staining was recorded for each section, allowing for the identification of
transplanted animals in which human nuclei were and were not detected. The results are reported
as the number of transplanted animals with detectable MotorGraft cells.
Analysis of endogenous motor neuron sparing was conducted from images from the ventral
and dorsal horns, and represent sparing up to 2mm rostral and caudal of the injury epicenter. The
number of motor neurons from the quadrants (images) was quantified using ImageJ software
(U.S. National Institute of Health; Bethesda, Maryland). It should be noted that the sections
quantified did not contain human positive cells. If a cell double stained positive for both ChAT
and Ku80 then that area of tissue was not used in quantification. At least 3 of the 5 slides for the
animal had to be quantifiable to be included in the analysis. The counts for the slides for each
animal were averaged. Statistical analysis was performed using a Student’s T-test (2 tailed
distribution and two sample equal variance).
Analysis of serotonergic sprouting was conducted from images from the ventral and dorsal
horns, which were taken at the same exposure, imported into ImageJ software (U.S. National
Institute of Health; Bethesda, Maryland), and inverted to grayscale images with the same
threshold applied to all images. The density of the staining was determined by multiplying the
56
grey value in pixels by the area measured. The results interpret serotonergic sprouting 1mm
rostral and 1mm caudal to the injury epicenter. Transplanted and nontransplanted groups were
averaged, and statistical analysis was performed using a student’s t-test (2 tailed distribution and
two sample equal variance).
6.8.3. SCI Morphometry:
Animals in groups I and II were used for morphometric analysis of the injury site. Sections that
have been used for other histological analyses (MotorGraft cell survival and IHC) were used to
examine the gross morphometry of the spinal cord at the injury epicenter and surrounding tissues.
The area of intact tissue, including that of pathologic tissue, was traced on the images using the
Olympus MicroSuite B3SV software to determine the area and perimeter values.
6.4. Electrophysiological results
Histology analysis of the transplanted and vehicle groups proved a regenerative and protective
effect of the Motorgraft transplant in the host. Genetic assays also show up-regulation of neuro-
protective and anti-inflammatory genes, while protein assays show significant higher levels of
neuron growth factors in transplanted animals as compared to vehicles (Data shown in following
sections).
Different parameters of the Interference Pattern Analysis were scrutinized looking for resolution
that allowed using them as a discriminatory tool in identifying beneficial treatments which
correlate with histological improvement. I included in the scrutiny parameters obtained from
Zero-Crossing Analysis, Turns-Amplitude Analysis (TAA), Power Spectral Analysis (PSA) and
Burst Analysis.
57
Although results obtained by these analyses support their respective findings, the only parameters
that were able to show significant differences between groups were Zero-Crossing and Turns per
second (T/S). I conclude that Electromyography studies should focus in these parameters as their
main functional outcomes.
The number of turns per second (T/S), one of the parameters obtained from a Turns-Amplitude
Analysis (TAA), is known to increase as there is an increase in the percentage of polyphasic
potentials [48]. Polyphasic potentials are abnormal electrical configurations of a motor unit that is
typically observed after nerve injury and/or neuropathology [49]. An increase in turns per second
was observed in both MotorGraft and vehicle control animals 3 or 4 weeks after transplant, which
is to be expected as all animals received spinal cord injuries prior to transplantation (Figure 13).
A B
Figure 13- IP analysis of EMG signal from intercostal muscles 2 (a) and 3 (b) presented as turns
per second. Data expressed as mean and standard error. Student t-test with *p<0.05, **p<0.005.
The timing of these polyphasic potentials is consistent with the literature that states that the
earliest manifestation of axonal denervation can be detected 1-4 weeks after nerve injury [49].
Turns per second in the vehicle control group increased with time at both the second (Figure 13a)
and third (Figure 13b) intercostal muscles, and these exceeded that of the MotorGraft transplanted
58
group at 5 weeks post transplantation (p<0.05). These data suggest that MotorGraft
transplantation decreases polyphasia, or abnormal electrical configurations in a state of motor
neuron loss.
6.5. Behavioral results
The BBB locomotor scale is often used to group animals into treatment groups following SCI [50,
51]. Thus, the BBB scores 5 days post injury were used for placement of animals into groups
such that all groups had similar BBB scores at time of treatment. As shown in Figure 14 BBB
locomotor scores increased over the course of the study to 11 and 12 for the MotorGraft and
vehicle groups, respectively 27 days after treatment. These data demonstrate that transplantation
of MotorGraft at T3 did not affect lower motor pool recovery, as was expected due to T3 motor
neurons not being involved in hindlimb locomotion.
Figure 14-BBB locomotor scores for MotorGraft treated and vehicle control animals with a T3
SCI. Testing was performed at 5 days post injury prior to MotorGraft transplantation for
pretreatment comparison (5D Post-injury/Pre-MG). Testing was preformed again at 1 week, 2
weeks, 20 days, and 27 days after MotorGraft transplantation (1 Wk Post-MG, 2 Wks Post-MG,
20 D Post-MG, and 27 D Post-MG, respectively). The locomotor capability of MotorGraft and
vehicle control treated animals did not differ. Data are expressed as mean ± standard error.
6.6. Histological results
6.1.6. MotorGraft cells survive following transplantation
IHC staining for human nuclear protein to identify the presence of MotorGraft cells was
performed on spinal cords of all animals. Human nuclei were detected in all but two (2) of the
59
MotorGraft transplanted animals analyzed. Typically there were a lot of human nuclei present, as
opposed to just a few, within a section of analyzed spinal cord tissue (Figure 15a-f). These data
demonstrate that MotorGraft survives following transplantation into the spinal cord. ChAT
immunohistochemical staining was performed to determine if transplanted MotorGraft cells
express markers of mature motor neurons. ChAT staining localized with MotorGraft and
surrounding cells (Figure 15g-i).
Figure 15-MotorGraft cells survive and begin to express ChAT. a, d) Human nuclei (green) were
detected in the spinal cords of transplanted animals. b, c, e, f) Hoechst nuclear counter stain
(blue) (b and e) colocalizes with human nuclear staining (c and f). g, h, i) ChAT positive staining
(red) is localized with MotorGraft (human nuclei in green) and surrounding cells (Hoechst in
blue). Images taken at 200X magnification.
All analyses comparing MotorGraft transplanted to vehicle control groups did not include the two
animals where MotorGraft cells were not detected
6.2.6. MotorGraft Transplantation Spares Endogenous Neurons
The average number of endogenous neurons in the MotorGraft transplanted group, rostral to the
60
injury epicenter, was 38 neurons with a standard error of +/- 3. The average number of neurons in
the vehicle control group, rostral to injury epicenter, was 29 neurons with a standard error of +/-
2. Rostral to the injury epicenter the MotorGraft transplanted group showed a significant (p <
0.01) sparing of neurons compared to the vehicle control group, (p<0.01) (Figure 16a). The
difference in neuronal sparing is evident in representative pictures of NeuN staining (Figure 16e,
f). The average number of neurons in the MotorGraft transplanted group, caudal to the injury
epicenter, was 36 neurons with a standard error of +/- 4. The average number of neurons in the
vehicle control group, caudal to the injury epicenter, was 37 neurons with a standard error of +/-
3. Caudal to the injury epicenter the MotorGraft transplanted group did not demonstrate statistical
significance in neuronal sparing (Figure 16a). Therefore, caudal to the injury site neuronal
sparing was not affected by the transplantation of MotorGraft, whereas MotorGraft spared
neurons rostral to the injury epicenter.
61
Figure 16-a) The average number of neurons in a quadrant of spinal cord cross sections from
vehicle control (ave= 29) and MotorGraft transplanted animals(ave=38). There were
significantly greater number of neurons in transplanted animals (ave=38). b) The average
number of motor neurons in a ventral horn of vehicle control (ave= 7) and MotorGraft
transplanted animals.(ave=12) rostral to the injury epicenter. c, d) Representative NeuN
staining (red) in transplanted (c) and vehicle control (d) spinal cord sections. e, f)
Representative ChAT staining (red) in transplanted (e) and vehicle control (f) spinal cord
sections. Images taken at 200X magnification
6.3.6. Transplantation Spares Endogenous Motor Neurons
The average number of endogenous ChAT positive cells in the MotorGraft transplanted and
vehicle control groups was compared to examine whether the neuronal sparing may have
included endogenous motor neurons. The average number of ChAT positive cells counted in a
single ventral horn of the MotorGraft transplanted group in sections that are rostral to the injury
epicenter was 12 cells with a standard error of +/- 2 cells (Figure 16b). The average number of
62
ChAT positive cells counted in a single ventral horn of the vehicle control group in sections that
are rostral to the injury epicenter was 7 cells with a standard error of +/- 2 cells (Figure 16b). The
number of ChAT positive cells rostral to the injury epicenter in the MotorGraft transplanted
group was significantly (p<0.05) greater than that of the vehicle control group, demonstrating that
MotorGraft transplantation results in sparing of endogenous motor neurons (Figure 16b). The
difference in motor neuron sparing is evident in representative pictures of ChAT staining (Figure
4c, d). There was no significant difference in the number of ChAT positive cells caudal to the
injury site despite the MotorGraft transplanted group having 13 +/- 2 cells as compared to the
vehicle control group having 9 +/- 2 cells (Figure 16b). Although there was no significant
difference caudal to the injury site, this suggests that there was a trend in the data.
6.4.6. Morphometry
The area of intact spinal cord tissue, whether it be normal or pathologic, was examined.
Morphometric analysis of the spared intact spinal cord tissue resulted in no significant difference
between MotorGraft transplanted and vehicle control groups (data not shown). These data
implicate that although there is significant sparing of neurons and motor neurons in particular,
there is not an effect on the overall area of the intact spinal cord tissue. The lack of difference in
the area of intact tissue reveals that the comparison in the number of neurons and motor neurons
is justified as the area of tissue was comparable.
6.5.6. Serotonergic Sprouting
Serotonergic fiber sprouting analysis demonstrates that the MotorGraft transplanted group has
significantly greater sprouting as compared to the vehicle control group at 2mm rostral to the
injury epicenter (Figure 17a). Although there was not statistical significance at 1mm rostral, 1mm
caudal, and 2mm caudal, the data suggests that there may be a trend to support increased
63
serotonergic sprouting with MotorGraft transplantation. These data suggest that MotorGraft may
have a trophic effect on the local environment.
Furthermore, as serotonin is thought to be involved in recruitment of more motor neurons with
afferent input after SCI [52], these data suggest that the electrophysiological properties of motor
units would be improved, just as was observed with MotorGraft transplantation (see Figure 13).
A
B C
Figure 17- Comparison of serotonergic fiber sprouting (Stained by 5-HT) in vehicle control and
MotorGraft transplanted groups. MotorGraft transplantation resulted in increased integrated
density of 5-HT fibers (red) 2mm rostral (+2) to the injury epicenter. Analysis is at 2mm and
1mm rostral (+2 and +1, respectively) and 2mm and 1mm caudal (-2 and -1, respectively) to the
injury epicenter. Images taken at 200X magnification. Data is expressed as mean ± standard
error. *p<0.05.
64
6.6.6. Gene Expression Analysis and Growth Factor Expression Analysis
Although not shown in this document, PCR analysis of transplanted animals detected a decrease
in host-expression of the pro-inflammatory and pro-apoptotic genes CD40, CRP, Gadd45,
Caspase 4, and TGFβ2 and increased expression of IL-10, which has anti-inflammatory functions.
There was also detected a significant increase in the growth factors NT-3 and NGF of the spinal
cord of animals as compared to vehicle animals, via ELISA assays, proving increased expression
of in vivo secretion of growth factors by the transplant.
6.7. Discussion
Reinnervation of a denervated muscle is characterized by an increase in the amplitude of the
signals. The surviving motor neurons sprout towards denervated muscle fibers, and their MUPs
increase both in amplitude and duration. Early findings are characterized by polyphasia due to
differences in conduction between newly innervated fibers and old ones. As time goes and the
connections in the end-plate mature, the duration of individual MUP decrease and so do the
amplitudes. MUP analysis or IP analysis indirectly can provide information about individual
MUPs.
I studied different parameters of the IPA to use as indication of re-innervation and overall
beneficial effects that correlated with histological improvement. I found that parameters related to
amplitude as mean amplitude per turn (A/T) and mean amplitude of the burst are too variable to
provide the necessary resolution to discriminate between groups, even after applying averaging
techniques across measurements and ensemble average across time. Power Spectral Analysis does
not show significance either. Turns per second from the Turns-Amplitude Analysis and Zero-
Crossings have found to be the most sensitive parameters to track regeneration.
65
At the end of the study, the transplanted group displayed lower turns per second and
zerocrossings. This data supports the hypothesis that a motor neural transplant enhances
reinnervation of the internal intercostals after a traumatic insult to the spinal cord. The results are
corroborated by the results from histology, gene expression analysis and growth factors
expression analysis.
6.8. Summary
Rats subjected to the model of injury described in section 5.6 were transplanted with MotorGraft
from California Stem Cell, Inc (Motor Neuron Progenitors) and were measured according to the
experimental protocol described in section 5.7 and section 6.3.
The transplant, as observed from histology, has significant beneficial effects in neuronal sparing
and sprouting. PCR analysis showed decreased expression of pro-inflammatory and pro-apoptotic
genes and increased expression of anti-inflammatory genes in transplanted animals, while ELISA
analysis detected increased levels of growth factors in vivo in the transplanted animals as
compared to vehicles. Altogether, it supports the beneficial role of the transplant in regeneration
and protection in the injured environment.
However, the behavioral BBB locomotor test does not detect a difference between groups,
probably because the injury does not target specifically locomotion. On the other hand, turns,
zerocrossings of the intercostal muscles were able to show accelerated regeneration. As a
summary, in the results shown in this Chapter supports the usefulness of the model of injury and
the techniques here developed as assessment tool of efficacy of therapies in the context of
denervation diseases or traumatic injuries to the spinal cord.
66
Chapter 7
Effects of electrical and mechanical stimuli in
neurons
7.1. Goal of this chapter
In previous chapters, I presented the development of a model of rat injury that mimics
denervation of the respiratory system due to disease or injury at the spinal cord level and
evaluates efficacy of regenerative treatments by analyzing the electromyography (EMG) signal. I
validated the technique and the model in Chapter 6 using a motor neuron progenitor transplant.
