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Corneal neurotization maintains corneal epithelial integrity and restores nerve-derived
peptides in a rat model of neurotrophic keratopathy
by
Kira Antonyshyn
A thesis submitted in conformity with the requirements for the degree of Master of Science
Institute of Medical Science
University of Toronto
ii
Corneal neurotization maintains corneal epithelial integrity and restores nerve-derived peptides in a rat model of
neurotrophic keratopathy
Kira Antonyshyn
Master of Science
Institute of Medical Science University of Toronto
2019
Abstract PURPOSE: Investigate effect of corneal neurotization on epithelial integrity and
restoration of nerve-derived peptides in denervated rat corneas.
METHODS: Three rat corneal conditions were assessed (n=45): denervation,
denervation + neurotization, normal innervation. Corneal epithelial and stromal
thicknesses were evaluated using Hematoxylin and Eosin staining. Corneal ulceration
and perforation were assessed under a Wood’s lamp and normal light, respectively.
Immunohistochemistry and western blot were used to evaluate Substance P (SP) and
calcitonin gene-related peptide (CGRP) presence.
RESULTS: Where significance is p<0.05, central epithelium was thinner in the
denervated compared to the denervated + neurotized and normally innervated corneas.
The denervated stroma was thinner than the normally innervated, but not the denervated
+ neurotized stroma. Neurotization protected the denervated cornea from ulceration and
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perforation. Neurotization restored the denervated cornea with SP and CGRP, co-
localized with axons innervating the cornea.
CONCLUSION: Corneal neurotization restores epithelial integrity and nerve-derived
peptides in the denervated cornea.
iv
Table of Contents
ABSTRACT ......................................................................................................................................... II
CONTRIBUTIONS ............................................................................................................................ VIII
LIST OF ABBREVIATIONS ................................................................................................................... IX
LIST OF FIGURES ................................................................................................................................ X
LIST OF TABLES ............................................................................................................................... XII
1.1 PREAMBLE ...................................................................................................................................2
1.2 THESIS ORGANIZATION .................................................................................................................3
1.3 CORNEAL ANATOMY AND PHYSIOLOGY ........................................................................................4
I - INTRODUCTION..........................................................................................................................4 II - CORNEAL ANATOMY ...............................................................................................................4 III - CORNEAL INNERVATION ..................................................................................................... 12 ........................................................................................................................................................ 15 IV - CORNEAL EPITHELIAL MAINTENANCE AND REPAIR ..................................................... 16 V - LIMBAL STEM CELLS ............................................................................................................ 19 VI - SCHWANN CELLS IN THE CORNEA .................................................................................... 22
1.4 NEUROTROPHIC KERATOPATHY .................................................................................................. 24
I. INTRODUCTION......................................................................................................................... 24 II. ETIOLOGY, DIAGNOSIS, PROGNOSIS .................................................................................. 24 III - MOLECULAR BASIS OF DISEASE ....................................................................................... 26 IV - ANIMAL MODELS OF NEUROTROPHIC KERATOPATHY ................................................. 28
1.5 TREATMENTS FOR NEUROTROPHIC KERATOPATHY AND CORNEAL NEUROTIZATION .................... 32
I – INTRODUCTION ....................................................................................................................... 32 II - MANAGEMENT AND TREATMENT OF NEUROTROPHIC KERATOPATHY ...................... 32 III - CORNEAL NEUROTIZATION AND OTHER METHODS OF RE-INNERVATION................. 34 IV - ANIMAL MODELS OF CORNEAL REINNERVATION AND NEUROTIZATION ................... 38 V - WOUND HEALING AFTER CORNEAL NEUROTIZATION .................................................... 41
1.6 THESIS AIMS AND HYPOTHESIS ................................................................................................... 43
v
AIM 1 .............................................................................................................................................. 43 AIM 2 .............................................................................................................................................. 43 AIM 3 .............................................................................................................................................. 44
2.1 INTRODUCTION .......................................................................................................................... 46
2.2 MATERIALS AND METHODS ........................................................................................................ 48
I – ANIMALS ..................................................................................................................................... 48 III – STERILE SURGERIES ................................................................................................................. 50 IV – CORNEAL HARVEST .................................................................................................................. 53 V – EVALUATION OF CORNEAL EPITHELIAL AND STROMAL THICKNESS ............................................. 53 VI – EVALUATION OF CORNEAL ULCERATION, SCARRING AND PERFORATION ................................... 53 VII - IMMUNOHISTOCHEMISTRY ......................................................................................................... 54 VIII - WESTERN BLOT....................................................................................................................... 55 IX – STATISTICS .............................................................................................................................. 55
2.3 RESULTS ..................................................................................................................................... 56
I - CORNEAL NEUROTIZATION PREVENTS CENTRAL EPITHELIAL THINNING IN THE DENERVATED CORNEAL MODEL OF NK .................................................................................................................. 56 II - CORNEAL NEUROTIZATION PROTECTS THE DENERVATED CORNEA FROM ULCERATION, SCARRING AND PERFORATION .......................................................................................................................... 58 III - CORNEAL NEUROTIZATION RESTORES SP AND CGRP IN THE DENERVATED CORNEA ................. 60 ........................................................................................................................................................ 62 ........................................................................................................................................................ 62
2.4 DISCUSSION ............................................................................................................................... 63
3.1 – SUMMARY OF RESULTS ............................................................................................................ 69
3.2 – FUTURE DIRECTIONS ................................................................................................................ 69
FUTURE DIRECTION 1 – CORNEAL EPITHELIUM .................................................................... 70 FUTURE DIRECTION 2 – TROPHIC SUPPORT .......................................................................... 70 FUTURE DIRECTION 3 – SCHWANN CELLS ............................................................................. 74 FUTURE DIRECTION 4 – LIMBAL STEM CELLS ....................................................................... 79 COLLABORATIONS FOR FUTURE DIRECTIONS ..................................................................... 88
3.3 – CONCLUDING REMARKS ........................................................................................................... 88
REFERENCES .................................................................................................................................... 90
vi
Acknowledgements
I would like to acknowledge my primary supervisor, Dr. Gregory Borschel, and my co-
supervisor, Dr. Tessa Gordon. Thank you for challenging me and teaching me the
fundamental skills to value and to pursue research in the future. Thank you for teaching
me how to turn failures into learning opportunities that result in successes. Your
continuous guidance and encouragement throughout the past two years has been
invaluable.
I would also like to acknowledge my thesis committee members, Drs Clara Chan, Derek
van der Kooy, and Freda Miller. Thank you for your guidance, mentorship, and, most
importantly, questions. They have helped me gain deeper insight into the value of the
research in this thesis and taught me never stop asking questions.
I would like to thank my mentors, Drs Joseph Catapano and Konstantin Feinburg. Thank
you for teaching me the skills necessary to excel in this MSc, for your encouragement,
and your eagerness to help me succeed.
The techniques, expertise and encouragement from my colleagues in the Borschel
laboratory has been instrumental in the completion of this thesis. Dr. Jennifer Zhang, Dr.
Kevin Zuo, Katelyn Chang, Dr. Kasra Tajdaran: thank you for supporting me in my
research pursuits, and beyond. To the undergraduate students we have had the pleasure
of working with, Kaveh Moeni, Jenny Cheung, Golsa Shafa, Kiana Tajdaran, Marina
Manoraj, thank you for your contributions.
I would also like to acknowledge Dr. Asim Ali and Dr. Brian Ballios for their valuable
ophthalmological perspectives and input throughout this MSc. Thank you, Elizabeth
Greczylo, for your efforts that were instrumental in the coordination of schedules.
vii
Thank you to my friends whose advice always put things into perspective. Thank you for
always being there when I needed a break, and for your understanding when I was not
able to take one.
Thank you especially to my family for their patience, love and support. Mom and Dad,
thank you for teaching me the value of hard work and perseverance, and for always
encouraging me to pursue my goals. To my brothers, Jeremy and Luka, thank you for
leading by example. Your commitment to your work motivates me, and your ability to
make light of every situation is uplifting. Thank you for helping me get to wherever I need
to go.
I would also like to thank the following programs which have supported my research:
Ontario Graduate Scholarship (OGS), The Hospital for Sick Children (Research Training
Award), University of Toronto (IMS Entrance Scholarship).
viii
Contributions
Kira Antonyshyn (author) solely prepared this thesis and completed all aspects of this
work in whole or in-part. The author acknowledges the following contributions by other
individuals:
Dr. Gregory Borschel (primary supervisor and program advisory committee member) –
mentorship; laboratory resources; planning and execution of experiments; thesis
preparation
Dr. Tessa Gordon (program advisory committee member) – mentorship; planning and
execution of experiments; thesis preparation
Dr. Clara Chan (program advisory committee member) – mentorship; guidance;
interpretation of results; thesis preparation
Dr. Derek van der Kooy (program advisory committee member) – mentorship; guidance;
interpretation of results; thesis preparation
Dr. Freda Miller (program advisory committee member) – mentorship; guidance;
interpretation of results; thesis preparation
Dr. Joseph Catapano – mentorship; planning of experiments; assistance in surgeries for
assessment of ulceration and scarring in Chapter 2
Dr. Jennifer Zhang – maintenance of the thy1-GFP+ colony; assistance with western blot
analysis for Chapter 2; laboratory manager
Dr. Konstantin Feinberg and Dr. Kaveh Moeni – assistance in microarray analysis and
immunohistochemistry in Chapter 3
Jenny Cheung – assistance with sectioning of the cornea using a cryostat used for
immunohistochemical analysis in Chapter 2 and 3
ix
List of Abbreviations
BCVA Best corrected visual acuity
BSA Bovine serum albumin
BDNF Brain derived neurotrophic factor
CGRP Calcitonin gene-related peptide
CP Common peroneal
EGF Epidermal growth factor
FNE Free nerve endings/intraepithelial nerve terminals
HBSS Hank’s balanced salt solution
IGF-1 Insulin-like growth factor-1 (IGF-1)
LP Limbic plexus
LSC Limbal stem cell
NGF Nerve growth factor
NK Neurotrophic Keratopathy
NT-3 Neurotrophin 3
NT-4 Neurotrophin 4/5
PBS Phosphate buffer solution
PDGF platelet-derived growth factor
PED Persistent epithelial defect
RT Room temperature
SBP Subbasal plexus
SC Schwann cell
SCcm Schwann cell conditioned media
SCpm Schwann cell pre-conditioned media
SD Standard deviation
SEP Subepithelial plexus
SN stromal nerve trunks
SP Substance P
TAC Transient amplifying cell
TRPV1 Transient receptor potential vanillin type 1
V1 Ophthalmomaxillary nerve
x
List of Figures
Figure 1-1 Corneal anatomy…………………………………………………………..…….5
Figure 1-2 Hemidesmosome anchoring complex…………………………………………9
Figure 1-3 Origin of corneal innervation…………………………………………….…….12
Figure 1-4 Pattern of corneal innervation………………………………………………...14
Figure 1-5 Similarities between non-myelinating Schwann cells and corneal basal
epithelial cells………………………………………………………………..…15
Figure 1-6 Key players in corneal epithelial maintenance and repair……………….…16
Figure 1-7 Limbal stem cell function……………………………………………………....20
Figure 1-8 Limbal stem cell deficiency versus neurotrophic keratopathy…………...…22
Figure 1-9 Symptoms of neurotrophic keratopathy ……………………………….….…24
Figure 1-10 Corneal esthesiometry………………………………………………………...26
Figure 1-11 Model of neurotrophic keratopathy……………………………………..….…31
Figure 1-12 Corneal neurotization……………………………………………………….…36
Figure 1-13 In vivo confocal microscopy of subbasal nerves before and after corneal
neurotization……………………………………………………………………37
Figure 1-14 Model of corneal neurotization…………………………………………….….40
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Figure 1-15 Corneal reinnervation after neurotization …………………………………...40
Figure 1-16 Corneal healing in the denervated cornea after corneal neurotization…..42
Figure 2-1 Rat models………………………………………………………………..……50
Figure 2-2 Experiment timeline……………………………………………………………52
Figure 2-3 Corneal epithelial and stromal thickness after corneal neurotization…….58
Figure 2-4 Corneal ulceration after corneal neurotization……………………………...59
Figure 2-5 Corneal scarring and perforation after corneal neurotization………..……60
Figure 2-6 SP and CGRP immunostaining………………………………………………61
Figure 2-7 Control immunostaining..………………………………………………...……62
Figure 2-8 SP western blot analysis………………………………………………...……62
Figure 3-1 Schwann cells ensheath axons in the cornea and limbus…………...……72
Figure 3-2 Schwann cells are located in close proximity to Sox9 positive cells…..…73
Figure 3-3 Proposed mechanism of action of corneal Schwann cells in response to
axonal injury……………………………………………………………………75
Figure 3-4 Preliminary microarray analysis…………………………………………...…79
Figure 3-5 Transgenic confetti mouse……………………………………………………80
xii
List of Tables
Table 1-1 – Causes/Etiology of neurotrophic keratopathy………………………...………25
Table 2-1 – Sample size and group distribution.………………………………….…………49
Table 2-2 – Primary and secondary antibodies………………………………….…………54
Table 3-1 – Antibody markers for future directions…………………………………..…….69
1
CHAPTER 1 Introduction and
Literature Review
2
1.1 Preamble
The cornea is the clear part of the eye through which you can see. Its transparency allows
for transmission of light to the retina for visual processing, and consequently allows for
normal vision. The tissue’s transparency is maintained via the dense, regular
arrangements of cells and extracellular components in the corneal layers and via
avascularity (DelMonte and Kim, 2011). The cornea is one of the most densely innervated
tissues of the body (Rózsa and Beuerman, 1982), and its innervation is critical for
maintenance of ocular surface health, transparency, and consequently vision. Corneal
sensory nerves are responsible for the protective blinking and tearing reflexes that
prevent corneal abrasion and expel foreign objects. These nerves provide the cornea with
trophic support and survival signals for the corneal epithelium via release of nerve-derived
peptides, that, at least in part, maintain and heal the corneal epithelium after injury (Müller
et al., 2003).