In the following chapters, I propose a new scaffold made of piezoelectric polymers as the
method to enhance neuronal plasticity via electrical stimulation. Piezoelectric polymers are an
ideal platform for combinational therapies in regeneration since they can combine electrical,
mechanical, topographical and chemical cues. This chapter starts by reviewing the effect that
different of these cues have in neuronal plasticity, focusing in the cues inherent to a piezoelectric
polymer (mechanical and electrical). Then, I review alternative means for delivery of electrical
fields to the site of injury and show the advantages of piezoelectric polymers over the others.
Finally, I present the significance and originality of my contribution.
67
7.2. Strategies for Spinal Cord Injury
Contrary to previous knowledge, nowadays it is known that the CNS has an intrinsic capability
for regeneration, which is not successful because of lack of enough stimulation cues as well as the
presence of inhibitory cues. Enhancing the former and blocking the latter is the rationale behind
regeneration of damaged pathways.
The problem of neuronal repair in the context of spinal cord injury can be simplified to the
processes of growth (trophism), guidance towards the target (tropism) and establishment of
functional synapses at the end of that process
Several strategies have been implemented in order to promote such growth, and modulation of the
following factors may also act as guidance cues. These include:
a) Chemical growth factors
b) Substrate mechanical support
c) Stiffness
d) Mechanical stimuli
e) Electric stimuli
The combined effects of several growth enhancement factors, based on well-characterized
features of nerve cell behavior are extremely important. These mechanisms, once understood,
would lead in time to important new approaches to growth enhancement and regeneration of
spinal cord injuries. Despite efforts, none of the approaches have yield significant results.
Nowadays, there is a special interest in looking for combinational treatments where scaffolds,
neurotrophic growth factors, cells and different biomaterials are combined in search of
combinational if not synergistic effects ([53], [54], [55], [56], [57] and recent review articles:[58],
[59],[60]).
68
7.3. Chemical growth factors
Neurotrophins enhance cell survival and axonal growth in vivo but they require a permissive
substrate to promote such a plastic effect. NGF, BDNF, NT-3 stimulates dendritic branching and
axonal growth. Blocking of inhibitory molecules like nogo has a similar effect. These growth
factors might be supplied or secreted by cells, or doped into substrates with an engineered release
time. (Reviewed from [61])
7.4. Structural support
Scaffolds that enhance axonal growth in the SC are divided in two groups: Implants of cells into
the wounded area and cell-free bridges made with biocompatible materials. The first group
includes grafts with Schwann cells, olfactory ensheating glia, genetically manipulated fibroblasts
and stem cells, among others. Immuno-reaction is a problem for those drafts that use ensheathing
cells from another human or animals. For grafts where the own person cells are used, purification
and expansion of the cell population will take more time than the required to start an effective
treatment for an acute SCI. The problem is also the poor knowledge about these natural
mechanisms of regeneration, which makes them difficult to manipulate [62].
The last point lead to the use of substrates, engineered to contain the necessary properties to
promote regeneration. For some time it has been known that polymeric substrates can enhance
growth of neurons and muscle cells due to their structural properties [63], [64], [65], [66], [67].
In the context of biocompatible materials, polymers can be divided between non- biodegradable
and biodegradable. The concerns for these materials implantable materials are the degradability,
reabsorption and biocompatibility. Long-term exposure would promote scar formation and be
69
treated as a foreign body [61]. Undegradable substrates are easily manufactured, more
hypdrophobic and hence, less cellularly adhesive, and pose a higher risk of inflammation ([68],
[69], [70]). On the other hand, the manufacture design of biodegradable polymers is usually
challenging and poses constraints in volume and degradation rates to prevent a significant change
on the PH of the extracellular matrix [71]. In both groups there are FDA approved polymers to
use in human implants like polyacrylonitrile polyvinylchloride (PAN=PVC),
poly(tetrafluoroethylene) (PTFE), poly(glycolic acid) (PGA) and poly(lactide-co-caprolactone)
(PLCL).
Some examples of nerve conduits with non-degradable polymers include PAN=PVC channels
used to deliver Schwann cells or olfactory ensheathing glia, PTFE channels and poly(2-
hydroxyethyl methacrylate) (PHEMA) [68].
Among the common degradable materials that have been studied for use in nerve guidance
channels in the CNS there is the polymer family of poly(a-hydroxyacids,) which include synthetic
polymers and copolymers such as PGA,PLCL, poly(lactic acid) (PLA) and poly(lactic acid-co-
glycolic acid) (PLGA) and natural occurring polymers like collagen and chitosan. The last group
is abundant but the manufacturing of scaffolds is difficult, often because of insolubility in the
most common solvents. Chitosan has also a low mechanical strength.
At this point there is not one polymer with clear advantages respect to the others, and so there is
much search for new materials as well as modification of known substrates to enhance their
mechanical and chemical properties, like doping of chemical factors onto the substrates or
copolymerization to increase hydrophilicity. The use of hydrogel materials it is wide spreading
since they can expand to fill the entire wound, which makes them very suitable in the context of
SCI ( [72],[73]; [74]).
70
7.5. Stiffness
The structural properties of substrates can enhance neuronal plasticity. Stiffness has been shown
to be a modulator of neuronal morphology. However, the effects of stiffness in neuronal
branching have produced conflicting results. Neurons grown on gels of the range of 300–3000 Pa
experience increased dendrite branching in stiffer gels [75]. While some studies support the same
increase in dendrite branching, others show decreased branching with stiffness. Cell density,
stiffness ranges, methods and cell age could account for the differences.
The mechanism under that effect seems to involve adhesivity of the substrate. Focal adhesion
kinases have been seen to change behavior on compliant substrates. The trans-membrane protein
tyrosine phosphatase a transduces mechanical forces via av/b3-integrin activation. Integrins have
been shown to play a role in mechano-sensing (Reviewed in [76]). Integrins are receptors of shear
stress in endothelial cells. Strength of integrin–cytoskeleton linkages is dependent on substrate
rigidity and fibrobast cultures grown on stiffer substrates overexpressed integrins.
Furthermore, anisotropy of the mechanical moduli can serve as a guidance cue as well. Stiffness
gradients have been shown to direct growth of axons, due to identified components in the cell
membrane that respond to mechanical traction. Dorsal root ganglia (DRG) from chick embryos
cultured in substrates with a gradient of stiffness between 60Pa and 365Pa grew neurites followed
the compliant side with a 300%, while neurons grown in isotropic substrates experienced a 20%
variation [77].
7.6. Mechanical stimuli
Although cells are mechano-sensitive entities, there are few studies on the effect of mechanical
71
stimuli on cell in terms of stress, whether stretch, compression or vibratory. Lately, studies on the
effects different mechanical stimuli have in a variety of cells as well as on mechanisms of
actuation have wide spread due to the advances of micro technology to control stimulation at the
cellular level while new optical approaches allow real time imaging of its effects. The field of
biomechanics is blossoming and, in the context of spinal cord injury, there is much need to
elucidate the response in growth that mechanical cues may have in cells of the Central Nervous
System (CNS).
The nature of the mechanical response of the cell depends much on the cell type, some of them
being intrinsically designed to respond to mechanical stimuli. It would be the case of many
sensory receptors, which are the sensory end-organs of primary neurons on the Peripheral
Nervous System (PNS). The responses of sensory receptors are not only specific to the type of
stimuli, which includes stretch, vibration and shear stress, but also to the spectral content and
amplitude of stimulation. Activation is generally mediated by mechanically gated ion channels.
Usually, opening of those channels depolarizes the cell membrane. If a threshold is reached, it
fires an action potential, although depolarization can also set the release of a neurotransmitter that
excites other cells, like in the case of cochlear hair cells.
Beyond sensory receptors, neurons have inherent mechano-sensitive properties themselves.
Controlled mechanical stimulation of sensory axons has shown specificity in their response to
static and vibration stimuli in the same manner their sensory receptors would, although end-
organs are required to fine-tune the threshold of activation [78]. The mechanism of mechano-
transduction seems to involve different ion channels depending on the type of stimuli. Rapidly
adapting currents are likely to have a strong contribution from [Na+] ion channels, while it is
suggested that slowly adapting currents are mediated by [Ca++
] ion channels [79]. In line with the
last study, [Ca++
] transients were affected by dynamic axial stretching [80].
72
If we focus on the cellular response to vibratory stresses alone, there are not many studies
dedicated to the neuronal population, apart from the study of sensory receptors and sensory
neurons. However, many studies address the effect of whole-body vibrations in different systems,
including the nervous system.
Although the actual stimuli experienced in the tissues depend on elastic matching and attenuation
factors within the body at different frequencies, the effects of whole-body vibration on the tissues
are still indicative of the response of a certain cell type to vibration. The frequency range to which
the human body is more sensitive is from 1 to 80Hz.
The interest on the impact that chronic vibration has on human body started back in the 1950s
within the context of work safety. The epidemiology of low back pain reveals a higher incidence
of spine disorders among professional drivers, higher even among those with longer driving
duties, which suggested vibration as a deleterious agent of spine health. The ISO 2631 standard,
from the International Standard Organization, was developed to evaluate the effect of human
exposure to whole-body vibration and to set thresholds delimiting injury [81].
Blurred vision, loss of balance, loss of concentration and other circulatory, digestive and neural
deficiencies are all examples of the deleterious effects of vibration in many systems[82]. On the
other hand, vibration in the 18-80Hz range has been shown to decrease low back pain in a manner
comparable to conventional physiotherapy [83] and [84].
We found other examples of the positive effects of vibration in the context of sports medicine,
where vibratory platforms seem to enhance physical performance. Whole-body vibration at 26 Hz
resulted in increased jumping height [85] and exercise in vibration plates at 40Hz increased
73
strength in naïve and non-naïve subjects more than resistance exercise alone [86].
Frequencies and amplitudes are the most relevant features when considering the positive or
negative effects of vibration, although axis of loading and duration of exposure are also
important.
Not many cellular studies exist at the neuronal level, but chronic vibration has been found to
affect rat neurons of the inferior vestibular nucleus (IVN) in a deleterious manner. Neurons
exposed for 5, 10 and 15 days, in 2h-sessions to vibration at 60Hz and 0.4mm in amplitude,
changed all their main features including an increase mean frequency of the background activity
and a more populated and chaotic firing pattern [82].
Another interesting finding on the effects of vibration in cells is the possible enhancement of
endocytosis. Rat cortical CryoCells increased antibody take upon vibration at 500um and 200Hz
for 3h, although some cell detachment was also observed[87].
Many studies have scrutinized the most deleterious frequencies involved in degeneration of the
intervertebral discs (IVDs), which correlates with low-back pain. Close to the resonant frequency
of the spine (4-5Hz), most degeneration of the IVD occurs although that is probably due to
mechanical energy transfer and not to vibratory frequency itself, while frequencies in the 20-
300Hz range seem to stimulate protein synthesis on IVDs and surrounding tissue, which is
regarded as beneficial contribution to IVD health.
Cultures of annulus cells loaded at 1.7MPa and 20Hz in sessions of 30 min for 9 days stimulated
protein synthesis and reduced protein degradation on the extracellular matrix while they did not at
0.3MPa and 1Hz[88]. IVDs subjected to axial vibrations on sessions of 10-60min, up-regulated
74
collagen type II (normally high) in the extracellular matrix at 80Hz, while at 8Hz decorin
(normally high) was up-regulated and at 40Hz was down-regulated. On the other hand, at 0.4-
0.5g, but not below 0.4g, collagen type I (normally low) was up-regulated, while versican
(normally high) was down-regulated, which, according to the authors, could suggest adverse
effects of vibration at those amplitudes [89].
Lately, there is much interest in the ability of vibration to build up muscle. Vertical stretch of
0.4mm amplitude on the range of 8-10Hz for 10/min day for 3 days has shown to enhance
number, length and average area of myotubes in C2C12 myoblasts [90]. On the other hand,
chondrocytes exposed to 1.4 g vibration at 400Hz arrested DNA synthesis [91]. Many studies
have demonstrated the positive effects of bodily vibration on muscle and bone maintenance [92-
95].
Although it would be important for the field of neuronal regeneration, there are few studies on
possible trophic effects of mechanical stimulation in the neural population. Disrupting
myelination produces in the stress–strain response of spinal cord tissue in uniaxial tension, which
suggests that both types of glia and myelin are important components of the structure–function
relationship in spinal cord tissue [96]. Stretch forces have been found to be a stimulatory cue in
axonal-growth. Axons can stretch up to 8mm/day and reach 10cm of elongation over 14days,
while getting a 35% increase in their calibers[97], but the impact of vibration on neuronal
plasticity remains a matter for study.
Recently, one study addressed the effect of nano-vibration, at 10 KHz, on neurite growth in PC12
cells, showing significant enhancement after several days. The mechanism behind this non-
physiological stimulation was attributed strictly to vibratory enhancement of nerve growth
factor[98], but still lacks an insight on the effects of vibration in a neuronal population.
75
As it has been review, vibration can induce changes in cells in terms of attachment, endocytosis,
cell viability, protein synthesis, firing patterns and plasticity. We could also consider [Na+] and
[Ca++
] mechano-sensitive channels as potential mediators in some of these phenomena.
Deleterious and beneficial effects of vibration over those features depend on the frequency,
amplitude and duration of stimulation.
As a first approach and admitting that thresholds might differ between cell types, amplitudes
above 1.4g seem to have deleterious effects on general viability of the cells and so would seem
for amplitudes between 0.4-0.5g but not below. Attachment is inversely correlated with amplitude
as well. Vibration on the frequency range of 20-300Hz is innocuous at worst and beneficial if we
account for protein synthesis, in concrete collagen presence in the extracellular matrix at 80Hz.
At very low frequencies (8-10Hz), vibration can play a trophic role.
As a summary, cells exposed to vibration should be ensured an amplitude and frequency range
that promotes well-being and maintenance of the culture. Not deleterious conclusions should be
inferred without more consideration, as even beneficial effects can be observed with the proper
settings.
7.7. Electrical Stimuli
Different cells are responsive to electrical fields in a manner that depends on the cell type.
Migration, proliferation and trophic responses in terms of dendritic branching and axonal growth
and guidance are a subject of study that has challenged the scientific community since the times
of Ramon y Cajal and Ingvar in 1920.
76
Endogenous electrical fields (EFs) are naturally occurring during the process of wound healing to
promote growth and protein absorption adsorption [99], [100]. Different studies have measured
the amplitude of the steady EF created in the injured tissue of amphibians and chick embryos.
Endogenous EFs generated upon injury are necessary for epithelium and corneal regeneration.
The amplitudes range between 40-50mV/mm in the cornea [101] and 100-150mV/mm in the skin
[102, 103], and some have been seen to persist for days. Reviewed from [104]and [105].
Additionally, migration of more than 15 cell types has been observed upon exposure of electrical
fields [106],[105],[107]. For example, endothelial cells have been observed to migrate towards
the cathode after exposure to EF above 100-200 mV/mm. It was also observed asymmetrical
distribution of filamentous actin in the cytoplasm, with a transient increase of 80% in the side of
the cathode [108]. Corneal epithelial cells under EF between 100–150 mV/mm for 16 hours
experienced asymmetric up-regulation of epidermic growth factor receptors, which is enhanced
by fibronectin and laminin [109]. EF’s of 120mV/mm also directed neuronal migration of
hippocampal neurons towards the cathode [110].
Direct electrical fields have an effect also in alignment. It has been shown that a direct electrical
field of 500mV/mm for 24 hours induces astrocyte alignment [111]. Electrodes providing an
extracellular voltage of 300-400uV/mm for 15 days post injury with a reverse polarity every 15
min to the injured spinal cord of dogs, has shown to change the density and orientation of
astrocytes[112].
Focusing in the neuronal context, it would seem reasonable that a cell type devoted to
transmission of electrical signals is susceptible to the effect of EFs. Indeed, neurons are
electrically sensitive. Electrical activity, either received directly from neurons or from electrodes,
can enhance dendritic growth and branching. The activity dependence of growth and branching of
77
dendritic arbors has also been noted [113]. Conversely, deprivation of synaptic input causes
dendritic shrinkage, and branch loss [114].
Neurons of the central nervous system (CNS) do not regenerate spontaneously. There is
increasing evidence that externally applied electric fields (EF’s) can induce neuronal growth and
guidance in the CNS. In vivo studies showed that regeneration of the lamprey spinal cord was
facilitated by a small steady current (10 µA, cathode at the distal end) across the lesion for 6 days
[115].
Axonal EF-mediated nerve regeneration in non-mammals has been reproduced in mammals [100,
116-127]. A weak, steady electric field ( 400 mV/mm) applied by electrodes implanted across
transected spinal neurons in guinea pigs induced axonal growth and penetration through the glial
scar within the lesion [128]. Follow-up studies showed that after 1 month of constant current
delivery, there was regeneration of the spinal cord, exhibited by growth cones, extensive
penetration and branching of axons within the lesion, as well as crossing of ascending axons into
the rostral segment [129].
Borgens and colleagues have studied EF-mediated responses of neurons and astrocytes in injured
guinea pig spinal cords [112, 129]. The same group used oscillating electrical fields with a
periodicity of 15 min for bidirectional regeneration in dogs suffering from spinal cord injury
[130] and in humans for Phase 1 clinical trials [131]. They based the periodicity setting in the
study of McCaig, which establishes a time-window during which cathode attraction is stimulated
but anodal retraction is avoided [132].
Different in vitro studies have proven the efficacy of EFs as low as 10mV/mm for directing
axonal growth and stimulating neurite outgrowth and directional branching. However, the effects
78
of polarity in guidance and branching between different studies are confusing and contradictory.
Cell type, charge of the substrate and if the process is axonal or dendritic seem to play a role in
the attraction or repulsion towards the cathode experienced by the neurites exposed to EFs [105].
Chick dorsal root ganglia (DRG) exposed above 40mV/mm showed higher growth rates towards
the cathode, [133], [134]. Spinal neurons of the Xenopus have faster growth rates towards
cathode and retraction from anode at 7-20mV/mm [135], increased sprouting towards the cathode
at 250mV/mm [136]and doubling of branching at 120-150mV/mm, with 80% of the branches
present in the cathodal side[137].
EF’s of 1000mV/mm applied to cultured spinal Xenopus neurons caused directional growth of
neurites toward the cathode and away from the anode if cultured in a negatively charged
substrate, but the opposite in a positively charged substrate, and the response was graded with
adhesivity. Thus, it seems that expression of adhesion molecules may interact with electrical
fields to modulate the guidance properties[105].
On the other hand, hippocampal neurons of rat embryos exposed to 28-219mV/mm had neurites
oriented perpendicular to the EF and a decreased number of neurites facing the cathode [125].
Another study with the same type of neurons, showed that dendrites are attracted to cathodes
while axons are not[138]. And rat PC12 cells growth was biased towards the anode [139] upon
exposure to EFs.
Finally, Zebrafish exposed to 100mV/mm were completely irresponsive to EFs[140] Therefore, it
seems that animal species might also have a role in the effect of neurons to direct electrical fields.
About the mechanism by which EFs exert their influence in neuronal plasticity, it has been found
79
that activation of [Na+]channels and voltage-gated [Ca
++] channels stimulates dendritic growth
and branching in various neurons, incuding cerebrocortical neurons [141, 142]. In the Xenoopus
spinal growth cones, attraction to the cathode seems to be mediated by the neurotrophin receptors
TrkB and Trkc, the nicotinic receptor nAChR, receptor tyrosine kinase,s phospholipase C,
different isoforms of kinase C , extracellullar Ca2++
and intracellular reserves of Ca2++
. Moreover,
cAMP could be a regulatory molecule modulating EF response along with Ca2++
[105].
Another study suggests the role that electrical fields have in protein adsorption and neurite
outgrowth. PC-12 cells grown on conductive films of polypyrrole were electrically stimulated
simultaneously to serum containing media and also pure fibronectin, which result higher levels of
fibronectin and protein absorption and also in longer neurite outgrowth, with respect to cells non-
electrically stimulated of electrically stimulated after protein exposure. This suggests that
absorption of fibronectin and immediate electrical stimulation are necessary for neurite outgrowth
[100].
Besides plasticity, steady state EFs have also an effect in neuronal excitability. EFs of less than
40mV/mmm have been seen to alter the threshold of action potentials evoked by orthodromic
stimulation in rat hippocampal slices [143].
There is much less study of neuronal plasticity by non-steady electrical fields. Electrical
stimulation at 20Hz for 1h of the completely transected sciatic nerve in the rat, showed higher
levels of re-innervation and a higher number of myelinated fibers in the distal nerve by histology
and electrophysiology [144]. Electrical stimulation at 20 Hz for 1 h during 7 days of the sciatic
nerve in vitro produced a 4 fold increase on the DRG neurite outgrowth. After a thoracic hemi-
laminectomy, stimulation at 20 Hz for 1 h during 7 days of the sciatic nerve increased axon
projections into the central lesion of the spinal cord. [145]. Electrical stimulation increased the
80
levels of intracellular cAMP, which makes it a candidate for the mediating mechanism under EF-
induced plasticity.
Because oscillating electric fields not have been studied much, there is little information on which
to select as appropriate stimulation frequencies for enhancement of neuroplasticity.
7.8. Electrically active polymers
While the above experiments were performed with metal electrodes, attractive alternatives are
being developed using quasi-permanent surface charge (electrets), polymers that generate electric
charge upon applied mechanical stress (piezoelectrics), and electrically conducting polymers.
The main advantage of a biocompatible polymer approach versus electrode implantation could be
effectiveness in stimulation delivery and minimization of infection risks. Many synthetic
materials are more biocompatible that metals, including inert metals. Some are even
biodegradable. They provide great flexibility of manufacture, which translates into localization of
EFs where they are most effective. Three-dimensional geometries can be used to deliver stimuli
to deep areas. Their mechanical properties better resemble the ones of the extracellular matrix.
They also can be easily doped with growth factors. Altogether, it makes electrically active
polymers the best platform for combinational therapies.
The most common conductive polymer is polypyrrole (PP). Films of PP have been used to study
neuronal plasticity [146, 147]. PC12 cells cultured on PP films and subjected to a steady field of
100mV for 2h showed a significant increase in neurite length [148]. Similar results were found
when PC12 cells were grown on glass coated with nano-particles of gold and subjected to pulsed
electrical fields [149]. Neural stem cells grew more neurites when their conductive polymer
81
matrix was electrically stimulated [147] Although PP is a non-degradable conducting polymer,
degradable alternative to PP have been studied. Pyrrole-thiophene oligomers have been linked
together with hydrolyzable esters [150] and blends of PP with chitosan or hyaluronic acid are
being investigated to create conducting materials with more biocompatible properties [151],
[152].
Electret materials and piezoelectric materials have the benefit in front of conductive polymers of
not requiring an external source for electrical stimulation. Examples of the effect of an electret is
found in the use of charged forms of poly(tetrafluoroethylene) (PTFE) which has resulted in a
higher number of myelinated axons upon injury with respect its non-charged form [153].
However, electrets have not caught much attention in the scientific community.
A potentially more powerful means of producing EF’s is via piezoelectric polymers. A
piezoelectric polymer generates a variation in the charge density of its surfaces in response to a
mechanical deformation. Thus, it can be used as an autonomous source of EF’s. Polymers with
piezoelectric (PZ) properties, such as polyvinylidene fluoride (PVFD), have been used to enhance
growth and myelination of peripheral nerve axons [154-156]. When grown on PZ-PVFD at
1200Hz for 72 hours, mouse neuroblastoma cells grew neurites at a rate 4 times faster than cells
growing on an ordinary, non-piezoelectric polymer [155]. The use of the piezoelectric copolymer
of PVDF, polyvinylidenefluoride-trifluoroethylene (PVDFTrFe), has also resulted in a higher
number of myelinated axons in the sciatic nerve after injury when compared to the
nonpiezoelectric form [99]. The generation of EF’s by the PZ materials is critical to their action,
since un-polarized, non-PZ polymers of identical composition do not enhance neurite outgrowth.
82
7.9. Significance
In this study, I tested the biocompatible piezoelectric PVFD for its effect on the outgrowth of
neurites in neurons of the central nervous system, and in specific, rat spinal cord neurons. Various
modifications of PVDF have been shown to provide suitable substrates for growth of neurons in
culture[157] but it has not been studied the effects that electricity delivered by a polymer has in
neurons of the central nervous system. Herein I show that spinal cells grown on stimulated
piezoelectric PVDF films generate significantly greater neurite branching compared to those
grown on non-stimulated and non-piezoelectric films. My studies include consideration of
different types of neurons (spinal cord neurons and cortical neurons) and different materials
(PVDF and polyvinylidene fluoride-trifluoroethylene, PVDF-TrFe). All of them show the same
trends, supporting the hypothesis that delivery of electrical fields via a piezoelectric polymer
enhances arborization in neurons of the central nervous system.
The novelty of this project is the development of a for spinal cord injury repair system that
integrates three methods in one platform: (1) the use of a polymer substrate and (2) the use of
electric fields (EF) (3) the use of vibration on cells.
83
Chapter 8
Piezoelectricity
8.1. Goal of this chapter
In Chapter 8, I first introduce the principle of piezoelectricity and the basic vocabulary related to
the field. Then, I explain the different classes and the constitutive equations describing the
behavior of piezoelectric materials. Finally, I present three of the most useful piezoelectric
polymers with promising potential in the biomedical field due to its biocompatibility and/or
biodegradability and I explain the choice of materials which I use in the experiments presented in
Chapter 10.
8.2. History
The direct piezoelectric effect was discovered by Jacques and Pierre Curie in 1880. The Curie
brothers were able to measure charge produced in the surface of materials like tourmaline, zinc
blende, quartz, topaz or cane sugar once subjected to mechanical stress. The name comes from
the Greek word piezo, which means pressure. Thus, piezoelectricity is the ability of some
materials to generate an electric potential in response to an applied mechanical stress. The
converse effect is true as well, as Lippmann predicted in 1881. Piezoelectric materials create
strain as response to an applied electrical potential. For that reason, piezoelectric materials are
84
used both as sensors and actuators.
Piezoelectricity was only an experimental curiosity and mathematical challenge until World War
I, when most popular piezoelectric applications were conceived and developed. Crystal resonators
started being used as frequency stabilizers in oscillators. In 1917 Paul Langevin developed the
sonar, a submarine detector and communication system, which uses both the direct and reverse
piezoelectric effects. Microphones, accelerometers, bender element actuators, speakers, sound
pick-ups and signal filters were also inventions of those times. After 1940, piezoelectric
applications became more focused in actuators, like piezo-ignition systems and relays. Nowadays,
the need for micro-electromechanical systems (MEMS) that can perform both detection and
analysis on a chip has in piezoelectric materials its best ally.
8.3. Piezoelectric classes
The piezoelectric effect dwells on the crystal structure of the material and the ability for charge
separation which that crystal structure entails. In a piezoelectric crystal, the positive and negative
electric charges are separated forming an electric dipole. A dipole p
is defined as two equal and
opposite charges ±q separated by the vector of distance d
.
dqp
Dipoles are usually randomly oriented, so that the material is electrically neutral in macroscopic
terms. Some physical processes, like uniaxially or biaxilly drawing and poling, can align the
dipoles so macroscopically the material loses its electrical neutrality. Poling is a procedure by
which a strong electric field is applied across the material, usually at elevated temperatures,
forcing a remnant polarization at 0V/m.
From the total 32 crystal classes, the 21 that have a non-Centro symmetric crystal structure are
85
piezoelectric, of which 11 classes are neutral at 0 volts and 10 classes contain a remnant
polarization .
The polar group is also pyroelectric, meaning that an applied temperature gradient produces an
electrical field. Ferroelectrics are a subset within pyroelectric materials. In pure dielectric
materials, the polarization P
is linearly related to electric field E
by the susceptibility of the
medium e
and the permittivity of the free space o . In ferroelectric materials, the polarization
P
follows a hysteresis loop, which presents a remnant polarization at 0V/m and which can be
reversed upon exposure to a strong electrical field.