The importance of corneal innervation is apparent in its absence. In absence of corneal
sensory innervation and hence, sensation, patients develop Neurotrophic Keratopathy
(NK). This disease is characterized by corneal epithelial breakdown, progressive corneal
scarring, and can eventually result in vision loss. NK treatment in is focused primarily on
ophthalmic management of symptoms primarily. However, these treatments fail to treat
the underlying cause of NK (Reviewed by Sacchetti and Lambiase, 2014; Semeraro et
al., 2014; Mastropasqua et al., 2017). Corneal neurotization is a surgical procedure that
re-innervates the NK cornea by nerves directed to the cornea via nerve autografts. In
patients, this procedure restores corneal sensation and innervation (Fung et al., 2018).
However, because ~15% of treated corneas fail to acquire protective corneal sensation
(Catapano et al., 2019), we have developed an animal model of NK and corneal
neurotization to investigate the effects of the neurotization surgery and its mechanisms.
In this model, corneal neurotization resulted in improved healing of the corneal epithelium
after injury, as compared to the denervated NK cornea. Further studies are required to
determine if corneal neurotization maintains epithelial integrity and restores the cornea
with the nerve-derived mediators necessary to preserve corneal health.
3
1.2 Thesis organization
This thesis is organized in “paper format” using modified peer-reviewed content and
unpublished data where indicated. Chapter 1 introduces corneal anatomy and physiology
in the healthy cornea, neurotrophic keratopathy (NK) and current NK treatments, including
corneal neurotization. Chapter 2 presents original research evaluating the effect of
corneal neurotization on corneal epithelial and stromal thickness and on the presence of
nerve-derived peptides in the denervated NK cornea. This chapter further compares the
effect of topical treatments, nerve growth factor and Schwann cell conditioned media, on
healing an epithelial injury in the denervated cornea with each other, and with the
previously published healing effect of corneal neurotization (Catapano et al., 2018). Part
of this chapter was reformatted from work published in the journal of IOVS (Catapano et
al., 2018). This work investigates corneal ulceration and scarring in the denervated and
neurotized corneas. In this chapter, the future directions specific to the presented results
are discussed. Chapter 3 summarizes the results in a broader context of the existing
literature and outlines ongoing and future directions.
4
1.3 Corneal Anatomy and Physiology I - INTRODUCTION
The corneal structure informs corneal function. Corneal sensory innervation is critical in
maintaining a clear and functional cornea. In this chapter, corneal anatomy and
physiology are summarized and an emphasis is placed on corneal innervation and its
influence on the surrounding structures.
II - CORNEAL ANATOMY
The cornea plays a critical role in vision. It protects against infection and injury by
providing a barrier between the external environment and internal ocular contents (Figure
1-1). The cornea is responsible for the majority of the total refractory power of the eye,
and consequently normal vision requires maintenance of corneal shape (Meek et al.,
2003; Meek and Knupp, 2015). Clarity is achieved by there being no blood vessels or
myelinated axons in addition to the layered arrangements of the corneal cells.
The cornea is comprised of five layers. The most anterior three layers are innervated by
corneal sensory nerves and two posterior layers are not supplied by any innervation.
From anterior to posterior these are the: i) corneal epithelium ii) Bowman’s layer iii) stroma
iv) Descemet’s membrane v) endothelium (Figure 1-1B).
5
i) Epithelium The corneal epithelium functions primarily as a protective barrier. In humans, this corneal
layer is about 50 μm thick and is composed of four to six layers of non-keratinized
stratified squamous epithelial cells. The two to three most superficial cell layers contain
flattened nuclei. The deeper wing (suprabasal) epithelial cells are polyhedral and the
basal epithelial cells are columnar in shape (Eghrari et al., 2015) (Figure 1-1C). Epithelial
cells adhere at tight junctions via proteins such as ZO-1, JAM-A, E-cadherin, occludin
and caludin-1 (Ban et al., 2003). The tight junctions between epithelial cells function to
Figure 1-1 Corneal anatomy. A. Outline of the anterior features of the globe with an
emphasis on the cornea (boxed) in relation to other anatomical features (Navaratnam et
al., 2015). B. A sagittal section of the cornea demonstrates the five corneal layers
(Navaratnam et al., 2015). C. A sagittal section of the corneal epithelial cell layer (i).
A
B
i.
ii.
iii.
iv.
v.
C
superficial cells
wing cells
basal cells
glycocalyx layer
tear film apical
microvilli
6
create a watertight seal which assists in preventing pathogenic organisms from
penetrating the cornea (Ban et al., 2003; DelMonte and Kim, 2011; Eghrari et al., 2015).
Human cultured corneal epithelial cells also secrete pro-inflammatory cytokines, including
IL-1β, IL-6, IL-8 and TNF- α (Zhang et al., 2003; Kumar et al., 2004; Zhang et al., 2005;
Kumar et al., 2006), which are known to assist in immune regulation and protection
against pathogens (Harder et al., 2000; Liu et al., 2014). In the basal corneal epithelium,
the Langerhan cells are involved in the corneal immune response (Hamrah and Dana,
2007).
The superficial surface of the corneal epithelium is covered by apical microvilli and
microplicae, a charged glycocalyx and a layer of tear film (Figure 1-1C). This 7 μm tear
film is composed of mucinous, aqueous and lipid layers, each contributing to the overall
function of the cornea. The tear film “smooths out micro-irregularities” of the superficial
anterior epithelial surface (DelMonte and Kim, 2011) and prevents desiccation, infection
and friction from opening and closing of the eyelids during blinking (Perry 2008). The lipid
layer of the tear film is the most superficial layer and is secreted by the Meibomian glands
at the rim of the eyelid in the tarsal plate (Perry, 2008; DelMonte and Kim, 2011; Eghrari
et al., 2015). The aqueous layer is secreted primarily by the lacrimal glands located in the
upper lateral region of the orbit, and partially by the accessory lacrimal glands in the
conjunctiva. Tear secretion from the lacrimal gland is stimulated by afferent innervation
from the trigeminal nerve and efferent innervation from parasympathetic and sympathetic
innervation (Dartt, 2011). The aqueous secretions from these glands that form this layer
of the tear film are isotonic and contain proteins such as lysozomes, lacritin and others
(McKown et al., 2009). The deepest, mucous, layer of the tear film is secreted by
conjunctival goblet cells. These cells are innervated by efferent sympathetic and
parasympathetic nerves. Secretion of this layer, predominantly containing the
polysaccharide MUC5AC, is stimulated by noxious stimulation of sensory nerves in the
cornea (Dartt, 2011). The glycocalyx sits between the superficial tear film and the corneal
epithelium to hydrate the corneal surface and protect it from infection (Dartt, 2011). It is
composed of soluble and membrane-spanning polysaccharides, such as MUC1, MUC4,
7
and MUC16, as well as those secreted by the lacrimal gland, goblet cells, and the corneal
squamous epithelium (Argüeso and Gipson, 2001; Gipson, 2004).
The lifespan of individual epithelial cells is approximately seven to ten days (Hanna et al.,
1961; Eghrari et al., 2015), and complete cellular turnover is estimated to occur in about
two weeks (Cenedella and Fleschner, 1990). The limbal epithelial stem cells (LSCs) are
located at the limbus, the junction of the cornea and sclera. During normal cellular
turnover, the LSCs proliferate, differentiate, and terminate eventually as corneal epithelial
cells. Basal and wing cells replenish the superficial epithelial cells as they undergo
apoptosis and slough into the tear film throughout normal desquamation. The former cells
migrate in an apical direction from posterior and peripheral to anterior and central cornea
(Eghrari et al., 2015). The β-4 integrin is differentially expressed on the membranes of
LSCs and epithelial cells in the basal, basolateral and apical membranes of the cornea
(Pajoohesh-Ganji et al., 2006; Stepp, 2006). As these cells differentiate to become wing
and suprabasal cells, their β-4 integrin expression is downregulated (Stepp, 2006; Stepp
et al., 2017). The epithelial cells expressing β-4 integrin are located in close proximity to
subbasal axons, and it is hypothesized that these epithelial cells wrap around the axons
in the corneal epithelium to act as surrogate Schwann cells (SCs) by providing axonal
support (Stepp et al., 2017). The basal epithelial cells adhere to the underlying the
epithelial basement membrane and stroma via hemidesmosomes (Torricelli et al., 2013;
Eghrari et al., 2015).
Hemidesmosomes together with anchoring fibrils and filaments, form an anchoring
complex with an extracellular matrix that is associated with the epithelial basement
membrane and stroma (Torricelli et al., 2013). The epithelial basement membrane
releases factors, transforming growth factor β-1 and platelet-derived growth factor
(PDGF), that modulate epithelial cell differentiation and apoptosis. Primarily, the
basement membrane functions to maintain adherence of the corneal epithelium to the
underlying Bowman’s layer and corneal stroma via the anchoring complexes (Torricelli et
al., 2013; Eghrari et al., 2015).
8
Three components are necessary to form the aforementioned hemidesmosome, and thus
the anchoring complex (Figure 1-2). These are 1) an intracellular plaque protein 2) a
transmembrane protein, and 3) basement membrane proteins that are associated with an
extracellular matrix (Torricelli et al., 2013). The intracellular plaque proteins link elements
of the intracellular cytoskeleton at the surface of the plasma membrane of the basal
epithelial cells (Figure 1-2A). The transmembrane proteins, including α6β4 integrin and
type XVII collagen, serve as cell receptors to connect the basal cell interior to the
extracellular matrix (Figure 1-2B) (Stepp et al., 1990; Torricelli et al., 2013). These
transmembrane proteins link the epithelial basement membrane with keratin intermediate
filaments and laminin in the extracellular matrix (Figure 1-2C) (Stepp et al., 1990; Dowling
et al., 1996; Stepp et al., 1996). Collagen VII is another major component of anchoring
fibrils that extends into the extracellular matrix to attach to anchoring plaques located in
the stroma (Figure 1-2D) (Gipson et al., 1987).