Under normal circumstances, even polarized materials do not display a net dipole moment. The
intrinsic dipole moment is neutralized by electric charge from the medium or from thermal
conduction that builds up on the surface of the material. When a mechanical stress is applied, the
symmetry of the polar crystal is disturbed and the variation of the dipole moment creates a charge
misbalance that generates a voltage across the material.
8.4. Constitutive equations for a piezoelectric material
Piezoelectric materials are electromechanical transducers. The constitutive equations describing
the mechanical and electrical behavior of the material must be extended to include the elastic
response to an applied electrical field and the electrical response to strain. Besides, piezoelectric
materials usually undergo processes like drawing and poling to render them piezoelectric
macroscopically. Those processes introduce asymmetries which break the isotropic configuration.
All of that must be taken into account in the equations of electro-mechanical coupling which
model the behavior of piezoelectric materials.
86
The electrical response of a generic anisotropic material is:
EP
1
EPED o
2
Where P
is the remnant polarization, E
is electric field, χ is the susceptibility matrix, D
is the
electric displacement, εo is the vacuum permittivity constant and ε is permittivity matrix.
For the development that comes afterwards, they can be described more conveniently in their
axial components:
jij
ji EP for i,j=1to3 3
j
jiji ED for i,j=1to3 4
Where 1
o
ij
ij 5
Hooke’s law for a generic material states that a mechanical stress produces a proportional strain.
Stress can be applied as compression, extension and shear, and thus, stress and strain are tensors.
TsS
6
Or alternatively,
iji
ij TsS for i,j=1 to 6, 7
Where S
is the strain tensor, T
is the stress tensor and s is the compliance matrix. The
compliance matrix s is symmetrical and so, only 21 of those 36 constants are independent.
Figure 18 depicts the nomenclature used. The stress for direction 1, 2 and 3 is tensile stress
applied in direction 1, 2 and 3 respectively, while 4,5 and 6 describe shear stress around direction
1 2 and 3 respectively. In that context, T4 would be shear stress around axis 1 and –T3 would be
87
compression stress on the axis 3.
Figure 18- Nomenclature of axis
In a piezoelectric material, a mechanical stress T
produces a polarization in the material that
contributes to the total displacement D
, along with the displacement created by the electrical
field E
itself. This contribution of T
is proportional by a factor of d, called the direct
piezoelectric constant. Equation 1.9 is the expression of the direct piezoelectric effect.
j
j
j
ijjiji TdED for i=1to3, j=1 to 6 8
Similarly, a piezoelectric material exposed to an electrical field E
generates a strain that adds to
the elastic response of the material to the stress T
. This contribution to the total strain S
is also
proportional to the constant d. Equation 1.10 models the inverse piezoelectric effect.
i
iiji
i
jij EdTsS for i=1to3, j=1 to 6 9
Or more compactly,
TdED
10
EdTsS t
11
A d tensor can be identified for each of the 20 classes of piezoelectric crystal structures. We
88
describe the d tensor by a matrix of 3 by 6, dij,, where i is the electrical field in the i direction
created by stress applied in the j direction. Due to symmetries, this matrix is symmetrical and
only 15 of its 18 coefficients are independent.
8.5. Piezoelectric polymers
A polymer is macromolecule consisting of repeating subunits or mers linked by covalent bonds,
which form chains that can arrange spatially in amorphous regions, crystalline regions or a mix of
both. The percentage and type of crystallinity depends not only of the material but also
temperature, pressure and time in cooling off.
Crystals of piezoelectric polymers are non-centro-symmetric and so they have a net dipole
charge. However, the crystalline regions are randomly oriented and overall, any polarization or
piezoelectric effect cancels out and the polymer is macroscopically neutral. Polymers can be
rendered macroscopically piezoelectric by drawing or poling the material.
Many polymers exhibit some piezoelectricity, like PVDF and several of its copolymers (PVDF-
TrFe), nylons (Nylon 7, Nylon 11), cellulose and polyureas (P(MDA/MDI), Polyurea 5, Polyurea
9). Their piezoelectric constants range from 20pC/N for d31 for PVDF at 20oC to 1.7 pC/N for
Polyurea 9 at 20oC, passing by -10pC/N for d36 for PLLA at 20
oC [158].
8.1.5. PVDF
Probably, the most commercially used piezoelectric polymer is Poly-vinylidene fluoride (PVDF).
PVDF is biocompatible aside of displaying one of the highest piezoelectric constant within the
polymeric group. The schematic of the PVDF molecule is shown in Figure 19.
89
Figure 19-PVDF Molecule
PVDF can crystalize in different conformations, namely α, β, ϒ and δ. The α-phase is obtained by
cooling off the melt at a normal rate of about 10oC/min. This gives a trans-gauche-trans-gauche
+
structure which makes the crystal non-polar. Slower cooling of the sample gives rise to the ϒ-
phase. Poling of the α-phase gives rise to δ-phase. None of these phases has significant polarity
since the fluor atoms at both sides of the carbon backbone which tend to cancel each other’s
polarity; therefore, none if these phases are piezoelectric.
The only piezoelectric form of PVDF, the β-phase, can be obtained by uniaxial stretching of the
α-phase at room temperature, which forces a reorientation of the bonds into an all-trans
configuration. In the β-phase, the molecules arrange in an orthorhombic lattice. Because all the
fluorine atoms are on the same side of the carbon backbone, there is net dipole moment. For
macroscopic results, is customary to pole as well.
The point group of the PVDF crystal in its β form is C2V (mm2). This group belongs to one of the
polar classes; therefore, PVDF is not only piezoelectric but pyroelectric and ferroelectric as well.
The piezoelectric constant pertaining to that group is
d=
000
00000
00000
333231
24
15
ddd
d
d
The axis nomenclature used in the piezoelectric matrix is the following. In order to display
90
piezoelectricity macroscopically, piezoelectric materials are often drawn and poled. Drawing
aligns the crystals in the stretching direction. That direction is named 1 by convention. The
direction of poling is usually perpendicular to the plane of the film and is named 3. Direction 2
would be the direction perpendicular to the other two
Figure 20- Axis nomenclature for the piezoelectric matrix
The d constant matrix of PVDF uniaxially stretched and poled is the same as the individual PVDF
crystal. Thus, the piezoelectric matrix for uniaxially stretched and poled PVDF has
piezoelectricity in both the shear and tensile axes.
8.2.5. PVDF-TrFe
Another piezoelectric polymer is the PVDF copolymer Polyvinylidene fluoride-trifluoroethylene
(PVDF-TrFe). PVDF-TrFe crystallizes directly into the all-trans configuration and because of
that, there is no need to stretch to align the crystals. Polarization is still needed in order to
synchronize all the crystal regions towards a known direction. The point group of PVDF-TrFe is
the same as for PVDF. As a result, the same type of d matrix applies.
8.3.5. PLLA
Poly-Lactic Acid (PLA) is also a piezoelectric polymer. PLA is biocompatible and biodegradable.
Because of these properties, it has been used extensively in sutures, scaffolds and in biomedical
devices for implantation. PLA is a chiral molecule and as such two conformations exist: Poly-L-
91
Lactic Acid (PLLA) and Poly-D-Lactic Acid (PDLA). The only form that can be rendered
piezoelectric is PLLA.
Figure 21- PLLA molecule. The asterisk indicates the chiral carbon atom.
Due to its chirality, PLLA arranges in a helix. There are two known crystal structures: the
pseudo-orthorhombic α-phase contains two chains in the unit cell, which arrange in a 10/3 helix
conformation, the β-phase is an orthorhombic unit cell and contains six chains, which have a 3/1
helix conformation.
Works by Fukada [158] and Tajitsu [159] show that uniaxially stretching PLLA films orients the
polymer chains and crystals, giving a piezoelectric constant responsive to shear. Processing
conditions have to be such that the crystals are formed in the α form, which is the piezoelectric
one. PLLA belongs to one of the non-polar groups and therefore is piezoelectric but not
pyroelectric.
The point group of PLLA crystal in its α-phase is D2 (222), but uniaxially stretched PLLA can be
identified with point group D∞ (422). Thus, the piezoelectric matrix for stretched PLLA shows
only shear piezoelectricity:
d=
36
36
00000
00000
000000
d
d
92
8.6. Summary
Piezoelectric polymers have smaller piezoelectric constants than other piezoelectric materials,
their versatility, wide temperature range of operation, elastic properties and behavioral properties
like biocompatibility and biodegradability make them an interesting alternative to their peers,
especially in the biomedical field.
In Chapter 8 I have presented three of the most important piezoelectric polymers, PVDF, PVDF-
TrFE and PLLA. PVDF and their copolymers are biocompatible and display a stronger
piezoelectric effect than PLLA. The advantage of PLLA dwells in its biodegradability. A
combinational platform based on piezoelectric biomaterials to provide electrical and mechanical
stimulatory cues for neuronal outgrowth would most benefit of the biodegradation capability of
PLLA.
In the experiments described in Chapter 10, I have used PVDF and PVDF-TrFE instead of PLLA
due to their higher piezoelectric constant. These experiments are intended to test the principle that
piezoelectric polymers are a practical mean to deliver electrical field and modulate neuronal
plasticity. I have proved their potential for combinational therapies and that further studies should
be devoted to fine tune their stimulation patterns to exploit neuronal regeneration cues.
93
Chapter 9
Experimental design and characterization of
piezoelectric materials
9.1. Goal of this chapter
Chapter 8 presented poly-vinylidene fluoride (PVDF), the most popular piezoelectric polymer in
the market due to their high piezoelectric constant and good mechanical properties. For those
reasons, included biocompatibility, PVDF is the materials of choice for the substrates I used in
the experiments presented in Chapter 10. First, I describe the manufacture process of the PVDF
films I fabricated for these set of experiments. I designed the process to confer these materials
with very specific crystalline, mechanical and piezoelectric properties to optimize their suitability
in cell culture. I have also included the results of characterization for those films. Then, I present
the experimental set-up and the characterization of VIBES, the vibratory device which was
developed to vibrate cell-plates with autonomy within the incubator. Finally, I present the
analysis tools I used to study arborization and alignment.
94
9.2. Fabrication of piezoelectric films
Films of polyvinylidene fluoride were fabricated from pellets with a molecular weight, Mn. of
180,000 (Soltex, Houston, TX). Films were extruded with a Brabender Extruder® at 185ºC at a
rate of 10 rpm and extruder speed of 70 rpm. First, films were shaped according to the ASTM
International standard D638 to produce uniform strain during stretching. To convert films from
crystalline phase α (paraelectric) to phase β (ferroelectric), they were stretched (uniaxially
oriented) and some were then polarized [160]. Stretching ratios up to 3.5:1 were achieved using a
custom film stretcher at 50oC and a drawing speed of 8 μm/second. After stretching, films were
cooled in place at 25oC for 12 hours to allow relaxation. Final thickness ranged between 17μm
and 22μm. The ferroelectric films were then polarized at 150 MV/m for 1 hour using contact
electrodes, which re-oriented the crystals by ferroelectric switching of the molecular dipoles and
made them piezoelectric. For non-piezoelectric control samples, films were identically
manufactured but not polarized. For simplicity, stretched films that were polarized and therefore
piezoelectric are denoted as PZ, and those not polarized (and hence non-piezoelectric) are
denoted as PV.
9.3. Characterization of materials
9.1.3. Rheolograph
Gold electrodes, with offset tabs, were sputtered on opposite sides of PVDF films. The relative
dielectric constant, εr , young modulus , c31, and piezoelectric coefficients d31 and e31, were
measured with a Rheolograph Solid® (ToyoSeiki, Japan) at a frequency of 10 Hz with a static
load of 5g at room temperature to keep the films from buckling during the compressive part of the
measurement cycle. Values of those constants for drawn and polarized PVDF films (PZ) and are
listed in Table 7. Both real and imaginary parts were measured, with no significant contribution
of the imaginary part. All errors are standard errors.
95
R[Young
Modulus[
I[Young
Modulus] R[d31] I[d31] R[e31] I[e31] R[εr] I[εr ]
(GPa) (GPa) (pC/N) (pC/N) (mC/m2) (mC/m2)
3.26±0.07 0.13±0.00 22.60±1.23 -0.84±0.09 73.90±3.99 0.17±0.06 11.30±0.72 -0.25±0.03
Table 7-Rheolograph measurements for PZ films fabricated as described in methods
Drawn and non-polarized PVDF films (PV) exhibited topographic, elastic and dielectric
properties equivalent to the polarized PVDF films (PZ) but had approximately zero
piezoelectricity (d31~0, e31~0).
9.2.3. Digital Scanning Calorimetry
The thermal characteristics of the samples, such as glass transition temperature (Tg) or melting
temperature(Tm), from extruded PVDF films were obtained using a DSC 2910 (TA Instruments,
Texas). All DSC measurements were performed at a rate of 10 °C/min for the heating ramp and
under a dry nitrogen gas atmosphere. The Glass transition temperature (Tg) for this polymer is
around -140oC [161] and it is not shown in Figure 22. S70 acronym represents the films extruded
at a speed of 70rpm which were used in the experiments hereafter described.
Figure 22- DSC graphic showing the melting temperature of PVDF.
96
9.3.3. Fourier Transform Infrared Spectroscopy
The crystal form of the drawn films was studied by Fourier Transform Infrared Spectroscopy
(FTIR) using a Nicolet Magna 760 IR spectrometer and analyzed using Omnic software (Fisher
Scientific). The piezoelectric strain and stress coefficients d31 and e31, the axial elastic modulus,
c31, and dielectric constant, εr, were measured with a Rheolograph Solid® (ToyoSeiki, Japan) at a
frequency of 10 Hz with a static load of 5g at room temperature to keep the films from buckling
during the compressive part of the measurement cycle.
The effect of polarization on FTIR spectra for PVDF films is shown in Figure 23. These results
confirm the transition from the α phase (paraelectric) in extruded un-drawn films to β-phase
(ferroelectric) in drawn films. The characteristic peaks of the α-phase and β-phase have been
marked for convenience.
Figure 23- FTIR spectra of non-polarized PVDF (PV, top) and piezoelectric PVDF (PZ, bottom).
The characteristic peaks of the α-phase and β-phase have been marked for convenience.