A
B
C
D
Corneal epithelial basement
membrane
Corneal epithelial basal cells
Bowman’s Layer & Stroma
9
Expression of the proteins associated with the anchoring complex change during corneal
epithelial wound healing (Fujikawa et al., 1984; Stepp, Zhu and Cranfill, 1996), and
abnormalities of the corneal epithelial basement membrane are associated with recurrent
epithelial breakdown (Payant et al., 1991; Colville et al., 1997; Resch et al., 2009). In vitro
organ culture studies of rabbit corneas implicate corneal innervation in the maintenance
of the expression of proteins required to form the anchoring complex (Nishida et al., 1996;
Yamada et al., 2005).
ii) Bowman’s Layer The Bowman’s layer is acellular and non-regenerating (DelMonte and Kim, 2011; Eghrari
et al., 2015). This 8-12 μm layer is composed of Type IV collagen, laminin, heparin sulfate
and fibronectin collagen fibrils (Jacobsen et al., 1984). In humans, this layer is well
developed compared to in rats and other species. However, there are no apparent
differences in the aforementioned anchoring structure (Hayashi et al., 2002).
iii) Stroma
The stroma functions to provide structural support and maintain transparency in the
cornea. In humans, the cornea is approximately 500 μm thick, the stroma comprising 80-
90% of the corneal thickness (Maurice, 1970; Boote et al., 2003). In the stroma, parallel
collagen fibers, fibrils, are arranged into parallel layers, or lamellae. The dense and
precise arrangement of the collagen fibrils, organized into orthogonal lamellae (Maurice,
1957, 1970; Jester et al., 1999; Hassell and Birk, 2010), reduces the opportunity for light
to scatter and thus promotes transparency. The arrangement of the lamellae varies with
stromal depth and, because the posterior cornea is more hydrated than the anterior,
Figure 1-2 Hemidesmosome anchoring complex. In the cornea, this links the basal
epithelial cells, epithelial basement membrane, and extracellular matrix and is composed
of A. intracellular plaque proteins in the basal epithelial cells, B. transmembrane proteins,
and C. basement membrane proteins connecting the basal epithelial cells, Bowman’s layer
and underlying stroma D. Anchoring fibril components link the complex to anchoring
plaques in the stroma. In this simplified diagram, not all molecules and proteins present in
the corneal areas of the complex are included.
10
refraction decreases as light passes through the stroma. Anteriorly the lamellae are
arranged into short, narrow sheets and extensively interconnected. Posteriorly, wide,
thick lamellae extend from limbus to limbus without interlamellar connections (Eghrari et
al., 2015).
There are 13 types of collagen fibrils in the stroma, the most common (58%) of which is
Type I collagen (Komai and Ushiki, 1991; Meek and Fullwood, 2001; Robert et al., 2001;
Fini and Stramer, 2005). Proteoglycans maintain the small distance between collagen
fibrils. Proteoglycans function to help regulate hydration and promote corneal clarity,
shape and volume (DelMonte and Kim, 2011).
Keratocytes reside between the lamellae, primarily in the anterior corneal stroma, and
synthesize molecules necessary to maintain stromal integrity, collagen fibers and matrix
metalloproteases (Jester et al., 1999; DelMonte and Kim, 2011). Keratocyte presence
can contribute to light scattering in the stroma, which is reduced by corneal water-soluble
crystallins (Jester et al., 1999; DelMonte and Kim, 2011; Eghrari et al., 2015). The
crystallins are lost from keratocytes during stromal repair after injury (Stramer and Fini,
2004). Keratocytes are activated by stromal injury and behave like fibroblasts in regulating
the healing response (Stramer and Fini, 2004). The keratocytes also synthesize matrix
metalloproteases that are critical in stromal remodeling after injury because of their
involvement in extracellular matrix remodeling, cell-matrix interaction, inflammatory cell
recruitment and cytokine activation (Fini and Stramer, 2005; Gabison et al., 2005). The
stroma also plays a role in immune regulation. Dendritic, and other bone marrow-derived
cells, such as macrophages, reside in the corneal stroma (Hamrah et al., 2003b) and
were shown to participate in immunity and inflammation in response to thermal cautery
of the rodent cornea (Hamrah et al., 2003a; Hamrah and Dana, 2007).
Blood vessels are absent in the stroma, preserving stromal and corneal clarity. This
avascularity is maintained by the balance of anti- and pro-angiogenic factors. For
example, the pro-angiogenic vascular endothelial growth factor (VEGF) function is
neutralized by its binding to corneal-secreted soluble VEGF receptor-1 (sVEGFR-1 or sflt-
11
1). Suppressing this receptor with antibodies or RNA interference in mice results in
angiogenesis, and vascularization of the cornea, and thus impaired corneal clarity
(Ambati et al., 2006).
iv) Descemet’s membrane The Descemet’s membrane is a 10 μm layer that functions as the basement membrane
for the corneal endothelium. The membrane is composed of two layers. The anterior
banded layer is composed of Type IV and VIII collagen fibrils and proteoglycans. The
posterior non-banded layer is laid down by endothelial cells. This membrane assists the
endothelium in maintaining corneal dehydration (Eghrari et al., 2015).
v) Endothelium The corneal endothelium comprises the deepest cellular layer of the cornea. It is a single
cell layer of flat, polygonal cells that contribute to approximately 4-5 μm of the corneal
thickness. The layer is essential in preserving corneal dehydration (DelMonte and Kim,
2011; Eghrari et al., 2015), which is important in the maintenance of corneal shape and
thus clarity (Zucker, 1966). The endothelial cells are adherent to the overlying Descemet’s
membrane via hemidesmosomes and to each other through lateral interdigitations, tight
junctions and gap junctions (DelMonte and Kim, 2011; Eghrari et al., 2015). The gap
junctions are involved in electrical coupling of endothelial cells (Polse et al., 1990)
Na+/K+-ATPase and intracellular carbonic anhydrase pumps are also located on the
lateral endothelial membranes and contribute to corneal dehydration (Stiemke et al.,
1991). Corneal dehydration is a passive pump-leak process as fluid moves from the hypo-
osmotic stroma and hypertonic aqueous humor. Although energy use is not directly
necessary, maintenance of the hydration gradient is reliant on energy-requiring ion
transport through the Na+/K+-ATPase and intracellular carbonic anhydrase pathways.
These pathways create a net efflux of ions from the corneal stroma to the aqueous humor,
facilitating fluid efflux from the cornea and thus corneal dehydration (Watsky et al., 1989;
DelMonte and Kim, 2011).
12
Endothelial cell density declines with age (DelMonte and Kim, 2011; Eghrari et al., 2015).
The surviving cells do not proliferate (Joyce, 2003) but their morphology adapts to occupy
the space left by the degenerated cells. This change in morphology with age is associated
with a reduced ability of the cornea endothelium to dehydrate the cornea (Polse et al.,
1990; Gambato et al., 2014). In endothelial failure, there is corneal edema due to a net
influx of ions and thus aqueous fluid into the cornea (DelMonte and Kim, 2011; Eghrari et
al., 2015).
III - CORNEAL INNERVATION
The corneal epithelium is the most densely innervated tissue in the body. The corneal
epithelial innervation is 300 to 600 times more dense than that of the skin epithelium, and
20-40 times than that in the tooth pulp (Rózsa and Beuerman, 1982; Guthoff et al., 2005;
Stepp et al., 2017). The majority of corneal sensory innervation originates at the trigeminal
ganglion (Figure 1-3). The innervating axons travel to the cornea through the
ophthalmomaxillary nerve (V1) and, once in the orbit, they travel by way of the long ciliary
nerves (Figure 1-3) (Müller et al., 2003), through the limbus to the cornea (Figure 1-4 A).
A small proportion of corneal sensory innervation is sympathetic and parasympathetic,
deriving from the superior cervical and ciliary ganglia, respectively (Figure 1-3). In both
humans and rats, the sympathetic and parasympathetic fibers are localized to the
peripheral cornea in small quantities (Toivanen et al., 1987; Marfurt et al., 1993; Marfurt
et al., 1998).
13
Corneal sensory nerve fibers travel radially from the limbus, the limbic plexus (LP; Figure
1-4A), through the deep stroma to superficial epithelium, creating a centripetal pattern of
corneal innervation (Figure 1-4, B) (Yang et al., 2018). The majority of innervation enters
the cornea from the LP by way of the stroma (Figure1-4 A). The innervating axons lose
their myelination within 1 mm of entering the stroma, thereby promoting corneal clarity.
Non-myelinating SCs in the stroma form a single layer of ensheathment around these
axons (Müller et al., 1996; Müller et al., 2003). Within the stroma, the nerves branch
extensively to form the sub-epithelial plexus (SEP) where the axons travel parallel to the
surface of the cornea before they penetrate the Bowman’s layer and form the sub-basal
plexus (SBP) between Bowman’s layer and the basal epithelium. Single intraepithelial
axons then branch from the nerve fibers of the SBP to travel anteriorly through the corneal
epithelium where they terminate as free nerve endings (FNE) (Figure 1-4).
The majority (~66%) of the sensory fibers innervating the cornea are unmyelinated C-
fibers that terminate as polymodal nociceptors in the epithelium. These axons have large
receptive fields attributed to their extensive axonal branching. The remaining are thinly
myelinating, Aδ fibers that terminate as cold-thermal and mechano-nociceptors
(Belmonte et al., 2004; Yang et al., 2018). The majority of the corneal nerve fibers
terminate in the corneal epithelium. The intraepithelial nerve fibers travel perpendicular
to the epithelial basement membrane (Figure 1-4 A,C) and terminate as free nerve
endings in basal and suprabasal/wing epithelial cell layers (Stepp et al., 2017; Yang et
al., 2018). A small population of nerve fibers in the cornea terminate in the stroma, some
in close proximity to stromal keratocytes (Muller et al., 1996; Seyed-Razavi et al, 2014).
In the peripheral nervous system, unmyelinated, unsheathed axons are termed free nerve
endings (Stepp et al., 2017). In the skin, free nerve endings are typically shorter in length
Figure 1-3 Origin of corneal innervation. Corneal sensory innervation (green)
originates at the trigeminal ganglion. The axons innervating the cornea travel to their
destination via the ophthalmomaxillary nerve (V1) which enters and, in the orbit, via long
ciliary nerves. The cornea receives some parasympathetic (blue) and sympathetic
(orange) innervation from the ciliary and superior cervical ganglions (respectively).
115-128
14
Figure 1-4 Pattern of corneal innervation. A. Sagittal-section of the cornea and limbus showing corneal
innervation. Via the limbic plexus (LP), the corneal innervation enters the stroma to form the sub-epithelial
plexus (SEP). From here axons penetrate the Bowman’s layer to form the sub-basal plexus (SBP)
beneath the corneal epithelium. Single axonal fibers branch and terminate as free-nerve endings in the
corneal epithelium. B (Muller et al., 2003), C (Rozsa and Beurman, 1982) Diagrammatic representation
of the distribution of the corneal innervation demonstrating the SEP that gives rise to the SBP, which
branch into intraepithelial nerve terminals also known as FNEs in the B whole cornea, and C cellular level
A
B C
SEP
SBP
FNE
FNEs
Stroma
SBP
SEP
Epithelium
15
than 100 μm, and, in contrast they are as millimeters long in the corneal epithelium
(Rózsa and Beuerman, 1982; Stepp et al., 2017). The plasma membrane of corneal
epithelial basal cells wraps around individual axons and groups of axons (Figure 1-5)
(Müller et al., 2003; Marfurt et al., 2010; Stepp et al., 2017) resembling the Remak
bundles of non-myelinating SCs wrapping around axons in unmyelinated peripheral
nerves (Shaheen et al., 2014; Stepp et al., 2017). These basal epithelial cells may act
as surrogate SCs to provide axonal support to the intraepithelial axons (Stepp et al.,
2017). Their terminals are dynamic, rearranging and re-growing in response to epithelial
cell turnover and injury in mice (Harris and Purves, 1989; Pajoohesh-Ganji et al., 2006;
Yu and Rosenblatt, 2007; Namavari et al., 2011; Stepp et al., 2017).
Figure 1-5 Similarities between non-myelinating Schwann cells and corneal basal epithelial cells. Schematic representations of cell membranes of A non-myelinating
Schwann cells and B corneal epithelial basal cells wrap around sensory axons. A. Cross-
section of a non-myelinating Schwann cell, sometimes referred to as a Remak bundle,
containing several axons. B. Cross-section of corneal epithelial basal cell membranes
wrapping around single axons or clusters containing several axons. In these schematics,
A and B are not shown at the same scale; axon (blue) diameters would normally be the
same in both A non-myelinating Schwann cells and B corneal epithelial basal cells.
(Reproduced from Stepp et al, 2017 with permission from Elsevier: License #
4595681499795)
16
IV - CORNEAL EPITHELIAL MAINTENANCE AND REPAIR
Functional corneal innervation is critical for maintenance and repair of the corneal
epithelium because corneal sensory nerves are an important source of trophic mediators
(Figure 1-6). Previous studies have demonstrated a trophic influence of the trigeminal
neurons on the corneal epithelium using co-cultures of corneal epithelial cells and
dissociated trigeminal neurons (Chan and Haschke, 1981, 1982; Ko et al., 2013;
Kowtharapu et al., 2014). The following nerve-derived peptides are suggested to play a
role in corneal epithelial maintenance and repair (Müller et al., 2003):
Figure 1-6 Key players in corneal epithelial maintenance and repair. Sagittal-section of the
cornea and limbus. Substance P (SP) and calcitonin gene-related peptide (CGRP) are co-
localized in corneal sensory nerves and promote epithelial cell (purple/pink) proliferation and
migration. Schwann cells (red) ensheath the axons (yellow) in the limbus and corneal layers
deeper than the epithelium. Nerve growth factor (NGF) is a neurotrophic factor that is thought
to promote epithelial differentiation and proliferation in the cornea, but its source is unknown.
NGF’s trkA receptor is found in high concentrations in the basal epithelium of the limbus where
the limbal stem cells (light blue) are found.
Legend
17
Substance P (SP) is a peptide which is present, at physiologically relevant levels, in the
normal healthy cornea (Figure 1-6) (Tervo et al., 1981; Elbadri et al., 1991; Marfurt and
Echtenkamp, 1995; Marfurt et al., 2001; Müller et al., 2003). Corneal epithelial cells
express receptor NK-1 which mediates SP function (Figure 1-6) (Nakamura, Ofuji, et al.,
1997; Mantyh, 2002; Yang et al., 2014). In culture, SP stimulates epithelial cell
proliferation (Reid et al., 1993; Garcia-Hirschfeld et al., 1994).
In vitro SP upregulates the expression of proteins that are necessary to maintain tight-
junctions in the corneal epithelium (Araki‐Sasaki et al., 2000; Ko et al., 2009). The peptide
is implicated in the formation corneal epithelial tight junctions, by upregulating the
expression of ZO-1 (Ko et al., 2009). In addition, SP, in conjunction with insulin-like growth
factor-1 (IGF-1), increases the expression of α5 integrins and E-cadherin, which is
required for epithelial adhesion to fibronectin in the extracellular matrix (Nakamura et al.,
1998; Chikama et al., 1999; Araki‐Sasaki et al., 2000). Administration of SP in vitro
upregulates integrin expression on the basal corneal epithelial cells and increases the
attachment of these cells to proteins of the epithelial-stromal anchoring complex (Nishida
et al., 1992, 1996; Yamada et al., 2005).
During corneal epithelial wound healing, SP in conjunction with IGF-1 stimulates epithelial
cell attachment to anchoring complex proteins of the newly wounded area (Juhasz et al.,
1993; Nakamura et al., 1998), thus aiding in epithelial cell migration. The synergistic
facilitation of epithelial cell migration by SP and IGF-1 has been confirmed both in vitro
and in vivo experiments (Nakamura et al., 1998; Chikama et al., 1999). SP, SP analogues,
and IGF-1 have all been used to treat persistent epithelial defects (PEDs) in human
patients (Brown et al., 1997; Kingsley and Marfurt, 1997; Chikama et al., 1998; Morishige
et al., 1999; Murphy et al., 2001; Yamada et al., 2008). It is therefore apparent that nerve
derived SP plays a role in maintaining epithelial integrity.
Calcitonin gene-related peptide (CGRP) is often co-localized with SP in corneal
sensory nerves (Figure 1-6) (Beckers et al., 1993; Marfurt, Murphy and Florczak, 2001;
Müller et al., 2003), and its receptors are abundant in the corneal and limbal epithelium
18
(Heino et al., 1995; Tran et al., 2000). The concentration of CGRP in human tears
increases following corneal epithelial injury (Mertaniem et al., 1995). After injury,
exogenous CGRP application has been shown to increase corneal epithelial cell DNA
synthesis and migration in some animal studies (Reid et al., 1993; Mikulec and Tanelian,
1996), but not in others. However, the studies that found no effect of the peptide on cell
migration, did not co-administer CGRP with other trophic peptides, such as SP (Nishida
et al., 1996). This suggests that CGRP may only work synergistically with SP or other
trophic factors to increase corneal epithelial cell migration. CGRP and SP may also have
an inflammatory role following corneal injury by stimulating IL-8 secretion (Tran et al.,
2000; Tran et al., 2000). Although CGRP alone may or may not elicit an effect on the
corneal epithelium, it could possibly potentiate the function of other trophic factors.
Thickness of the corneal epithelium (Alper, 1975), and proliferation of corneal epithelial
and LSC after injury (Park et al., 2006; Yin et al., 2011; Ueno et al., 2012) are influenced
by corneal innervation. Corneal wound healing is significantly impaired in animal models
of corneal hypoesthesia, including diabetes mellitus 1 and neurotrophic keratopathy (NK)
(Mikulec and Tanelian, 1996; Nagano et al., 2003; Nakamura et al., 2003). Nerve-derived
peptides, including SP and CGRP, have demonstrated trophic influences on the corneal
epithelium during normal epithelial cell turnover and are increased in concentration
following injury of the normally innervated cornea (Mertaniem et al., 1995). SP promotes
corneal wound healing in rats with type I diabetes mellitus by activating the epidermal
growth factor (EGF) signaling pathway (Yang et al., 2014) that is implicated in the turnover
of corneal epithelial cells (Nakamura, Nishida, et al., 1997; Peterson et al., 2014; Rush et
al., 2014) and wound healing (Zieske et al., 2000). In support of these findings, a study
demonstrated that the receptor for EGF was not activated in corneas of type I diabetic
rats and, in turn, which could account for the impaired healing of the debrided corneal
epithelium (Xu nd Yu, 2011). Re-activation of this pathway with SP promotes corneal
wound healing in rats with type I diabetes mellitus (Yang et al., 2014), providing further
evidence for a role of the EGF signaling pathway in the normal turnover and healing of
the corneal epithelium.
19
Other neuropeptides and neurotransmitters that have been studied in the cornea
include norepinephrine, acetylcholine and vasoactive intestinal polypeptide. Ample
evidence suggests these factors as well as SP and CGRP play a critical role in the
maintenance and repair of the corneal epithelium (Figure 1-6) (Müller et al., 2003) In the
cornea, several neurotrophins, including nerve growth factor (NGF) (Figure 1-6),
neurotrophin 3 and 4/5 (NT-3 and NT-4), and brain derived trophic factor, and their
receptors have been confirmed at the transcription and protein levels (Lambiase et al.,
1998). NGF in particular is of interest due to its effectiveness as a topical agent in treating
persistent epithelial defects (PEDs) in animal models (Lambiase et al., 2000; Esquenazi
et al., 2005) and in patients (Lambiase et al., 1998; Bonini et al., 2000; Tan et al., 2006;
Lambiase, Sacchetti and Bonini, 2012; Sacchetti et al., 2017). NGF stimulates epithelial
cell proliferation and differentiation in vitro (F. E. Kruse and Tseng, 1993; You, Kruse and
Völcker, 2000), and although the source of NGF in the cornea unknown, several have
been suggested in the literature, including corneal epithelial cells, corneal nerves (Figure
1-6) and tears (Müller et al., 2003). The SCs may also be a source (Figure 1-6), although
this has yet to be established.
V - LIMBAL STEM CELLS
Corneal epithelial homeostasis is dependent on the distinct population of stem cells that
are located within a stem cell niche in the Palisades of Vogt of the limbus (Fig. 1-7 A-i)
(Dua et al., 2005; Ahmad, 2012). Corneal epithelial wound healing is significantly impaired
in the absence of the limbal epithelium (Huang and Tseng, 1991). Limbal epithelial stem
cells (LSCs) increase and then decrease one and four days after injury, respectively
Cotsarelis et al., 1989; Lehrer e al., 1998; Park et al., 2006). Corneal epithelial cells also
increase and decrease but a day later than the LSCs (Park et al., 2006). The corneal
epithelial cells in turn, migrate centripetally to replenish the corneal epithelium in response
to both desquamation and corneal injury (Thoft and Friend, 1983; Collinson et al., 2004;
Di Girolamo et al., 2015). The XYZ Hypothesis proposes that, in order to maintain corneal
epithelial homeostasis, cell loss (Z) must be balanced by cell replacement through stem
cell proliferation and differentiation (X) and migration to the central cornea (Y) (Thoft and
20
Friend, 1983). In animal models, LSCs are often characterized as slow-cycling, retaining
BrdU, and they demonstrate the morphology of a typical stem cell (Romano et al., 2003;
Chen et al., 2004; Schlötzer-Schrehardt and Kruse, 2005; Bentley et al., 2007). These
LSCs differentiate into transient amplifying cells (TACs) that are fast-dividing cells in the
limbal basal epithelium and in the peripheral cornea (Fig. 1-7A). TACs are further
differentiated into post-mitotic and terminally differentiated corneal epithelial cells in the
more central cornea (Fig. 1-7A) (Schlötzer-Schrehardt and Kruse, 2005).
Figure 1-7 Limbal stem cell function. A. (i) Limbal stem cells (LSCs; light blue),
located in their niches in the basal limbal epithelium, (ii) proliferate and differentiate into
transient amplifying cells (navy blue) that migrate towards the central cornea in the basal
epithelium, above the Bowman’s Layer (green). (iii) These cells further differentiate into
post-mitotic (purple) and, finally, terminally differentiated corneal epithelial cells (pink).
B. Diagram of LSC markers as LSCs transition from resting LSCs to transient amplifying
cells prior to becoming terminally differentiated into corneal epithelial cells, based on
published literature (Di Iorio et al., 2005; Barbaro et al., 2007; Menzel-Severing et al.,
2018)
B
LSC niche
Migration Proliferation
Differentiation
21
Stem cells in various tissues, including bone marrow can be identified by their ability to
take up Hoechst 33342 dye (Goodell et al., 1996). Likewise, the LSCs have likewise been
identified with this dye (Ueno et al., 2012). Whilst there are no definitive LSC markers,
ABCB5 (Ksander et al., 2014; Frank and Frank, 2015), ABCG2, Hes1, p63, p75NTR and
trkA have been accepted as distinguishing markers for the LSCs in vivo (Schlötzer-
Schrehardt and Kruse, 2005; Takács et al., 2009). More recently, groups have identified
other markers, such as C/EBPδ, Bmi1, SOX9, ΔNp63α that have been shown to identify
resting LSCs and ΔNp63α that also identifies activated LSCs/early TAC cells, and finally,
ΔNp63β, ΔNp63γ and Wnt/β-catenin that identifies TACs (Di Iorio et al., 2005; Barbaro et
al., 2007; Menzel-Severing et al., 2018). Thereby, the differential expression of LSC
markers is able to identify and follow the state of activation and differentiation of LSCs
(Figure 1-7B).