97
9.4. Cell culture
9.1.4. Mixed Spinal Cord (SC) Cell Cultures
Spinal cords were dissected from embryonic day 16 rats, and cells were dissociated by
mechanical trituration and seeded in 24 well plates at a density of 50,000 cells/cm2. For cells
grown in PVDF-TrFe films the density used was 100,000 cells/cm2. Cells were cultured in
Neurobasal medium (Gibco) containing 2% B27 supplement (Gibco), 69µg/ml L-glutamine,
25µM beta-mercaptoethanol, and 1% penicillin/streptomycin. Cells were grown for 5 days at
standard incubation conditions [162]. After plating, 24 hours were allotted for cells to attach
before onset of stimulation.
9.5. Setup and Stimulation Protocol
Polymer films were cleaned and sterilized with 70% ethanol prior to use and placed into 24 well
culture dishes with cross-linked poly(dimethyl siloxane) (PDMS) (Sylgard, Dow Corning, MI) O-
ring anchors were placed on top of the films. The O-rings secured the films to the bottom of the
well as seen in Figure 24. Films were coated for 1 hour with 0.2 mg/ml Poly-D-Lysine (PDL) in
borate buffer at 37oC and washed three times with phosphate-buffered saline (PBS).
Figure 24- Set-up of the well plate for seeding.
Cells were tested using four sets of film conditions: unstimulated piezoelectric polymer (US-PZ),
stimulated piezoelectric polymer (S-PZ), unstimulated polymer (US-PV), and stimulated polymer
(S-PV). Cells were seeded and rested for 24 hours before starting mechanical stimulation.
98
Stimulated plates were subjected to vibration from VIBES (Figure 25). VIBES is a custom
vibrating platform as shown in Figure 24 that can be sterilized and placed in the incubator. It
provides a vertical vibration to plates for up to 42 hours before recharging, with selectable
frequency and amplitude. Un-stimulated plates were cultured in the incubator on top of PDMS
mats to isolate them from the vibration of the incubator, and VIBES was isolated itself with the
same type of mats. All films were placed with the dipole orientation up.
Figure 25- VIBES: Polycarbonate base for stimulation of well plate cell cultures.
The stimulation protocol consisted of a mean vibratory frequency at 50 Hz and fixed vibratory
amplitude of approximately 0.3g, to produce a varying electric field on the surfaces of the
polymer. Wells used in the experiment were arranged in a radial configuration around the center
of the plate. For a single experiment, each condition was run at least in duplicate wells. For
experiments with SC neurons grown on PVDF materials vibration was applied continuously for
96 hours.
Variations in vibration frequency and amplitude between wells were approximately 0.5% and
12%, respectively. A MSI® sensor was used to characterize the well plate by gluing it to the
bottom of one of the wells (Figure 26)
99
Figure 26- Mechanical characterization of well-plate stimulation system: contour plot of
frequency (left) and amplitude (right) across the well plate.
9.1.5. Electrical Field Measurement
The piezoelectric response was measured on films coated with gold electrodes sputtered onto
both surfaces. The variation in voltage between the surfaces of the film when vibrated at different
amplitudes was measured using a voltage amplifier and a Hewlett Packard digitizing
oscilloscope. Calculation of charge density such created was made using the dielectric properties
of the film.
Polarization magnitude, P
, can be estimated from the charge density on the surface of the
material, , according to equation (1), where d̂ is the unit vector in the direction of polarization,
in this case, the thickness of the polymer.
dP ˆ
12
The electric displacement, D
, can be described by (2), where o is the permittivity of vacuum
and E
is the electrical field.
PED o
, 13
Due to the boundary conditions, on the surface of the polymer, D
is constant in the direction
normal to the surface. The contribution to D
within the material thus is P
, and outside, the
contribution is E
. Thus,
100
o
PE
14
Therefore, vibratory oscillations modify the net polarization of the piezoelectric material in such a
way that EFs on the PZ film surface changes its magnitude in synchrony with them.
To correlate the electrical field sensed by the cells upon vibration with the volume and
frequencies of VIBES, one of the fabricated piezoelectric films with sputtered electrodes is placed
in the well in the same arrangement used for cell culture.
Knowing the voltage across electrodes as measured by the voltage amplifier and the dielectric
constant of the material, the following relation between volume of VIBES and electrical fields
was found. Calibration of VIBEs was made both in empty wells and in wells with deionized
water. Results in Figure 27 account for the latter.
Figure 27: Mechanical characterization of well plate stimulation at 80Hz, 50Hz and 20Hz.
Therefore, in the presented experiments, when vibrated at 30 points, VIBES at 50 Hz, the S-PV
film exhibited an oscillating electrical field of 50,000 mV/mm peak, corresponding to a voltage
difference between surfaces of 52 mV peak, or a polarization variation of 44.24pC/cm2 peak.
101
9.6. Immunocytochemistry
After 5 DIV (or 4DIV in the case of cortical neurons), neuronal cultures were fixed with 4% PBS
for 30 min at 37 °C. Neurons were permeabilized in blocking solution (5% normal goat serum,
0.02% sodium azide, and 0.1% Triton X-100 in PBS). Neurons were immunostained using a
1:500 dilution of anti-MAP2 (Chemicon, Temecula, CA, USA.) for 1–2 h at room temperature
and then incubated for 1–2 h with a secondary antibody conjugated to fluorophore (Jackson
ImmunoResearch, West Grove, PA, USA).
9.7. Cell analysis and imaging
9.1.7. Neuronal Cell Cultures
Neurons were imaged on an Olympus Optical IX50 microscope (Tokyo, Japan) with a Cooke
Sensicam CCD cooled camera and fluorescence imaging system and ImagePro software
(MediaCybernetics, Silver Spring). Images were inverted to black cells on a white background to
visualize dendrites with greater accuracy. For each well, an average of 15 neurons was analyzed.
Pictures were randomly selected by scanning the well from left to right and from top to bottom
until enough neurons had been acquired.
Analysis was done exclusively on isolated neurons, with sufficient space to grow. Excluded from
the analysis were those that were part of a cluster, binary system, or overlapped with another
neuron, and those with less than a 180o
spatial range. This method was intended to allow the
processes to reach their maximum expansive potential. Processes are designated generically as
neurites.
Division by order was performed using a centrifugal labeling scheme, where processes attached
to the soma have a number of 1 and the number increases at each branch point, as illustrated in
102
Figure 28.
Figure 28- Centrifugal labeling scheme. Processes attached to the soma (green circle) have an
order of 1. At each branch point the order is increased by one.
For neurite analysis, we used Bonfire software, developed in the Firestein laboratory at Rutgers
University [163]. Bonfire is a semi-automatized software program that assists in neurite
branching analysis by interfacing between the plugin NeuronJ [164] of Image J (NIH, Bethesda,
MD, USA) and Neuron Studio [165]. Bonfire uses MATLAB scripts to calculate different metrics
that characterize neurite arborization. It computes number of branch points, terminal points, and
number of processes, in total and broken down by order. It also performs Sholl analysis, with the
possibility of breaking the curve down into branch order as well.
Sholl analysis is a method used to quantify the morphology of dendritic fields. It is performed by
counting the number of neurites that cross each circle from a set of successive concentric circles
drawn around the centroid of the soma of the neuron. We performed Sholl analysis with an inner
ring at 9.3 μm and at ring intervals of 6 μm from the first ring.
Neuronal density was done by counting images at 20X and using the corrective factor 7
cells/mm2. At least 5 images per well were averaged as described in [166].
103
9.8. Statistical analysis
9.1.8. Neuronal Cell Cultures
Statistics were calculated using GraphPad Instat Software (San Diego, CA, USA). The non-
parametric test of Mann–Whitney was performed followed by the appropriate post hoc test. [167],
[168], [169],[170].
9.9. Summary
This chapter has reported the materials and methods for the experiments I describe in Chapter 10.
It has been included both the design, fabrication and characterization I made of the materials, the
design and characterization of the stimulation, the methods of culture for neurons and the
analyses performed in the data to study arborization, elongation and alignment.
104
Chapter 10
Growth of cells on piezoelectric polymer films
10.1. Goal of this chapter
In Chapter 10, I report the effects of piezoelectricity and vibration in the branching features of rat
spinal cord neurons in terms of number of branch points, terminal points, number of processes
and length. Morphology of the neurite arbor is shown by Sholl analysis. I also show the
distribution by order of the process for all these metrics. Then, I present the effect that
piezoelectricity and vibration have on cell density. In all these experiments, the appropriate
controls allowed studying vibration, topography and piezoelectricity independently. Finally, I
discuss the implications of all these results in terms of the potential mechanisms of electrical
sensory.
10.2. Effects of piezoelectric polyvinylidene fluoride in spinal cord
neurons
The effect that vibration of a piezoelectric polymer as polyvinylidene fluoride (PVDF) has in
neuronal branching has been tested using vibrated polarized PVFD (S-PZ), non-vibrated and
polarized PVDF (US-PZ), vibrated non-polarized PVDF (S-PV) and non-vibrated non-polarized
PV (S-PV).
105
Stretched and polarized PVDF displays piezoelectricity upon mechanical deformation. Hence, S-
PZ displays piezoelectricity, US- PZ does not. But vibration itself may affect cells and that needs
to be accounted for. While US-PZ controls for the effects that polarized PZ can have in initial
cell-attachment, S- PV controls for the effects of solely vibration on the cells.
Hereafter I reported the effects of piezoelectricity and vibration on spinal cord neurons. PVDF
was obtained from extrusion, stretched to a ratio of 3.5:1 and polarized at 150MV/m to induce
piezoelectricity, with a piezoelectric constant of d31~19pC/N. Rat spinal cord neurons E16 were
plated at a density of 50,000 cells/cm2 density on films coated for 1h in Poly-D-lysine (PDL).
Stimulation of the plates started after 24 hours of seeding and produced and stress of 0.3g and an
electric field of 50,000mV/mm. Stimulation was delivered continuously for 4 days and cultures
were fixed and stained with MAP2 at 5 DIV.
A B
C D
Figure 29- Spinal cord cultures immunostained with a MAP2 antibody after 5 DIV in the four
conditions: (A) US-PV, (B) S-PV, (C) US-PZ and (D) S-PZ. Scale bar=30 µm.
Representative images of immune-stained neurons for each of the four conditions are shown in
106
Figure 29.
10.1.2. Branching, terminal points and process number per cell
Arborization of neurite fields were quantified according to 3 metrics: (1) number of branch
points, (2) terminal points, and (3) number of processes. Overall results are presented in Figure
30, where the left graph shows the effect of vibration on PV and the right graph shows the effect
of vibration on PZ.
As seen in Figure 30A, vibration of the non-PZ films reduced all of the arborization measures
substantially (25-50%) but not significantly. When applied to PZ films (Figure 30B), vibration
increased all 3 measures: branch points, terminal points, and processes (p<0.001). These data
support the positive effect of piezoelectric growth stimulation of neurites, independent of strictly
mechanical cues, such as structural film properties and vibration
A B
Figure 30- Comparison of branching features between (A) US-PV and S-PV and (B) US-PZ and
S-PZ. *** p<0.001, **** p<0.0001 (Unpaired t-test/Mann-Whitney test). Standard error
depicted.
The results of PV and PZ have been split in two graphs because of possible differences in coating
due to polarization. PZ films were placed with the net dipole facing up. This might have an effect
in the coating of PZ films with the positively charged PDL, which would not be present in the
107
coating of PV films.
10.2.2. Average number of processes per cells split by order
If divided by order, branching in the piezoelectric polymers is significantly bigger. Analysis of
arborization, in term of number of neurites, as a function of branching order is shown in Figure
31. Firstly, vibration did not significantly affect neurite number, at any order, as seen in Figure
31A, although trends were similar to that in Figure 30A. Vibration of PZ films, however,
enhanced branching at all order levels, as seen in Figure 31B. The neurons grown on stimulated
PZ has significantly more primary, secondary, and higher order processes than those grown on
non-stimulated PZ.
A B
Figure 31- Comparison of average number of processes per cell between (A) US-PV and S-PV
and (B) US-PZ and S-PZ. * p<0.05, *** p<0.001, **** p<0.0001 (Unpaired t-test/Mann-Whitney
test). Standard error depicted.
10.3.2. Average length of process per cell by order
Vibration had no significant effect on the length of neurites found in neurons grown on either PV
or PZ materials as shown in Figure 32.
A B
108
Figure 32- Comparison of average number of processes per cell between (A) US-PV and S-PV
and (B) US-PZ and S-PZ. (Unpaired t-test/Mann-Whitney test). Standard error depicted.
10.4.2. Sholl Analysis
The morphology of the arbor field is studied by Sholl analysis. As seen in (Figure 33A, vibration
did not affect intersections of neurons grown on PV films. Neurons grown on the vibrated PZ
film, in contrast, (Figure 33B), exhibited around 85% more intersections. These results again
support the stimulatory effect of piezoelectric activity on neurite outgrowth and branching.
A B
Figure 33- Comparison of Sholls analysis of the total number of neurite intersections between
(A) US-PV and S-PV and (B) US-PZ and S-PZ. Bar indicates significance * p<0.05 (Unpaired t-
test/Mann-Whitney test). Standard error depicted.
109
10.5.2. Neuronal density
Figure 34 shows the effect that vibration and piezoelectricity have in neuronal density after four
days of stimulation. There is a significant increase in neuronal density between US-PZ and S-PZ
but not in the PV materials, suggesting that piezoelectricity might increase the viability of cells,
reduce apoptosis or enhance the expression of integrins and that effect is independent of
vibration.
A B
Figure 34- Comparison of neuronal densities for PV substrates (A) and PZ substrates (B) **
p<0.01 (Unpaired t-test/Mann-Whitney test). Standard error depicted.
10.3. Discussion
Rat spinal neurons were subjected to alternating electrical fields applied directly from their
culture substrate for a total of 4 days, the polarization variation being of 44.24pC/cm2 at its peak.
In order to establish an independent effect of piezoelectric activity on neurite growth, it is
necessary to rule out confounding variables that may affect growth. These include both structural
features of the substrate and the mechanical vibration. It is well known that the first variable
directly influences neurite growth since neurons are exceedingly sensitive to substrate
morphology at the nano-to micro scale [171-173]. The surface of PZ film is fibrillar, with
uniaxially oriented nanofibers on the order of 10 nm, produced during the stretching process as
characterized by [174]. This type of surface morphology provides a permissive substrate for cell
growth and provides cues for growing neurons [175].
110
To eliminate the structural cues as a systematic variable, I restricted the analysis to substrates that
were fabricated identically, coming from the same sample of stretched and polarized PVDF and
differing only in their treatment in the incubator: vibrated or not. These two substrates, labeled
US-PZ and S-PZ in Figure 30B, Figure 31B, and Figure 33B, yielded neurons with significantly
different morphological attributes. Arborization, as measured by branch points, more than
doubled, and the number of terminal processes and intersections increased by approximately 80%
in neurons grown on the S-PZ substrate as compared to those grown on the US-PZ.
To identify whether vibration played an independent role in regulating neuritogenesis, I tested its
effects on neurons grown on non-piezoelectric films. The PZ effect couples a mechanical input to
an electrical output. To establish a baseline effect of mechanical vibration on neuronal plasticity, I
measured the morphological attributes of neurons under the isolated influence of mechanical
vibration, i.e. on polymers not induced with PZ activity via polarization. Results from these two
conditions, labeled S-PV and US-PV, are seen in Figure 30A, Figure 31A, and Figure 33A. As
seen in Figure 30A, vibration does not increase growth but rather reduces all 3 metrics, although
this effect does not reach a statistical significance at the P<0.05 level. The results here presented,
not only point toward the possible detrimental role of vibrations on stimulating growth but also
suggest that the EF produced by the 50 Hz piezoelectric effect may overcome its negative effects
on neurite growth. S-PZ substrates did not show a significant decrease due to vibration.
Contextualization of this result is difficult, as few studies have investigated effects of vibration on
cultured cells. One study on the effect of nano-vibration, at 10 KHz, on neurite growth in PC12
cells showed significant enhancement after several days. The mechanism behind this non-
physiological stimulation was attributed strictly to vibratory enhancement of nerve growth
factor[98]. However, the different range of frequency and cell type may account for the
111
discrepancies Altogether, my results support the idea that EF’s produced by vibrating PZ films
enhance neurite branching at all order levels in neurons on spinal cord neurons
Factors that may affect arborization include cell density, neuronal density, growth factor
concentration, glutamate concentration, substrate stiffness and calcium concetration. Some of
them work in combination and/or have mechanistic feedback loops (reviewed from [75]). Figure
34B shows that neuronal densities increased significantly (more than 100%) in S-PZ substrates as
compared to those grown on US-PZ. However, there is a non-significant decrease in neuronal
density between US-PV and S-PV as shown in Figure 34A. Those findings suggest a possible
role of piezoelectricity in the activation of cell adhesion proteins.
Mechanical properties of substrates have been reported to activate the integrin population [176],
[177]. Integrins show increase expression in neurons grown in stiffer substrates and integrin
expression has been to cause increased neurite branching [178]. Whether the branching observed
in these experiments as a result of piezoelectricity is due to integrin over-expression partly or
entirely remains to see.
One likely mediator of the EF-induced neurite branching are increases in internal calcium,
[Ca++
]I. The crucial role of [Ca++
]I in cellular movement associated with neurite growth is well
known [179], and its moment-to-moment level within microdomains is a primary indicator of cell
growth [180]. Turning direction of growth cones correlates well with local [Ca++
]I as shown by
the action of chemotactic agents, such as netrin, that increase Ca++
selectively within the turning
side of the growth cone. Direct evidence that [Ca++
]i , and specifically voltage-gated Ca
++
channels, play a major roles in EF galvanotropism was shown in mouse neuroblastoma cells,
whose neurite extension and growth cone elongation toward the cathode correlated directly with
cathode-directed elevation in Ca++
levels and depolarization [181]. Calcium dynamics also
112
regulate the activation of integrins, suggesting another potential mechanism of neuronal
outgrowth via Ca++
dynamics. Another likely mediator of neurite growth is cAMP, as suggested
in a study where outgrowth from both DRGs in vitro and spinal sensory nerves in vivo was
enhanced by electrical stimulation at 20 Hz [145].
10.4. Summary
The results here presented show that PZ stimulation of central neurons (spinal cord) promotes
their outgrowth in culture. Using image analysis, I showed that EFs from PZ promote branching
of neurites, increasing their number at all order levels. The total range of the neurite field
increases also upon exposure to piezoelectricity, although the average process length of the
processes is not affected by it. These phenomena are independent of vibration and topography of
the substrates. The observation of increased neuronal density upon piezoelectricity exposure
provides some mechanistic insight of the phenomena into the possible role of integrins
The results from these experiments also show that mild vibrations at 50Hz to neurons of the
central nervous system do not have a significant effect in outgrowth, although the trends suggest
that vibration might decrease branching. Vibrations at 50Hz have not shown to have any effect in
neurite length, total extension of the neurite field or neuronal density.
113
Chapter 11
Conclusion
I have developed: (1) an injury model that allows assessment of treatments for injuries and
diseases that lead to muscle denervation, and (2) a novel treatment paradigm based on
piezoelectric polymers. This injury model is appropriate for spinal cord injuries produced by
contusion and for motor neuron diseases affecting lower motor neurons like spinal muscular
atrophy, amyotrophic lateral sclerosis, progressive muscular atrophy, pseudobulbar palsy and
myasthenia gravis. The injury model I proposed is a bilateral contusion at the thoracic level which
denervates the intercostal muscles. The use as model of injury of such a relevant system like
respiration, encourages the study of problems related to the autonomic and somatic systems in the
context of spinal cord injury. Although I have used a contusion injury, as it is the most relevant
scenario, the model could be easily extended to hemisection with the additional advantage that
the same animal could be used as their own controls.
The technique of assesment of function involves injurying the spinal cord and studying the
electromyographic signal of the denervated intercostal muscle with time. I found that the most
sensitive parameters to identify functional improvement are the turns/second and zero/seconds of
the Interference Pattern Analysis. The signal was studied with EMGvet, a new software I
developed to account for specifics of the electromyographic signal of rats.
114
Many in vivo studies have demonstrated the power of electrode stimulation to enhance neurite
growth and promote axonal regeneration. Oscillating electrical fields delivered by electrodes have
achieved clinical success with spinal cord regeneration [182], and thus, development of new,
more versatile electrical interfaces is an important task. In vitro studies have described the effect
of electrical fields in mammalian neurons but have focused mostly in the peripheral nervous
system. Herein I have shown that electrical stimulation can be delivered directly to cells via a
piezoelectric polymer. The use of polymer substrates for applying electrical charge in vivo may
have advantages over electrodes because polymers can also provide structural and chemical
growth enhancement while delivering electrical fields intimately within the cellular environment.
Piezoelectric polymers represent a versatile platform by which to promote neuronal growth, since
they not only can deliver EFs efficiently, they can be doped with chemical growth factors, and
arranged as scaffolds, providing a complete complement of physical and chemical growth
enhancers.
Herein, I have presented a multidisciplinary approach to the problem of Spinal Cord Injury. I
have provided researchers of the field with a refined set of tools that include software, techniques
and models to test more effectively future therapies. I have also studied the effect in neuronal
branching of piezoelectricity and I have presented the potential of piezoelectric biomaterials for
combinational therapies leading to regeneration of the central nervous system. Importantly, the
work here presented establishes a foundation on which future work in SCI research can be
performed
115
Chapter 12
Bibliography
1. NSCISC Annual Report for the Model Spinal Cord Injury Care Systems 2006, N.S.C.I.S. Center, Editor. 2006.
2. Anderson, K.D., Targeting recovery: priorities of the spinal cord-injured population. J Neurotrauma, 2004. 21(10): p. 1371-83.
3. Inskip, J.A., et al., Autonomic assessment of animals with spinal cord injury: tools, techniques and translation. Spinal Cord, 2008. 47(1): p. 2-35.
4. Basso, D.M., M.S. Beattie, and J.C. Bresnahan, A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma, 1995. 12(1): p. 1-21.
5. Barth, T.M., T.A. Jones, and T. Schallert, Functional subdivisions of the rat somatic sensorimotor cortex. Behavioural Brain Research, 1990. 39(1): p. 73-95.
6. Kunkel-Bagden, E., H.-N. Dai, and B.S. Bregman, Methods to Assess the Development and Recovery of Locomotor Function after Spinal Cord Injury in Rats. Experimental Neurology, 1993. 119(2): p. 153-164.
7. Soblosky, J.S., et al., Ladder beam and camera video recording system for evaluating forelimb and hindlimb deficits after sensorimotor cortex injury in rats. Journal of Neuroscience Methods, 1997. 78(1-2): p. 75-83.
8. Wong, J.K., K. Sharp, and O. Steward, A straight alley version of the BBB locomotor scale. Experimental Neurology, 2009. 217(2): p. 417-420.
9. Vrinten, D.H. and F.F.T. Hamers, [`]CatWalk' automated quantitative gait analysis as a novel method to assess mechanical allodynia in the rat; a comparison with von Frey testing. Pain, 2003. 102(1-2): p. 203-209.
10. Maurissen, J.P.J., et al., Factors affecting grip strength testing. Neurotoxicology and Teratology. 25(5): p. 543-553.
11. Schallert, T., et al., CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury. Neuropharmacology, 2000. 39(5): p. 777-787.
12. Metz, G.A.S. and I.Q. Whishaw, Skilled reaching an action pattern: stability in rat (Rattus norvegicus) grasping movements as a function of changing food pellet size. Behavioural Brain Research, 2000. 116(2): p. 111-122.
13. Johansson, R.S., Å.B. Vallbo, and G. Westling, Thresholds of mechanosensitive afferents in the human hand as measured with von Frey hairs. Brain Research, 1980. 184(2): p. 343-351.
14. Stålberg, E., et al., Multi-MUP EMG analysis -- a two year experience in daily clinical work. Electroencephalography and Clinical
116
Neurophysiology/Electromyography and Motor Control, 1995. 97(3): p. 145-154. 15. Fuglsang-Frederiksen, A., The utility of interference pattern analysis. Muscle &
Nerve, 2000. 23(1): p. 18-36. 16. Finsterer, J., EMG-interference pattern analysis. Journal of Electromyography
and Kinesiology, 2001. 11(4): p. 231-246. 17. Willison, R.G., Analysis of electrical activity in healthy and dystrophic muscle in
man. Journal of Neurology, Neurosurgery & Psychiatry, 1964. 27(5): p. 386-394. 18. Nandedkar, S.D., D.B. Sanders, and E.V. Stålberg, Automatic analysis of the
electromyographic interference pattern. Part I: Development of quantitative features. Muscle & Nerve, 1986. 9(5): p. 431-439.
19. Nandedkar, S.D., D.B. Sanders, and E.V. Stålberg, Automatic analysis of the electromyographic interference pattern. Part II: Findings in control subjects and in some neuromuscular diseases. Muscle & Nerve, 1986. 9(6): p. 491-500.
20. Zalewska, E. and I. Hausmanowa-Petrusewicz, Effectiveness of motor unit potentials classification using various parameters and indexes. Clinical Neurophysiology, 2000. 111(8): p. 1380-1387.
21. Fuglsang-Frederiksen, A., T. Smith, and H. Hgenhaven, Motor unit firing intervals and other parameters of electrical activity in normal and pathological muscle. Journal of the Neurological Sciences, 1987. 78(1): p. 51-62.
22. Richard D. Ball, M., PhD, Basics of Needle Electromyography: An AAEE Workshop. 1985, AAEE: Rochester, MN.
23. Xu, Y., et al., Needle electromyography of the rectus abdominis in patients with amyotrophic lateral sclerosis. Muscle & Nerve, 2007. 35(3): p. 383-385.
24. Bruce, E.N., ed. Biomedical Signal Processing and Signal Modelling. ed. Wiley. 2001.
25. Nandedkar, S.D., D.B. Sanders, and E.V. Stålberg, Simulation and analysis of the electromyographic interference pattern in normal muscle. Part I: Turns and amplitude measurements. Muscle & Nerve, 1986. 9(5): p. 423-430.
26. Nandedkar, S.D., D.B. Sanders, and E.V. Stålberg, Simulation and analysis of the electromyographic interference pattern in normal muscle. Part II: Activity, upper centile amplitude, and number of small segments. Muscle & Nerve, 1986. 9(6): p. 486-490.
27. Nandedkar, S.D. and D.B. Sanders, Measurement of the amplitude of the EMG envelope. Muscle & Nerve, 1990. 13(10): p. 933-938.
28. De Troyer, A., P.A. Kirkwood, and T.A. Wilson, Respiratory Action of the Intercostal Muscles. Physiological Reviews, 2005. 85(2): p. 717-756.
29. De Troyer, A. and S. Kelly, Chest wall mechanics in dogs with acute diaphragm paralysis. Journal of Applied Physiology, 1982. 53(2): p. 373-379.
30. De Troyer, A., A. Legrand, and T.A. Wilson, Respiratory mechanical advantage of the canine external and internal intercostal muscles. The Journal of Physiology, 1999. 518(1): p. 283-289.
31. Legrand, A., et al., Rostrocaudal gradient of electrical activation in the parasternal intercostal muscles of the dog. The Journal of Physiology, 1996. 495(Pt 1): p. 247-254.
32. Le Bars, P. and B. Duron, Are the external and internal intercostal muscles synergist or antagonist in the cat? Neuroscience Letters, 1984. 51(3): p. 383-386.
33. Wilson, T.A., et al., Respiratory effects of the external and internal intercostal muscles in humans. The Journal of Physiology, 2001. 530(2): p. 319-330.
34. Hamberger, G.E., ed. De Respirationis Mechanismo et usu Gennino. Vol. . 1749, Cristoph Croeker: Iena.
35. Greer, J.J. and T.P. Martin, Distribution of muscle fiber types and EMG activity in
117
cat intercostal muscles. Journal of Applied Physiology, 1990. 69(4): p. 1208-1211.
36. Kelsen, S.G., et al., Structure of parasternal intercostal muscles in the adult hamster: topographic effects. Journal of Applied Physiology, 1993. 75(3): p. 1150-1154.