The activation of LSCs promotes corneal epithelial wound closure (Mort et al., 2012;
Ljubimov and Saghizadeh, 2015). Whilst their numbers have been suggested to decrease
in denervated corneas of mice (Ueno et al., 2012), patients with limbal stem cell deficiency
(Dua et al., 2000; Menzel-Severing et al., 2018) and rabbits with surgically removed limbal
epithelium in innervated cornea (Huang and Tseng, 1991) exhibit corneal vascularization
and conjunctivalization which are not typical symptoms of NK (Figure 1-8). These
symptoms are also elicited in the normally innervated cornea by damaging the LSC niche
with localized alcohol application in mice but, removing LSCs surgically in the innervated
cornea without damaging the niche, resulted in dedifferentiation of the corneal epithelial
cells and repopulation of the LSC niche (Nasser et al., 2018). This study indicates that
corneal epithelial homeostasis is dynamic, relying on functioning corneal epithelial cells,
LSCs and the LSC niche.
22
In contrast to the sensory nerve innervation of corneal epithelial cells being essential for
maintaining their function, the role in limbal function is not understood. Some studies
indicate that innervation is important for limbal function. The sensory nerve endings in the
limbus are compact nerve endings that are located in close proximity to the Palisades of
Vogt (Al-Aqaba et al., 2018). The authors noted that these endings are typical of rapidly-
adapting low-threshold mechanoreceptors which are encapsulated by differentiated
Schwann and/or endoneurial/perineurial-related cells. The LSCs may be directly
correlated with this innervation because the severity of the symptoms of LSC deficiency
in patients correlated with reduced corneal sub-basal nerve density and increased nerve
tortuosity (Chuephanich et al., 2017).
The evidence that 1) LSCs function to maintain corneal epithelium, 2) the disease
symptoms of LSC deficiency and NK differ, and 3) the ill-defined relationship between
LSCs and sensory nerve innervation, suggests that NK patients are not LSC deficient or
that the LSC niche is damaged. Rather, LSC function may be impaired.
VI - SCHWANN CELLS IN THE CORNEA
Both myelinating and unmyelinating Schwann cells (SCs) are present in the limbus.
Within 1 mm of entering the corneal stroma, the axons lose their myelination. In the
cornea, unmyelinating SCs continue to ensheath the axons in the sub-epithelial and
subbasal plexus (Müller et al., 2003), but are not present in the corneal epithelium. It is
Figure 1-8 Limbal stem cell deficiency vs neurotrophic keratopathy. In limbal stem
cell deficiency, patients exhibit A. conjunctivalization and neovascularization of the cornea,
but in Neurotrophic Keratopathy patients exhibit B. epithelial breakdown, progressive
scarring and vision loss (photograph from patient at The Hospital for Sick Children). (A
Modified from Journal of Investigative Dermatology Symposium Proceedings, Sun and
Lavker, Corneal Epithelial Stem Cells: Past, Present, and Future, pages 202-207, copyright
(2004) with permission from Elsevier: License # 4595681016500)
23
suggested the basal epithelial cells act as surrogate SCs, by responding to epithelial and
intraepithelial axonal injury via mechanisms similar to those induced by SCs in response
to axonal injury and during Wallerian Degeneration (Stepp et al., 2017). Although the
presence of SCs is noted, little is known about their role in the limbus and/or cornea, and
the SC response to corneal denervation remains to be determined.
SCs have been extensively studied in other tissues (Jessen et al., 2015; Jessen and
Mirsky, 2016; Carr and Johnston, 2017). These cells provide axonal support in the form
of myelination or the formation of Remak (non-myelinating) bundles (Jessen, Mirsky and
Lloyd, 2015; Harty and Monk, 2017). SCs contribute to the repair process after nerve
injury by releasing neurotrophic factors such as NGF, brain-derived neurotrophic factor,
NT-3, VEGF and others (Meier et al., 1999; Fontana et al., 2012; Brushart et al., 2013).
It is suggested that axonal injury stimulates the de-differentiation of SCs (Stoll and Müller,
1999). De-differentiated SCs have been shown to play a critical role in tissue regeneration
in the rodent digit tip (Johnston et al., 2016) and to promote healing of an epithelial injury
in skin (Johnston et al., 2013). SCs have been found to support hematopoietic stem cells
in the bone marrow (Yamazaki et al., 2011; Yamazaki and Nakauchi, 2014), and a
relationship between SCs and stem cells in other tissues has been hypothesized due to
their anatomical proximity (Carr and Johnston, 2017).
SCs have a critical function in other tissues and injury models, and their physical presence
in the cornea and limbus suggests they may play an important role in maintaining limbal
and corneal health. It’s important to note that if corneal epithelial basal cells act as
surrogate SCs, as has been suggested by Stepp et al. (2017), that they too may play an
important role in maintaining corneal health as we proposed above.
24
1.4 Neurotrophic Keratopathy
I. INTRODUCTION
In this subchapter, neurotrophic keratopathy (NK), the corneal disease investigated in this
thesis, is discussed in detail.
II. ETIOLOGY, DIAGNOSIS, PROGNOSIS
In the absence of corneal sensory innervation, patients lack protective reflexes and the
nerve-derived trophic mediators that support corneal epithelial maintenance and healing.
Consequently, these patients develop NK that is characterized by corneal anesthesia or
hypoesthesia, recurrent or persistent epithelial defects (PEDs), progressive stromal
scarring and can eventually lead to blindness (Bonini et al., 2003; Ramaesh et al., 2007;
Sacchetti and Lambiase, 2014) (Figure 1-9). NK can be congenital or develop as a result
of corneal nerve injury. Corneal nerve injury can be a result of viral infection, chemical
burns, intracranial trigeminal nerve injury or corneal surgery. Systemic diseases that
affect the peripheral nervous system, such as diabetes, multiple sclerosis or leprosy, can
also cause axonal degeneration in the cornea, resulting in NK (Table 1-1).
Figure 1-9 Symptoms of neurotrophic keratopathy. In neurotrophic keratopathy,
patients exhibit A. corneal epithelial ulcerations and breakdown, which can lead to B. progressive scarring and vision loss. Photographs from the Hospital for Sick Children.
25
Congenital Trigeminal hypoplasia Riley-Day Syndrome
Goldenhar-Gorlin Syndrome Mobius syndrome/corneal hypoesthesia Familial corneal hypoesthesia
Cranial Nerve V (Trigeminal) Nerve Palsy
Surgery (trigeminal neuralgia) Neoplasm Aneurysm Facial trauma Intracranial tumor
Infections Herpes simplex Herpes zoster Leprosy
Systemic diseases Diabetes Vitamin A deficiency Multiple sclerosis
Ocular surface pathologies/trauma
Contact lens wear Surgeries resulting in ciliary nerve trauma
- Laser-assisted in situ keratomileusis (LASIK)
- Corneal incision - Lamellar and penetrating keratoplasty
(transplant) Corneal dystrophies
Toxicity Topical anaesthetics Chemical burn Timolol Betaxolol Trifluridine Sulfacetamide
Table 1-1 Causes/etiology of neurotrophic keratopathy
The prevalence of NK is five in 10,000 people worldwide (Sacchetti and Lambiase, 2014).
Because patients have absent corneal sensation, they often fail to present for care. As a
result, several patients remain undiagnosed until scarring of the cornea results in visual
decline. For a clinical diagnosis of NK, patients must present with both corneal epithelial
ulcerations and absent or significantly impaired corneal sensation. Corneal sensitivity is
traditionally measured with Cochet-Bonnet esthesiometry by recording the patient
response to pressure on the ocular surface using a nylon filament of variable length (0-6
cm) (Norn, 1975; Sacchetti and Lambiase, 2014; Semeraro et al., 2014; Mastropasqua
et al., 2017, 2018; Versura et al., 2018). First, sensitivity is measured at the
26
esthesiometer’s full length of 6 cm, and its length is decreased in steps until the patient
reports sensation or contact. This is repeated in the central cornea as well as the four
quadrants of the cornea: superior, temporal, inferior, nasal (Figure 1-10). Normal corneal
sensation is considered as between 5-6cm, whereas patients with corneal anesthesia or
hypoesthesia tend to report sensation between 0-3cm (Dua et al., 2018). More recently,
CO2 gas esthesiometry has been developed to record patient response to a burst of CO2
gas. The latter esthesiometer enables a non-contact evaluation of mechanical, chemical
and thermal neuroreceptors in the cornea by varying pressure, CO2 concentration and
temperature, respectively (Tesón et al., 2012). In vivo confocal microscopy can also be
used to evaluate and document the absence of corneal sensory innervation in the affected
cornea(s) of NK patients (Villani et al., 2014). Corneal epithelial ulcerations can be
detected with topical fluorescent dye, which stains the underlying basement membrane
or stroma, and slit lamp microscopy (Sacchetti and Lambiase, 2014).
Clinically, according to the Mackie (1995) classification, NK is classified into three stages
based on the severity of the disease. Stage I is characterized by superficial punctate
keratopathy that over time can progress to stromal scarring, and stage II by PEDs. Stage
III involves corneal ulcerations that progress to extensive stromal injury, including
perforation and stromal melting (Mackie 1995). The cause and severity of the disease
determine the prognosis of, and treatment for, NK patients.
III - MOLECULAR BASIS OF DISEASE
Figure 1-10 Corneal esthesiometry. Corneal sensation is measured in A the central cornea, and B the peripheral cornea in four quadrants.
B
A B B
B
27
Corneal sensory nerves are an important source of trophic mediators as described above
in Chapter 1.3.IV (Figure 1-6). Studies have shown that the absence of corneal
innervation, in animal models, results in the thinning and breakdown of the corneal
epithelium, and impairs corneal wound healing (Sigelman and Friedenwald, 1954; Alper,
1975). Within 24 hours of trigeminal ganglion ablation in a mouse model of NK,
degeneration of the corneal axons is apparent, and the corneal surface starts to become
cloudy (Ferrari et al., 2011). In the absence of corneal sensory innervation, such as in
NK, corneal epithelial maintenance and healing after injury are impaired (Sigelman and
Friedenwald, 1954; Alper, 1975). This impairment may arise from a depletion of factors
such as those listed below (Figure 1-6):
Substance P (SP), described in Chapter 1.3.IV, is decreased in tears of patients with
impaired corneal sensation (Yamada et al., 2000). Subcutaneous injections of capsaicin,
which acts to deplete SP from axon terminals, has, in previous studies with mice, resulted
in corneal changes consistent with corneal hypoesthesia and NK (Fujita et al., 1984;
Shimizu et al., 1987). SP co-localizes with sensory axons in the normal, healthy cornea
(Tervo et al., 1981, 1982; Lehtosalo, 1984), and functions to stimulate epithelial cell
proliferation (Reid et al., 1993; Garcia-Hirschfeld, Lopez-Briones and Belmonte, 1994),
upregulate epithelial cell migration, in conjunction with insulin-growth factor 1 (IGF-1)
(Nakamura, Nishida, et al., 1997; Chikama, Nakamura and Nishida, 1999) and increase
the expression of tight-junction proteins in the corneal epithelium (Araki‐Sasaki et al.,
2000; Ko, Yanai and Nishida, 2009). Thus, the corneal changes consistent with NK in the
depletion or blocking of SP (Fujita et al., 1984; Shimizu et al., 1987), is presumably due
to the impaired epithelial cell proliferation, migration, and adhesion.
Calcitonin gene-related peptide (CGRP) co-localizes with SP in corneal sensory nerves
(Beckers et al., 1993; Marfurt, Murphy and Florczak, 2001; Müller et al., 2003; He and
Bazan, 2016) and, as described in Chapter 1.3, acts synergistically with other
neuropeptides and neurotrophins to facilitate epithelial cell migration (Nishida et al., 1996;
28
Nakamura et al., 1997; Tran et al., 2000; Tran et al., 2000). CGRP depletion in absent
corneal innervation can thus contribute to NK symptoms.
Nerve Growth Factor (NGF) is a neuropeptide suggested to be released from a variety
of possible sources: corneal sensory neurons (Lambiase et al., 1998), the corneal
epithelium (Lambiase et al., 2000), corneal tears (Vesaluoma et al., 2000), and corneal
SCs (Chapter 1.3.VI). NGF’s TrkA receptors are localized in the basal epithelium of the
limbus (Touhami et al., 2002; Qi et al., 2008). In the limbal epithelium exists the Palisades
of Vogt, the LSC niche (Ahmad, 2012), suggesting that NGF may act on these LSCs to
exert its healing effect on the cornea. NGF promotes corneal wound healing by
stimulating epithelial proliferation and differentiation (Blanco-Mezquita et al., 2013). In NK,
there is an impairment in epithelial healing following corneal injury, thus implicating
innervation in the NGF-mediated LSC-dependent epithelial healing (Sacchetti et al.,
2017).