37. Tsering, C., Demonstration of Segmental Arrangement of Thoracic Spinal Motor Neurons Using Lipophilic Dyes. Developmental Neuroscience, 1992. 14(4): p. 308-311.
38. Stirling, R.V., et al., The segmental precision of the motor projection to the intercostal muscles in the developing chicken embryo. Anatomy and Embryology, 1995. 191(5): p. 397-406.
39. Izumi, A. and M.Y. Kida, Segmental distribution of the motoneurons innervating trunk muscles in the spinal cord of the cat and rat. Neuroscience Research, 1998. 30(3): p. 247-255.
40. Giraudin, A., et al., Intercostal and Abdominal Respiratory Motoneurons in the Neonatal Rat Spinal Cord: Spatiotemporal Organization and Responses to Limb Afferent Stimulation. Journal of Neurophysiology, 2008. 99(5): p. 2626-2640.
41. Üstüner, T., et al., Rat intercostal nerves as experimental nerve grafts. Microsurgery, 1996. 17(3): p. 128-130.
42. Zhang, Y.W., J. Denham, and R.S. Thies, Oligodendrocyte Progenitor Cells Derived from Human Embryonic Stem Cells Express Neurotrophic Factors. Stem Cells and Development, 2006. 15(6): p. 943-952.
43. Kerr, D.A., et al., Human Embryonic Germ Cell Derivatives Facilitate Motor Recovery of Rats with Diffuse Motor Neuron Injury. The Journal of Neuroscience, 2003. 23(12): p. 5131-5140.
44. Lu, P., et al., Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury. Experimental Neurology, 2003. 181(2): p. 115-129.
45. Keirstead, H.S., et al., Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitor Cell Transplants Remyelinate and Restore Locomotion after Spinal Cord Injury. The Journal of Neuroscience, 2005. 25(19): p. 4694-4705.
46. Sharp, J., et al., Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitor Cell Transplants Improve Recovery after Cervical Spinal Cord Injury. STEM CELLS, 2010. 28(1): p. 152-163.
47. Rossi, S.L., et al., Histological and functional benefit following transplantation of motor neuron progenitors to the injured rat spinal cord. PLoS One, 2010. 5(7): p. e11852.
48. Finsterer, J., EMG-interference pattern analysis. J Electromyogr Kinesiol, 2001. 11(4): p. 231-46.
49. Feinberg, J., EMG: myths and facts. HSS J, 2006. 2(1): p. 19-21. 50. Keirstead, H.S., et al., Human embryonic stem cell-derived oligodendrocyte
progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J Neurosci, 2005. 25(19): p. 4694-705.
51. Cloutier, F., et al., Transplantation of human embryonic stem cell-derived oligodendrocyte progenitors into rat spinal cord injuries does not cause harm. Regen Med, 2006. 1(4): p. 469-79.
52. Lee, J.K., et al., Effect of spinal cord injury severity on alterations of the H-reflex. Exp Neurol, 2005. 196(2): p. 430-40.
53. Bamber, N.I., et al., Neurotrophins BDNF and NT-3 promote axonal re-entry into the distal host spinal cord through Schwann cell-seeded mini-channels. European Journal of Neuroscience, 2001. 13(2): p. 257-268.
118
54. Fouad, K., et al., Combining Schwann Cell Bridges and Olfactory-Ensheathing Glia Grafts with Chondroitinase Promotes Locomotor Recovery after Complete Transection of the Spinal Cord. The Journal of Neuroscience, 2005. 25(5): p. 1169-1178.
55. Nomura, H., et al., Extramedullary Chitosan Channels Promote Survival of Transplanted Neural Stem and Progenitor Cells and Create a Tissue Bridge After Complete Spinal Cord Transection. Tissue Engineering Part A, 2008. 14(5): p. 649-665.
56. Taylor, S.J. and S.E. Sakiyama-Elbert, Effect of controlled delivery of neurotrophin-3 from fibrin on spinal cord injury in a long term model. Journal of Controlled Release, 2006. 116(2): p. 204-210.
57. Tobias, C.A., et al., Alginate Encapsulated BDNF-Producing Fibroblast Grafts Permit Recovery of Function after Spinal Cord Injury in the Absence of Immune Suppression. Journal of Neurotrauma, 2005. 22(1): p. 138-156.
58. Benowitz, L. and Y. Yin, Rewiring the injured CNS: Lessons from the optic nerve. Experimental Neurology, 2008. 209(2): p. 389-398.
59. Busch, S.A. and J. Silver, The role of extracellular matrix in CNS regeneration. Current Opinion in Neurobiology, 2007. 17(1): p. 120-127.
60. Lu, P. and M.H. Tuszynski, Growth factors and combinatorial therapies for CNS regeneration. Experimental Neurology, 2008. 209(2): p. 313-320.
61. Horner, P.J. and F.H. Gage, Regenerating the damaged central nervous system. Nature, 2000. 407(6807): p. 963-970.
62. Bunge, M.B., Bridging areas of injury in the spinal cord. Neuroscientist, 2001. 7(4): p. 325-339.
63. Bos, R.R., et al., Degradation of and tissue reaction to biodegradable poly(L-lactide) for use as internal fixation of fractures: a study in rats. Biomaterials, 1991. 12(1): p. 32-6.
64. Cai, J., et al., Permeable guidance channels containing microfilament scaffolds enhance axon growth and maturation. J Biomed Mater Res A, 2005. 75(2): p. 374-86.
65. Cam, D., S.H. Hyon, and Y. Ikada, Degradation of high molecular weight poly(L-lactide) in alkaline medium. Biomaterials, 1995. 16(11): p. 833-43.
66. Evans, G.R., et al., Clinical long-term in vivo evaluation of poly(L-lactic acid) porous conduits for peripheral nerve regeneration. J Biomater Sci Polym Ed, 2000. 11(8): p. 869-78.
67. Evans, G.R., et al., In vivo evaluation of poly(L-lactic acid) porous conduits for peripheral nerve regeneration. Biomaterials, 1999. 20(12): p. 1109-15.
68. Belkas, J.S., M.S. Shoichett, and R. Midha, Peripheral nerve regeneration through guidance tubes. Neurological Research, 2004. 26(2): p. 151-160.
69. Mackinnon, S.E., et al., Peripheral Nerve Allograft: An Assessment of Regeneration across Pretreated Nerve Allografts. Neurosurgery, 1984. 15(5): p. 690-693.
70. Merle, M., et al., Complications from silicon-polymer intubulation of nerves. Microsurgery, 1989. 10(2): p. 130-133.
71. Park, A. and L.G. Cima, In vitro cell response to differences in poly-L-lactide crystallinity. J Biomed Mater Res, 1996. 31(1): p. 117-30.
72. Nomura, H., C.H. Tator, and M.S. Shoichet, Bioengineered strategies for spinal cord repair. Journal of Neurotrauma, 2006. 23(3-4): p. 496-507.
73. Willerth, S.M. and S.E. Sakiyama-Elbert, Approaches to neural tissue engineering using scaffolds for drug delivery. Advanced Drug Delivery Reviews, 2007. 59(4-5): p. 325-338.
119
74. Zhong, Y. and R.V. Bellamkonda, Biomaterials for the central nervous system. Journal of The Royal Society Interface, 2008. 5(26): p. 957-975.
75. Previtera, M., et al., Regulation of Dendrite Arborization by Substrate Stiffness is Mediated by Glutamate Receptors. Annals of Biomedical Engineering, 2010. 38(12): p. 3733-3743.
76. Geiger, B., J.P. Spatz, and A.D. Bershadsky, Environmental sensing through focal adhesions. Nat Rev Mol Cell Biol, 2009. 10(1): p. 21-33.
77. Sundararaghavan, H.G., et al., Neurite growth in 3D collagen gels with gradients of mechanical properties. Biotechnology and Bioengineering, 2009. 102(2): p. 632-643.
78. Usoskin, D., et al., En masse in vitro functional profiling of the axonal mechanosensitivity of sensory neurons. Proceedings of the National Academy of Sciences, 2010. 107(37): p. 16336-16341.
79. Bhattacharya, M.R.C., et al., Radial stretch reveals distinct populations of mechanosensitive mammalian somatosensory neurons. Proceedings of the National Academy of Sciences, 2008. 105(50): p. 20015-20020.
80. Nishimura, S., et al., Responses of single-ventricular myocytes to dynamic axial stretching. Progress in Biophysics and Molecular Biology. 97(2-3): p. 282-297.
81. Hill, T.E., G.T. Desmoulin, and C.J. Hunter, Is vibration truly an injurious stimulus in the human spine? Journal of Biomechanics, 2009. 42(16): p. 2631-2635.
82. Sarkisyan, S.G., Effect of Long-Lasting Vibration on the Impulse Activity of Neurons of the Inferior Vestibular Nucleus. Neurophysiology, 2005. 37(1): p. 29-35.
83. Rittweger, J., et al., Treatment of Chronic Lower Back Pain with Lumbar Extension and Whole-Body Vibration Exercise: A Randomized Controlled Trial. Spine, 2002. 27(17): p. 1829-1834.
84. Desmoulin, G.T., N.I. Yasin, and D.W. Chen, Spinal Mechanisms of Pain Control. The Clinical Journal of Pain, 2007. 23(7): p. 576-585 10.1097/AJP.0b013e3180e00eb8.
85. Cardinale, M. and J. Wakeling, Whole body vibration exercise: are vibrations good for you? British Journal of Sports Medicine, 2005. 39(9): p. 585-589.
86. RøNNESTAD, B.R., Comparing the Performance-Enhancing Effects of Squats on A Vibration Platform With Conventional Squats in Recreationally Resistance-Trained Men. The Journal of Strength & Conditioning Research, 2004. 18(4): p. 839-845.
87. Jacobsen, K.X. and W.A. Staines, Vibration enhancement of slide-mounted immunofluorescence staining. Journal of Neuroscience Methods, 2004. 137(1): p. 71-77.
88. Kasra, M., et al., Effect of dynamic hydrostatic pressure on rabbit intervertebral disc cells. Journal of Orthopaedic Research, 2003. 21(4): p. 597-603.
89. Desmoulin, G.T., C.R. Reno, and C.J. Hunter, Free Axial Vibrations At 0 to 200 Hz Positively Affect Extracellular Matrix Messenger Ribonucleic Acid Expression in Bovine Nucleus Pulposi. Spine, 2010. 35(15): p. 1437-1444 10.1097/BRS.0b013e3181c2a8ec.
90. Wang, C.-Z., et al., Low-magnitude vertical vibration enhances myotube formation in C2C12 myoblasts. Journal of Applied Physiology, 2010. 109(3): p. 840-848.
91. Liu, J., et al., Biosynthetic response of cultured articular chondrocytes to mechanical vibration. Research in Experimental Medicine, 2001. 200(3): p. 183-193.
92. Holguin, N., et al., Re: Short applications of very low-magnitude vibrations
120
attenuate expansion of the intervertebral disc during extended bed rest Reply. Spine Journal, 2010. 10(4): p. 364-365.
93. Judex, S., S. Gupta, and C. Rubin, Regulation of mechanical signals in bone. Orthodontics & Craniofacial Research, 2009. 12(2): p. 94-104.
94. Xie, L.Q., C. Rubin, and S. Judex, Enhancement of the adolescent murine musculoskeletal system using low-level mechanical vibrations. Journal of Applied Physiology, 2008. 104(4): p. 1056-1062.
95. Garman, R., et al., Low-level Accelerations applied in the absence of weight bearing can enhance trabecular bone formation. Journal of Orthopaedic Research, 2007. 25(6): p. 732-740.
96. Shreiber, D., H. Hao, and R. Elias, Probing the influence of myelin and glia on the tensile properties of the spinal cord. Biomechanics and Modeling in Mechanobiology, 2009. 8(4): p. 311-321.
97. Bryan, D.J., et al., Enhanced peripheral nerve regeneration through a poled bioresorbable poly(lactic-co-glycolic acid) guidance channel. J Neural Eng, 2004. 1(2): p. 91-8.
98. Ito, Y., et al., Effects of Vibration on Differentiation of Cultured PC12 Cells. Biotechnology and Bioengineering, 2011. 108(3): p. 592-599.
99. Fine, E.G., et al., Improved nerve regeneration through piezoelectric vinylidenefluoride-trifluoroethylene copolymer guidance channels. Biomaterials, 1991. 12(8): p. 775-80.
100. Kotwal, A. and C.E. Schmidt, Electrical stimulation alters protein adsorption and nerve cell interactions with electrically conducting biomaterials. Biomaterials, 2001. 22(10): p. 1055-1064.
101. Reid, B., et al., Wound healing in rat cornea: the role of electric currents. Faseb Journal, 2005. 19(3): p. 379-386.
102. Jaffe, L.F. and J.W. Vanable Jr, Electric fields and wound healing. Clinics in Dermatology. 2(3): p. 34-44.
103. Chiang, M., E.J. Cragoe, and J.W. Vanable, Intrinsic electric fields promote epithelization of wounds in the newt, Notophthalmus viridescens. Developmental Biology, 1991. 146(2): p. 377-385.
104. Robinson, K.R. and P. Cormie, Electric field effects on human spinal injury: Is there a basis in the in vitro studies? Developmental Neurobiology, 2008. 68(2): p. 274-280.
105. McCaig, C.D., et al., Has electrical growth cone guidance found its potential? Trends in Neurosciences, 2002. 25(7): p. 354-359.
106. Fang, K.S., et al., Epidermal growth factor receptor relocalization and kinase activity are necessary for directional migration of keratinocytes in DC electric fields. Journal of Cell Science, 1999. 112(12): p. 1967-1978.
107. Nuccitelli, R., A Role for Endogenous Electric Fields in Wound Healing, in Current Topics in Developmental Biology, G.P. Schatten, Editor. 2003, Academic Press. p. 1-26.
108. Li, X. and J. Kolega, Effects of Direct Current Electric Fields on Cell Migration and Actin Filament Distribution in Bovine Vascular Endothelial Cells. Journal of Vascular Research, 2002. 39(5): p. 391-404.
109. Zhao, M., J.V. Forrester, and C.D. McCaig, A small, physiological electric field orients cell division. Proceedings of the National Academy of Sciences of the United States of America, 1999. 96(9): p. 4942-4946.