IV - ANIMAL MODELS OF NEUROTROPHIC KERATOPATHY
There are several animal models of NK that involve complete or impaired corneal
denervation. The precise anatomy of corneal innervation was established in rabbit and
macaque monkey by direct observation (Zander and Weddell, 1951a) after which the
response to nerve injury was studied to establish the origin of the sensory innervation
(Zander & Weddell,1951b). Several models of injury were used: corneal autografting,
corneal section (keratotomy), section or avulsion of the infraorbital nerve, ciliary nerve
transection, superior cervical sympathetic ganglion extirpation, and Gasserian
(trigeminal) ganglion destruction by partial removal of the temporal lobe of the cortex.
Destruction of the trigeminal ganglion or ciliary nerve transection, but not infraorbital nerve
transection or superior cervical ganglion extirpation, was shown to result in complete
corneal denervation. Nerve transection caused such severe NK symptoms that all of the
animals had to be sacrificed. The ganglionic destruction was also associated with high
mortality, but with a protective eyelid suture, tarsorrhaphy, in the surviving animals,
complete absence of sensation was reported after three weeks with a small number of
29
regenerating fibers in the cornea (Zander and Weddell, 1951b). Hence, both injury models
are not appropriate NK animal models. Intracranial transection of the trigeminal ganglion
in monkeys as compared to transection at lower levels of the corneal innervation was
shown to be successful in immediate denervation of the cornea (Alper, 1975). Either intra-
or extra-dural approaches in rats resulted in their mortality 24 hours later due to
intracranial bleeding from vessels located near the trigeminal ganglion (Sigelman and
Friedenwald, 1954). In contrast, a transpalatal intra-oral approach to cauterize and
thereby injure the ophthalmic branch (V1) of the trigeminal ganglion was successful in
denervating 60% of the corneas. Success with transpalatal thermo-coagulation of the
medial trigeminal ganglion was reported in rats (Keen et al., 1982) and rabbits (Beuerman
and Schimmelpfennig, 1980; Schimmelpfennig and Beuerman, 1982). Circumferential
transection of all nerves to the rabbit cornea via limbus incisions while being less invasive,
resulted in partial, and not complete, corneal denervation and in subsequent reinnervation
(Chan-Ling et al., 1987). Immediate success with all these various techniques with the
exception of the radial nerve transection was not followed by long-term assessments of
corneal nerve regeneration.
Systemic and subcutaneous capsaicin injection in neonatal animals results in impaired
corneal sensation, ulceration, neovascularization, and opacification and, hence, has also
been used as a model of NK (Keen et al., 1982; Fujita et al., 1984; Gallar et al., 1990;
Ogilvy et al., 1991; Marfurt et al., 1993; Donnerer et al., 1996; Nakamura et al., 2003;
Lambiase et al., 2012). Nociceptive nerve endings, such as the A-δ and C unmyelinated
sensory fibers that terminate in the corneal epithelium (Zander and Weddell, 1951a; Tervo
and Tervo, 1981; Rózsa and Beuerman, 1982), are stimulated by capsaicin via their
binding to transient receptor potential vanillin type 1 (TRPV1) (Hiura et al., 2002).
Capsaicin binding to TRPV1 in neonatal rats induces retrograde neuronal death of
sensory neurons in the trigeminal ganglion (Hiura et al., 2002). Topical capsaicin
administration has also been used in adult models of corneal hypoesthesia (Gallar et al.,
1990). Capsaicin treatment resulted in SP depletion and desensitization of corneal C-
polymodal nociceptors and some Aδ nociceptors (Belmonte and Giraldez, 1981;
Donnerer et al., 1996), but not of non-specific acetylcholinesterase-positive nerves.
30
Therefore, the latter nerves are believed to have functional properties other than chemical
nociception (Hiura and Nakagawa, 2004, 2012; Nakagawa et al., 2009).
Although less invasive, capsaicin administration as a method of corneal denervation has
several disadvantages. Gallar et al., (1990) demonstrated corneal epithelial wound
healing was impaired by combined retrobulbar and topical capsaicin administration, but
neither method of administration impaired healing alone. No study has investigated the
density of corneal innervation following topical capsaicin application but, neonatal
capsaicin administration in rats resulted in immediate corneal denervation (Ogilvy et al.,
1991; Marfurt et al., 1993; Hiura and Nakagawa, 2005). However, several weeks following
the neonatal treatment the affected cornea was reinnervated despite persistent keratitis
at the same time point (Ogilvy et al., 1991; Marfurt et al., 1993; Hiura and Nakagawa,
2004, 2005). This re-innervation was attributed to sprouting of the remaining axons after
neonatal capsaicin administration (Marfurt et al., 1993; Hiura and Nakagawa, 2005).
Neonatal capsaicin application was less successful than V1 transection in achieving
corneal denervation (Keen et al., 1982).
Ablation of corneal innervation using a stereotactic frame is a promising technique
because there is less chance for human error in achieving corneal denervation.
Transcranial (Tervo et al., 1979; Nagano et al., 2003) and transpalatal (Wong et al., 2004)
stereotactic ablation of V1 via thermo-coagulation has been performed successfully in
rats. Corneal denervation was confirmed with absent blink reflexes and clinical signs of
NK (Tervo et al., 1979; Nagano et al., 2003; Wong et al., 2004). Tervo et al., (1979)
observed complete corneal denervation up to eight days post-operatively. More recently,
Ferrari et al (2011) described a model of transcranial ablation of V1 in mice. In this model,
electrocautery was used to successfully denervate the cornea as evaluated by absence
of blink reflex, decreased epithelial proliferation and documented corneal denervation.
However, the study did not quantitatively assess nerve density before and after V1 nerve
ablation (Ferrari et al., 2011). Transcranial and transpalatal corneal denervation
procedures, although successful, do not allow for visualization of the base of the
trigeminal ganglion and ophthalmic branch to confirm V1 injury.
31
To optimize the visualization of injury to corneal innervation, Yamaguchi et al. (2013)
describe exposing the posterior orbit and transecting the long ciliary nerves. Transection
of these long ciliary nerves resulted in significant corneal denervation. However, the
absence of innervation has only been documented only up to two weeks after transection
(Yamaguchi et al., 2013).
Our laboratory developed a novel rat model of NK (Figure 1-11) in which complete corneal
denervation is observed four weeks after the initial procedure. In this rat model,
denervation is achieved through stereotactic electrocautery of V1. The stereotactic
procedure is repeated three weeks after initial ablation to prevent spontaneous
regeneration of native sensory nerves. Stromal and sub-basal nerve fibers were
quantified and were significantly decreased as compared to normally innervated corneas.
The four week denervation period is the longest documented in an animal model, and
allows for evaluation and comparison between our rat model of NK with and without
corneal neurotization treatment (J. Catapano et al., 2018), as described in Chapter 1.5.
Figure 1-11 Model of neurotrophic keratopathy (NK). Corneal denervation in our
model of NK is achieved by stereotactic ablation of the ophthalmomaxillary nerve (V1),
originating from the trigeminal ganglion (TG). A. Stereotactic set-up used for corneal
ablation B. Intracranial ablation of V1.
A B V1
TG
32
1.5 Treatments for Neurotrophic Keratopathy and Corneal Neurotization
I – INTRODUCTION
Conventional NK treatment focuses on preventing the progression of corneal damage but
fails to provide a long-term solution to the disease. Corneal neurotization in NK patients
is a novel surgical procedure that treats the underlying cause of NK by restoring corneal
innervation, and thus sensation, to the NK cornea.
II - MANAGEMENT AND TREATMENT OF NEUROTROPHIC KERATOPATHY
Early diagnosis is critical for NK patients, yet due to the absence of sensation, thus pain,
patients often fail to present for care until the disease progresses to corneal scarring and
vision loss (Reviewed by Ramaesh et al., 2007; Davis and Dohlman, 2014; Semeraro et
al., 2014; Versura et al., 2018). Initial treatment aims to prevent the corneal epithelium
from injury and desquamation and to promote healing with the use of lubricants and
pharmacological agents. The first line of defense for Stage I NK involve preservative-free
artificial tears (Reviewed by Davis and Dohlman, 2014; Semeraro et al., 2014).
Once NK progresses to Stage II, and PEDs are present, treatment is focussed on
epithelial healing and prevention of worsening ulcerations. In the presence of existing
punctate keratopathy, a scleral (Grey et al., 2012) or corneal lens, or protective glasses
can be used to protect the corneal surface (Donnenfeld et al., 1995; Ramaesh et al.,
2007). Because inflammation has been shown to diminish epithelial healing in the
absence of sensory innervation (Cavanagh et al., 1979), anti-inflammatory treatments,
such as topical corticosteroids have been suggested in treating NK (Ramaesh et al.,
2007; Sacchetti and Lambiase, 2014). Several studies suggest an improvement in the
rate of epithelial wound healing in response to topical steroid treatments in both animals
and humans (Ashton and Cook, 1951; Gasset et al., 1969; Phillips et al., 1983). However,
this improvement is dependent on the dose of treatment (Ashton and Cook, 1951; Gasset
33
et al., 1969; Sugar et al., 1985). Steroids have also been shown to inhibit stromal healing
and endothelial recovery and thus, may increase risk of stromal melting or perforation
(Ashton and Cook, 1951; Gasset et al., 1969). Non-steroidal anti-inflammatory agents, in
contrast, neither impair nor improve corneal wound healing (Srinivasan, 1982; Hersh et
al., 1990). To avoid the toxic effect of topical steroid treatments, lubricants such as
preservative-free artificial tears are continued in conjunction with prophylactic antibiotics
(Semeraro et al., 2014; Mastropasqua et al., 2017).
Once NK progresses to stage III, surgical interventions are used to preserve ocular
integrity for persistent and recurrent corneal ulcerations. To prevent epithelial ulceration
from desiccation and desquamation, partial or complete tarsorrhaphy, surgical closure of
the eyelids, is performed. Eyelid injection with botulinum toxin can provide a protective
ptosis, namely the falling of the eyelid, to cover the insensate cornea (Kirkness et al.,
1988). In cases involving stromal injury or corneal perforation, conjunctival flaps are
raised to cover the corneal abrasion and sutured in place (Gundersen and Pearlson,
1969). Surgical amniotic membrane transplants on the corneal surface promote epithelial
healing in cases of refractory corneal ulcers (Lee and Tseng, 1997; Khokhar et al., 2005).
Perforations of less than 3 mm are closed with cyanoacrylate glue (Fogle et al., 1980;
Ramaesh et al., 2007; Sacchetti and Lambiase, 2014; Semeraro et al., 2014). Corneal
transplantation may be required in patients with more severe stage III NK to restore
corneal architecture. However, the success of these corneal grafts is poor in this NK
patient population. This is due to the nature of the disease, namely the absence of corneal
innervation and thus the trophic influence of corneal sensory nerves, that often results in
recurrent ulceration and scarring of the corneal graft (Jhanji et al., 2011; Lambley et al.,
2014).
Recent interventions have been targeted at utilizing biological agents to restore the
trophic mediators normally provided by corneal sensory innervation. Topical SP, in
conjunction with IGF-1, has been used to treat corneal ulcers in patients presenting with
PEDs (Brown et al., 1997; Chikama et al., 1998; Yamada et al., 2008), and SP was shown
to work synergistically with IGF-1 to heal corneal ulcers in animal models of spontaneous
34
chronic corneal epithelial defects in dogs (Murphy et al., 2001), NK achieved by corneal
denervation in rats (Nagano et al., 2003) and capsaicin-induced NK in rats (Nakamura et
al., 2003). Growth factors, such as NGF, increase corneal and limbal epithelial cell
proliferation in vitro (Kruse and Tseng, 1993), and promote epithelial healing after corneal
injury in rats (Lambiase et al., 2000). Topical application of exogenous NGF has been
used to treat corneal epithelial ulcers in patients with NK. NGF eye drops are well
tolerated in patients (Ferrari et al., 2014) and, in response to NGF treatment, patients
suffering from NK and other ocular surface diseases demonstrated improved corneal
sensitivity and reduced corneal ulceration in clinical trials (Lambiase et al., 1998; Bonini
et al., 2000; Tan et al., 2006; Lambiase et al., 2012; Sacchetti et al., 2017). However, this
topical NGF treatment primarily targets patients and animal models that present with
corneal hypoesthesia. In a case of congenital NK, topical application of exogenous NGF
significantly improved epithelial healing but, this treatment alone was insufficient to
prevent progressive stromal opacification (Tan et al., 2006). Autologous serum contains
nerve-derived trophic factors and growth factors such as SP, IGF-1 and NGF (Matsumoto
et al., 2004). It is used clinically to treat NK patients and seems to restore epithelial
integrity and improves healing of PEDs, further implicating improved treatment efficacy of
a combination of these factors (Tsubota et al., 1999; Matsumoto et al., 2004; Soni and
Jeng, 2016). Platelet rich plasma and umbilical cord serum have similarly been shown to
improve corneal epithelial health, perhaps more effectively than autologous serum (Soni
and Jeng, 2016; Giannaccare et al., 2017).
The aforementioned treatment options fail to address the underlying cause of NK (lack of
innervation) and require daily ophthalmic management and frequent physician follow-up.
Despite optimal ophthalmic management, patients continue to develop ulcerations which
can lead to progressive scarring and eventually vision loss.
III - CORNEAL NEUROTIZATION AND OTHER METHODS OF RE-INNERVATION
The first known attempt at surgical re-innervation of the cornea was reconstruction of the
damaged ophthalmic nerve (Samii, 1981). The procedure that was used involves a
35
craniotomy to expose the damaged intracranial ophthalmic nerve and subsequently
transecting and coapting a sural nerve graft to the extracranial occipital nerve. The
authors reported improved vision and corneal health, but no improved corneal sensation
post-operatively. Further, the injured ophthalmic nerve is difficult to access, and the
operative approach causes significant patient morbidity.
Neurotization is a surgical procedure in which a healthy donor nerve is used to restore
motor and/or sensory function in a tissue lacking innervation. This procedure is successful
for a range of clinical conditions including brachial plexus injuries (Wahegaonkar et al.,
2007), facial paralysis (Hontanilla, Marre, and Cabello 2014) and more. Terzis et al (2009)
described neurotization as a means of treating NK. This procedure involves reinnervating
the NK cornea with the patients’ contralateral supratrochlear and supraorbital nerves. The
procedure involves a bicoronal incision which is necessary to dissect enough length from
the donor nerves to re-innervate the contralateral cornea. The donor nerves are tunnelled
to the contralateral eye, separated into fascicles, and sutured to the conjunctival sac
adjacent to the corneal limbus. Six patients suffering from NK for a mean (± standard
deviation) of 7 ± 8.56 years were treated and demonstrated improvement in corneal
sensation, visual acuity and corneal health post-operatively at 2.80 +/- 2.17 years post-
operatively (Terzis et al., 2009). In one recent case, Gennaro et al., 2019 adapted Terzis’
direct technique to use the homolateral infraorbital nerve to directly reinnervate the cornea
in combination with facial reanimation procedure (Gennaro et al., 2019). Few other
reports have published using Terzis’ technique to treat NK (Allevi et al., 2014; Jacinto et
al., 2016). This may be due to the disfiguration and invasiveness associated with the
bicoronal incision required in this technique.
To eliminate the need for a bicoronal incision, Terzis’ direct corneal neurotization
technique was modified by Borschel, Zuker, and Ali at The Hospital for Sick Children
(Figure 1-12) (Elbaz et al., 2014; Bains et al., 2015; Fung et al., 2018; Catapano et al.,
2019). In this procedure, the surgeons co-apt the patients’ sural nerve graft to the
supratrochlear or other healthy donor nerve, contralateral to the NK cornea. The sural
autograft is subcutaneously tunnelled into the patients’ affected eye and separated into
36
fascicles which are then sutured directly into the corneal stroma (Elbaz et al., 2014; Bains
et al., 2015; Fung et al., 2018; Catapano et al., 2019). Although the modified procedure
involves harvesting the sural nerve graft from the patients’ lower leg, sural nerve graft
harvest was associated with minimal morbidity in both adults (Miloro and Stoner, 2005;
Hallgren et al., 2013) and paediatric patients (Lapid et al., 2007). Upon evaluation of 19
eyes that underwent the modified indirect corneal neurotization, there was a significant
improvement in corneal sensation 24 +/- 16.1 (mean +/- SD) months post-operatively.
Figure 1-12 Corneal neurotization. End-to-end nerve repair is performed. The patient’s
sural nerve autograft is attached to the supratrochlear nerve. The graft is separated into
fascicles, and the fascicles are sutured around the NK affected cornea. Illustrated by
Farheen Ali.
These patients also demonstrated an improved best corrected visual acuity (BCVA) and
corneal epithelial health (Catapano et al., 2019). In another study, our group
demonstrated reinnervation of the cornea after corneal neurotization in NK patients using
in vivo confocal microscopy (Figure 1-13) (Fung et al., 2018). The indirect corneal
neurotization technique developed by Borschel, Zuker, and Ali has been adopted by
several centers across the world (Ting et al., 2018; Weis et al., 2018). Other variations of
37
indirect corneal neurotization involve the use of great auricular nerve grafts (Benkhatar et
al., 2018; Jowett and Pineda, 2018) and cadaveric acellular nerve allografts (Leyngold et
al., 2019) have been reported.
Figure 1-13 In vivo confocal microscopy of subbasal nerves before and after corneal neurotization A. Patient’s cornea six months before corneal neurotization and
B. corresponding in vivo confocal microscopy image with absent corneal nerves in
subbasal cornea. C. Patient’s cornea six months after corneal neurotization, arrows
pointing to nerve fascicles from autograft and D. corresponding in vivo confocal
microscopy image demonstrating re-innervating axons in subbasal corneal layer.
(Reproduced from Fung et al, 2018 with permission from Elsevier: License
#4595690930627)
38
Variable improvement in post-operative BCVA was documented in most reports of
corneal neurotization. Leyngold et al (2019) reported a significant BCVA improvement
from 20/70 to 20/20 in one case. However, this patient underwent the procedure nine
months after NK diagnosis before incidence of corneal scarring (Leyngold et al., 2019).
In comparison, patients treated by other groups were diagnosed with NK at least five
years prior to corneal neurotization and many presented with PEDs and scarring pre-
operatively (Benkhatar et al., 2018; Jowett and Pineda, 2018; Catapano et al., 2019).
Although Leynold et al (2019) demonstrated success with the use of an acellular nerve
allograft in this case-report, nerve autografts are the gold standard for nerve
reconstruction following nerve damage (Patel et al., 2018). Terzis et al (2009) reported
only one of six patients presenting with a high corneal sensation post-operatively
measured by Cochet-Bonnet esthesiometry, and low corneal sensation (<20 mm) in two
patients. In contrast, Catapano et al (2019) found that 11 and 13 of 19 eyes presented
with high central and peripheral corneal sensation, respectively. Only one patient in this
study presented with low corneal sensation post-operatively. The more efficient indirect
re-innervation by Borschel, Zuker and Ali, compared to direct corneal neurotization, may
be due to patient age, surgical technique and the number of axons in the donor autograft.
Although promising, about 15% of patients treated did not reach levels of what is
considered protective corneal sensation (>20 mm) and continue to suffer from NK
symptoms (Catapano et al., 2019).
IV - ANIMAL MODELS OF CORNEAL REINNERVATION AND NEUROTIZATION
Previous animal models have exclusively focussed on corneal reinnervation from native
corneal nerves. These models investigated corneal re-innervation in HSV-1 keratitis
(Martin, 1996; Lambiase et al., 2008), diabetic keratopathy (Chikamoto et al., 2009; Yin
et al., 2011), lamellar flap surgery (Namavari et al., 2011; Chaudhary et al., 2012), corneal
abrasion (Li et al., 2011), and corneal transplantation (Omoto et al., 2012). Infectious and
metabolic diseases such as HSV-1 keratitis (Martin 1996; Lambiase et al. 2008), diabetic
keratopathy (Chikamoto et al., 2009; Yin et al., 2011) result in diffuse damage to corneal
innervation. However, a portion of corneal nerves remain intact. Damage to portions of
39
corneal innervation can also occur due to lamellar flap surgery (Namavari et al., 2011;
Chaudhary et al., 2012), corneal abrasion (Li et al., 2011), and corneal transplantation
(Omoto et al., 2012). Partially intact corneal innervation provides a potential source of
corneal reinnervation and thus, these reinnervation models are not applicable in models
of complete corneal denervation.
Catapano et al (2018) developed the first and only animal model of corneal neurotization
using a donor nerve (Figure 1-14), modelled after the technique in NK patients (Elbaz et
al., 2014; Bains et al., 2015). In the rat model, corneal denervation was achieved by
stereotactic ablation of the ophthalmomaxillary nerve (V1), and sural and common
peroneal (CP) nerve autografts were used to re-innervate the denervated cornea. The
procedure significantly increased nerve fiber density in the denervated and reinnervated
cornea as compared to denervation alone (J. Catapano et al., 2018) (Figure 1-15).
Although normally innervated and neurotized corneas exhibited the same nerve density
quantitatively, the nerve density of the neurotized cornea was not as uniform and did not
display the whorl pattern of the normally innervated subbasal nerve plexus. This may be
due to the differences during development and in adulthood in the corneal architecture.
These include differences in the arrangement of stromal lamellae and/or the presence or
absence of axonal guidance molecules. Further, in the rat NK model, only a small
proportion of regenerating nerve fibers from the donor autografts reinnervated the cornea
(J. Catapano et al., 2018). The sural and CP nerve autografts contain both myelinated
and unmyelinated nerve fibers, thus perhaps the cornea regulates which and what
number of nerves reinnervate the NK cornea. The possible reasons for differences in the
pattern of corneal innervation and reinnervation, and why only a small proportion of fibers
reinnervate the cornea from the grafts need to be explored further. Additionally, the
reinnervated corneas were clear suggesting that the reinnervating axons were
unmyelinated. The animal model of corneal neurotization provides an ideal method to
study the effects of corneal reinnervation in NK and a means to understand how to
improve outcomes for NK patients who have not benefited from the procedure.
40
Figure 1-14 Model of corneal neurotization. A. Corneal neurotization is performed
through coaptation of the common peroneal and sural nerves to the infraorbital nerve in
the rat. The donor autografts are guided to reinnervate the contralateral NK cornea. After
the procedure, a tarsorrhaphy is performed to prevent damage to the nerve grafts and to prevent
corneal ulceration in our models (Illustrated by Kasra Tajdaran). Above, B. the nerve grafts
overlay the cornea, C. the nerves are then brought below the conjunctiva and sutured
directly to the cornea, D. the conjunctiva is subsequently closed over the grafts to promote
revascularization.