110. Yao, L., et al., Small applied electric fields guide migration of hippocampal neurons. Journal of Cellular Physiology, 2008. 216(2): p. 527-535.
111. Alexander, J.K., B. Fuss, and R.J. Colello, Electric field-induced astrocyte
121
alignment directs neurite outgrowth. Neuron Glia Biology, 2006. 2: p. 93-103. 112. Moriarty, L.J. and R.B. Borgens, An oscillating extracellular voltage gradient
reduces the density and influences the orientation of astrocytes in injured mammalian spinal cord. Journal of Neurocytology, 2001. 30(1): p. 45-57.
113. Haas, K., J.L. Li, and H.T. Cline, AMPA receptors regulate experience-dependent dendritic arbor growth in vivo. Proceedings of the National Academy of Sciences of the United States of America, 2006. 103(32): p. 12127-12131.
114. Sorensen, S.A. and E.W. Rubel, The level and integrity of synaptic input regulates dendrite structure. Journal of Neuroscience, 2006. 26(5): p. 1539-1550.
115. Borgens, R.B., E. Roederer, and M.J. Cohen, Enhanced spinal cord regeneration in lamprey by applied electric fields. Science, 1981. 213(4508): p. 611-7.
116. McCaig, C.D., et al., Controlling cell behavior electrically: Current views and future potential. Physiological Reviews, 2005. 85(3): p. 943-978.
117. Kerns, J.M., et al., Electrical stimulation of nerve regeneration in the rat: the early effects evaluated by a vibrating probe and electron microscopy. Neuroscience, 1991. 40(1): p. 93-107.
118. Song, B., et al., Nerve regeneration and wound healing are stimulated and directed by an endogenous electrical field in vivo. Journal of Cell Science, 2004. 117(20): p. 4681-4690.
119. Prasad, S., et al., Electric field assisted patterning of neuronal networks for the study of brain functions. Biomedical Microdevices, 2003. 5(2): p. 125-137.
120. Brushart, T.M., et al., Electrical stimulation promotes motoneuron regeneration without increasing its speed or conditioning the neuron. Journal of Neuroscience, 2002. 22(15): p. 6631-6638.
121. Grill, W.M., et al., At the interface: Convergence of neural regeneration and neural prostheses for restoration of function. Journal of Rehabilitation Research and Development, 2001. 38(6): p. 633-639.
122. Shen, N.J. and S.C. Wang, Using a direct current electrical field to promote spinal-cord regeneration. Journal of Reconstructive Microsurgery, 1999. 15(6): p. 427-431.
123. Rajnicek, A.M., K.R. Robinson, and C.D. McCaig, The direction of neurite growth in a weak DC electric field depends on the substratum: Contributions of adhesivity and net surface charge. Developmental Biology, 1998. 203(2): p. 412-423.
124. Schmidt, C.E., et al., Stimulation of neurite outgrowth using an electrically conducting polymer. Proceedings of the National Academy of Sciences of the United States of America, 1997. 94(17): p. 8948-8953.
125. Rajnicek, A.M., N.A. Gow, and C.D. McCaig, Electric field-induced orientation of rat hippocampal neurones in vitro. Exp Physiol, 1992. 77(1): p. 229-32.
126. McCaig, C.D., Nerve branching is induced and oriented by a small applied electric field. J Cell Sci, 1990. 95 ( Pt 4): p. 605-15.
127. Lu, J. and P. Waite, Advances in spinal cord regeneration. Spine, 1999. 24(9): p. 926-930.
128. Borgens, R.B., The role of natural and applied electric fields in neuronal regeneration and development. Prog Clin Biol Res, 1986. 210: p. 239-50.
129. Borgens, R.B. and D.M. Bohnert, The responses of mammalian spinal axons to an applied DC voltage gradient. Exp Neurol, 1997. 145(2 Pt 1): p. 376-89.
130. Borgens, R.B., et al., An imposed oscillating electrical field improves the recovery of function in neurologically complete paraplegic dogs. J Neurotrauma, 1999. 16(7): p. 639-57.
131. Shapiro, S., et al., Oscillating field stimulation for complete spinal cord injury in
122
humans: a Phase 1 trial. Journal of Neurosurgery-Spine, 2005. 2(1): p. 3-10. 132. McCaig, C.D., Spinal neurite reabsorption and regrowth in vitro depend on the
polarity of an applied electric field. Development, 1987. 100(1): p. 31-41. 133. Marsh, G. and H.W. Beams, In vitro control of growing check nerve fibers by
applied electric currents. Journal of Cellular and Comparative Physiology, 1946. 27(3): p. 139-157.
134. Jaffe, L.F. and M.M. Poo, Neurites grow faster towards the cathode than the anode in a steady field. J Exp Zool, 1979. 209(1): p. 115-28.
135. Hinkle, L., C.D. McCaig, and K.R. Robinson, The direction of growth of differentiating neurones and myoblasts from frog embryos in an applied electric field. The Journal of Physiology, 1981. 314(1): p. 121-135.
136. Patel, N. and M.M. Poo, Orientation of neurite growth by extracellular electric fields. J Neurosci, 1982. 2(4): p. 483-96.
137. McCaig, C.D., L. Sangster, and R. Stewart, Neurotrophins enhance electric field-directed growth cone guidance and directed nerve branching. Developmental Dynamics, 2000. 217(3): p. 299-308.
138. Davenport, R.W., et al., A sensory role for neuronal growth cone filopodia. Nature, 1993. 361(6414): p. 721-724.
139. Cork, R.J., et al., The growth of PC-12 neurites is biased towards the anode of an applied electrical field. Journal of Neurobiology, 1994. 25(12): p. 1509-1516.
140. Cormie, P. and K.R. Robinson, Embryonic zebrafish neuronal growth is not affected by an applied electric field in vitro. Neuroscience Letters, 2007. 411(2): p. 128-132.
141. George, J., et al., Sodium Channel Activation Augments NMDA Receptor Function and Promotes Neurite Outgrowth in Immature Cerebrocortical Neurons. Journal of Neuroscience, 2009. 29(10): p. 3288-3301.
142. Zheng, J.Q. and M.M. Poo, Calcium signaling in neuronal motility. Annual Review of Cell and Developmental Biology, 2007. 23: p. 375-404.
143. Bikson, M., et al., Effects of uniform extracellular DC electric fields on excitability in rat hippocampal slices in vitro. The Journal of Physiology, 2004. 557(1): p. 175-190.
144. Vivó, M., et al., Immediate electrical stimulation enhances regeneration and reinnervation and modulates spinal plastic changes after sciatic nerve injury and repair. Experimental Neurology, 2008. 211(1): p. 180-193.
145. Udina, E., et al., Electrical stimulation of intact peripheral sensory axons in rats promotes outgrowth of their central projections. Experimental Neurology, 2008. 210(1): p. 238-247.
146. Collazos-Castro, J.E., et al., Bioelectrochemical control of neural cell development on conducting polymers. Biomaterials, 2010. 31(35): p. 9244-9255.
147. Ghasemi-Mobarakeh, L., et al., Electrical Stimulation of Nerve Cells Using Conductive Nanofibrous Scaffolds for Nerve Tissue Engineering. Tissue Engineering Part A, 2009. 15(11): p. 3605-3619.
148. Schmidt, C.E., et al., Stimulation of neurite outgrowth using an electrically
conducting polymer. Proceedings of the National Academy of Sciences of the United States of America, 1997. 94(17): p. 8948-8953.
149. Park, J.S., et al., Electrical Pulsed Stimulation of Surfaces Homogeneously Coated with Gold Nanoparticles to Induce Neurite Outgrowth of PC12 Cells. Langmuir, 2009. 25(1): p. 451-457.
150. Rivers, T.J., T.W. Hudson, and C.E. Schmidt, Synthesis of a novel, biodegradable electrically conducting polymer for biomedical applications. Advanced Functional Materials, 2002. 12(1): p. 33-37.
123
151. Collier, J.H., et al., Synthesis and characterization of polypyrrole-hyaluronic acid composite biomaterials for tissue engineering applications. Journal of Biomedical Materials Research, 2000. 50(4): p. 574-584.
152. Wan, Y., H. Wu, and D.J. Wen, Porous-conductive chitosan scaffolds for tissue engineering, 1 - Preparation and characterization. Macromolecular Bioscience, 2004. 4(9): p. 882-890.
153. Valentini, R.F., et al., Polymer electret guidance channels enhance peripheral nerve regeneration in mice. Brain Res, 1989. 480(1-2): p. 300-4.
154. Sabatini, A.M. and P. Dario, Measuring Technique for Characterizing the Electrical-Properties of Piezoelectric Tubular Devices. Electronics Letters, 1993. 29(24): p. 2096-2097.
155. Valentini, R.F., et al., Electrically charged polymeric substrates enhance nerve fibre outgrowth in vitro. Biomaterials, 1992. 13(3): p. 183-90.
156. Aebischer, P., et al., Piezoelectric nerve guidance channels enhance peripheral nerve regeneration. ASAIO Trans, 1987. 33(3): p. 456-8.
157. Young, T.H., et al., Immobilization of L-lysine on microporous PVDF membranes for neuron culture. J Biomater Sci Polym Ed, 2009. 20(5-6): p. 703-20.
158. Fukada, E., History and recent progress in piezoelectric polymers. IEEE Trans Ultrason Ferroelectr Freq Control, 2000. 47(6): p. 1277-90.
159. Tajitsu, Y., Piezoelectricity of chiral polymeric fiber and its application in biomedical engineering. IEEE Trans Ultrason Ferroelectr Freq Control, 2008. 55(5): p. 1000-8.
160. Takase, Y., J.I. Scheinbeim, and B.A. Newman, EFFECTS OF ANNEALING ON THE POLARIZATION SWITCHING OF PHASE-I POLY(VINYLIDENE FLUORIDE). Journal of Polymer Science Part B-Polymer Physics, 1990. 28(9): p. 1599-1609.
161. Mark, ed. Polymer Data Handbook. 1999, Oxford Univ. Press. 162. Du, Y., et al., Astroglia-mediated effects of uric acid to protect spinal cord
neurons from glutamate toxicity. Glia, 2007. 55(5): p. 463-72. 163. Kutzing, M.K., et al., Automated Sholl Analysis of Digitized Neuronal Morphology
at Multiple Scales. J Vis Exp, 2010(45): p. e2354. 164. Meijering, E., et al., Design and validation of a tool for neurite tracing and
analysis in fluorescence microscopy images. Cytometry Part A, 2004. 58A(2): p. 167-176.
165. Wearne, S.L., et al., New techniques for imaging, digitization and analysis of three-dimensional neural morphology on multiple scales. Neuroscience, 2005. 136(3): p. 661-680.
166. X. Jiang, P.C.G., B. Li, Y. Du, M.K. Kutzing, M.L. Previtera, N.A. Langrana and B.L. Firestein, Cell growth in response to mechanical stiffness is affected by neuron-astroglia interactions. The Open Neuroscience Journal, 2007. 1: p. 7–14.
167. Akum, B.F., et al., Cypin regulates dendrite patterning in hippocampal neurons by promoting microtubule assembly. Nat Neurosci, 2004. 7(2): p. 145-152.
168. Charych, E.I., et al., Activity-Independent Regulation of Dendrite Patterning by Postsynaptic Density Protein PSD-95. The Journal of Neuroscience, 2006. 26(40): p. 10164-10176.
169. Carrel, D., et al., NOS1AP Regulates Dendrite Patterning of Hippocampal Neurons through a Carboxypeptidase E-Mediated Pathway. The Journal of Neuroscience, 2009. 29(25): p. 8248-8258.
170. Chen, M., et al., A Novel Role for Snapin in Dendrite Patterning: Interaction with Cypin. Mol. Biol. Cell, 2005. 16(11): p. 5103-5114.
171. Kim, I.A., et al., Effects of mechanical stimuli and microfiber-based substrate on
124
neurite outgrowth and guidance. Journal of Bioscience and Bioengineering, 2006. 101(2): p. 120-126.
172. Yao, L., et al., Orienting neurite growth in electrospun fibrous neural conduits. J Biomed Mater Res B Appl Biomater, 2009. 90(2): p. 483-91.
173. Schulze, S., et al., Morphological Differentiation of Neurons on Microtopographic Substrates Fabricated by Rolled-Up Nanotechnology. Advanced Engineering Materials, 2010. 12(9): p. B558-B564.
174. Linda C. Sawyer, D.T.G., and Gregory F. Meyers, Polymer Microscopy: Characterization and Evaluation of Materials. 3rd ed. 2008: Springer
175. Webster, T.J., et al., Nano-biotechnology: carbon nanofibres as improved neural and orthopaedic implants. Nanotechnology, 2004. 15(1): p. 48-54.
176. Georges, P.C. and P.A. Janmey, Cell type-specific response to growth on soft materials. Journal of Applied Physiology, 2005. 98(4): p. 1547-1553.
177. Yeung, T., et al., Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell Motility and the Cytoskeleton, 2005. 60(1): p. 24-34.
178. Moresco, E.M.Y., et al., Integrin-Mediated Dendrite Branch Maintenance Requires Abelson (Abl) Family Kinases. The Journal of Neuroscience, 2005. 25(26): p. 6105-6118.
179. Yao, L., et al., Multichanneled collagen conduits for peripheral nerve regeneration: design, fabrication, and characterization. Tissue Eng Part C Methods, 2010. 16(6): p. 1585-96.
180. Gomez, T.M. and J.Q. Zheng, The molecular basis for calcium-dependent axon pathfinding. Nature Reviews Neuroscience, 2006. 7(2): p. 115-125.
181. Bedlack, R.S., Jr., M. Wei, and L.M. Loew, Localized membrane depolarizations and localized calcium influx during electric field-guided neurite growth. Neuron, 1992. 9(3): p. 393-403.
182. Doleski, S., et al., NGF release from thermo-responsive collagen-polyNIPAam polymer networks supports neuronal cell growth and differentiation. J Biomed Mater Res A, 2010. 94(2): p. 457-65.
183. Çakmak, M., et al., Effect of paralysis of the abdominal wall muscles by botulinum A toxin to intraabdominal pressure: an experimental study. J Pediatric Surgery, 2006. 41(4): p. 821-825.