Normal innervation Corneal denervation Corneal denervation + neurotization
A B C
41
Figure 1-15 Corneal reinnervation after neurotization. Demonstration in a Thy-1 GFP+
rat that expresses a green fluorescent protein (GFP) in axons of A. the normal innervation
of the cornea; B. loss of GFP positive axons four weeks after corneal denervation, and
C. reinnervation of the NK cornea, four weeks after denervation. (Catapano et al., 2018)
V - WOUND HEALING AFTER CORNEAL NEUROTIZATION
There is a well-documented impairment in epithelial healing after injury in the NK cornea
(Mikulec and Tanelian, 1996; Nagano et al., 2003; Nakamura et al., 2003). Many
conventional NK treatments aim to potentiate corneal epithelial cell proliferation and thus
healing using topical agents as discussed in Chapter 1.5.II. Several animal models of NK
and diabetic keratopathy also demonstrate an impairment in corneal healing following
epithelial injury (Araki et al., 1993; Xu and Yu, 2011; Ljubimov and Saghizadeh, 2015),
which is improved with the use of topical agents such as NGF (Lambiase et al., 2000),
SP and IGF-1 (Nagano et al., 2003; Nakamura et al., 2003), and corticosteroids (Ashton
and Cook, 1951; Gasset et al., 1969; Phillips et al., 1983). Corneal neurotization in human
patients has decreased the incidence of clinically evaluated PEDs, suggesting an
improvement in epithelial cell proliferation and healing (Terzis et al., 2009; Catapano et
al., 2019). Catapano et al. (2018) demonstrated that corneal neurotization significantly
improves epithelial healing in the rat denervated cornea (Figure 1-16 A). Corneal
epithelial injury/de-epithelialization was stained with fluorescein, which was observed
under a UV Wood’s lamp. Four days after de-epithelialization over the entire cornea,
corneal neurotization completely re-epithelialized the denervated cornea, mirroring the
healing observed in normally innervated corneas. In contrast, complete re-
epithelialization of the denervated cornea was absent in the untreated cornea (J.
Catapano et al., 2018) (Figure 1-16 B). The marked improvement in corneal wound
healing in our NK rat model that received the corneal neurotization, was attributed to the
re-innervating axons of the graft. However, the mechanism(s) by which corneal
reinnervation influences epithelial health and proliferation after injury, remains unknown.
42
Figure 1-16 Corneal healing in the denervated cornea is improved with corneal neurotization. A. Corneal neurotization (middle row) improved corneal healing over time
in the NK cornea compared to corneal denervation alone, shown in the bottom row. B.
Area of corneal healing was quantitatively assessed and there was a significant
improvement in healing in the neurotized group compared to the denervated group four
days after corneal epithelial debridement. (Catapano et al., 2018).
Day 0 Day 1 Day 2 Day 3 Day 4
0 1 2 3 4 Days after injury
43
1.6 Thesis Aims and Hypothesis
The primary aim of this thesis is to investigate the effect of corneal neurotization (corneal
reinnervation using peripheral nerve autografts) on corneal epithelial maintenance of a
denervated cornea in a model of neurotrophic keratopathy (NK). Corneal neurotization is
a surgical procedure that has restored corneal sensation and innervation in the majority
of NK patients, but, 15% of patients undergoing the procedure failed to reach levels of
protective corneal sensation (Catapano et al., 2019). Our laboratory has developed a rat
model of NK and corneal neurotization which can be used to better understand the effects
of the procedure. Using the rat models, the aims of the experiments described in Chapter
2 of this thesis are:
AIM 1 – To investigate the effect of corneal neurotization on corneal epithelial and stromal
thickness of a denervated cornea in a rat model of NK.
Rationale. The cornea is responsible for two thirds of the refraction of light that passes
through it. Hence, maintaining and/or restoring corneal thickness is important for visual
acuity. In NK, there is thinning of the corneal epithelium (Alper, 1975; Ferrari et al., 2011)
due to an impairment of epithelial cell proliferation, migration and adhesion (Sigelman
and Friedenwald, 1954; Müller et al., 2003). Clinically, NK can be associated with stromal
inflammation as well as stromal ulceration (melting) and perforation (Dua et al., 2018).
However, the effect of denervation on the stroma and stromal thickness is not well
studied. Stromal thickness in the absence of corneal abrasion (protected by a
tarsorrhaphy) has not yet been evaluated in corneal denervation or corneal reinnervation.
Hypothesis. Corneal neurotization prevents or reverses corneal epithelial thinning and
stromal thickness abnormalities in the denervated cornea.
AIM 2 – To investigate the effect of corneal neurotization on epithelial ulceration, and
corneal scarring and perforation after corneal denervation in the same rat model of NK.
44
Rationale. Impairment of epithelial cell proliferation (Reid et al. 1993; Garcia-Hirschfeld
et al., 1994), migration (Mikulec and Tanelian, 1996; Nakamura, Nishida, et al., 1997;
Chikama et al., 1998) and adhesion (Araki et al., 1993; Ko et al., 2009) leads to more
severe, and characteristic symptoms of NK in patients. These include corneal epithelial
breakdown or ulceration and progressive corneal scarring. In some cases, NK can result
in perforations of the cornea (Semeraro et al., 2014; Mastropasqua et al., 2017). The
question remains as to whether reinnervation by peripheral nerves can reverse these
changes.
Hypothesis. Corneal neurotization protects the denervated cornea from epithelial
ulceration, scarring, and perforation.
AIM 3 – To evaluate if, in the denervated cornea, nerve-derived peptides Substance P
(SP) and calcitonin gene-related peptide (CGRP) are restored after corneal neurotization.
Rationale. Nerve-derived peptides SP and CGRP are critical in maintaining the integrity
of the corneal epithelium by promoting corneal epithelial cell proliferation, migration, and
adhesion in the cornea (Sigelman and Friedenwald, 1954; Müller et al., 2003).
Subcutaneous capsaicin injection, that blocks SP function, in mice results in typical
symptoms of NK, including corneal opacification (Fujita et al., 1984; Shimizu et al., 1987).
Furthermore, concentrations of nerve-derived peptides are decreased in tears of patients
with reduced corneal sensation (Yamada et al., 2000).
Hypothesis. Following corneal neurotization, the reinnervating axons restore the nerve-
derived peptides, SP and CGRP, to the denervated cornea.
45
CHAPTER 2 Manuscript
Chapter 2.2.V and 2.3.II is modified from the following: Catapano, J. (2018). Corneal Neurotization Improves Ocular Surface Health in a Novel Rat Model of Neurotrophic Keratopathy and Corneal Neurotization, IOVS, 4345-4354.
46
2.1 INTRODUCTION Corneal sensory innervation preserves corneal transparency and shape, allowing for
transmission of light to the retina for visual processing. Corneal sensory nerves not only
protect the corneal surface through blinking and tearing reflexes, but also release trophic
factors that maintain corneal integrity and aid in corneal healing after injury. In the
absence of sensory innervation of the cornea, patients develop neurotrophic keratopathy
(NK). This disease is characterized by corneal epithelial breakdown (ulceration),
progressive scarring and eventual vision loss (Bonini et al., 2003; Ramaesh et al., 2007;
Sacchetti and Lambiase 2014). Standard treatments with topical agents and protective
lenses (Sacchetti and Lambiase, 2014; Semeraro et al., 2014; Mastropasqua et al., 2017;
Versura et al., 2018), fail to treat the underlying cause of NK, namely the absence of
protective corneal sensation.
The absence of innervation in NK leads to epithelial cell abnormalities and failure of the
cornea to protect itself from the effects of trauma, drying and further infection. The corneal
epithelium is the most superficial layer of the cornea, and thus forms a protective barrier
against the external environment. Maintenance of corneal epithelial thickness and corneal
clarity is essential for normal vision. In both animal models and patients, NK results in
corneal epithelial thinning and ulceration, and can lead to eventual corneal scarring and
vision loss (Alper, 1975; Semeraro et al., 2014; Mastropasqua et al., 2017). The thinning
of the corneal epithelium in animal and patients appears to be the result of impaired
corneal epithelial adhesion and proliferation during normal epithelial turnover,
demonstrated in animal models (Sigelman and Friedenwald, 1954; Alper, 1975). This
impairment is presumably due to loss of nerve-derived trophic support in NK (reviewed
by Sigelman and Friedenwald 1954). Epithelial thinning affects the refraction of the light
passing through the cornea, resulting in lower visual acuity (Reinstein et al., 2008).
Additionally, the morphology of corneal epithelial cells is altered in the rabbit denervated
corneas with the cells exhibiting swelling (Gilbard and Rossi, 1990) and, in the surface
epithelial cells, the loss of microvilli (Alper, 1975). It is important to note that the
47
morphology of corneal epithelial cells may be different between species when translating
information learned from animal models pertaining to humans (Henriksson et al., 2009).
Epithelial cell loss and abnormalities lead to diminished release of epithelial cell-derived
soluble factors, which are involved in stromal healing and remodeling in response to injury
or infection (Sacchetti and Lambiase, 2017). In the normally innervated corneal stroma,
nerve fibers travel parallel to stromal lamellae, the precise organization of which is
essential for corneal clarity (Maurice, 1957, 1970; Jester et al., 1999; Hassell and Birk,
2010), before branching to eventually terminate in the corneal epithelium. A minority of
corneal nerve fiber endings are found within the stroma. Some of these fibers terminate
in close proximity to keratocytes, specialized fibroblasts involved in stromal healing after
injury (Muller et al., 1996; Seyed-Razavi et al., 2014). Corneal infection and injury can
increase stromal ulceration (melting), and can lead to stromal scarring and perforation.
This can result in corneal opacification and eventually corneal blindness (Reviewed by
Dua et al., 2018). In severe cases, corneal perforations are treated with cyanoacrylate
glue (Fogle, Kenyon and Foster, 1980; Semeraro et al., 2014), conjunctival flaps
(Gundersen and Pearlson, 1969), or keratoplasty (Jhanji et al., 2011; Lambley et al.,
2014).
Substance P (SP) and calcitonin gene-related peptide (CGRP) are co-localized with
axons in the cornea. The initial impairment in epithelial cell proliferation (Reid et al. 1993;
Garcia-Hirschfeld et al., 1994), migration (Mikulec and Tanelian, 1996; Nakamura,
Nishida, et al., 1997; Chikama et al., 1998) and adhesion (Araki et al., 1993; Ko, Yanai
and Nishida, 2009), that leads to more severe symptoms of NK in animal models, has
been attributed to loss of these nerve-derived peptide factors the functions of which have
been confirmed in vitro. NK treatments in patients have, in the past, focused on restoring
trophic support through the use of these peptides administered topically in combination
or separately (Brown et al., 1997; Chikama et al., 1998; Yamada et al., 2008). A recently
developed surgical technique of corneal neurotization by a peripheral nerve via
implantation of an autograft in NK patients restored innervation in the corneas of NK
patients with resulting increased sensation and improved corneal health. (Elbaz et al.,
48
2014; Bains et al., 2015; Fung et al., 2018; Catapano et al., 2019). However, a small
percentage of patients fail to reach standards for normal corneal sensation (Catapano et
al. 2019).
In this study, we asked the question of whether corneal neurotization first, restores the
nerve-derived peptides SP and CGRP in the denervated cornea as a model of NK, and
second, whether it improves corneal epithelial integrity. We used our recently developed
rat model of NK, in which the cornea is denervated by transection of the
ophthalmomaxillary nerve, and of corneal neurotization in which the denervated cornea
is reinnervated by the contralateral infraorbital nerve via a peripheral nerve autograft. We
used hematoxylin and eosin staining of corneal sections to compare epithelial and stromal
thickness in denervated, reinnervated and normally innervated corneas. Fluorescein
staining was used to assess the extent of ulceration under UV light, and scarring and
perforation were assessed under normal light. Third, the presence of SP and CGRP in
the cornea of the three groups was determined and measured with immunohistochemistry
and Western blotting, respectively. Our findings demonstrate that corneal neurotization
promotes epithelial integrity in the denervated NK cornea by preventing epithelial thinning,
ulceration, scarring and perforation. Furthermore, the re-innervating axons restore SP
and CGRP in the denervated cornea.
2.2 MATERIALS AND METHODS I – Animals
Female Sprague Dawley rats (250-350g) were used in all experiments. All experiments
were approved by The Hospital for Sick Children Laboratory Animal Services and
adhered to the guidelines of the Canadian Council on Animal Care as well as the ARVO
Statement for the Use of Animals in Ophthalmic and Vision Research. Rats were
maintained in a 12:12 hour light:dark cycle in a temperature- and humidity-controlled
environment. Rats received ad libitum water and standard rat chow (Purina, Mississauga,
ON, Canada).
49
II – Sample size and group distribution
Rats (total n = 45) were split into three groups: one control group (normally innervated
cornea) and two experimental groups i) corneal denervation (NK model) and ii) corneal
denervation + corneal neurotization (Table 2-1). Seven rats per group (n=21) were used
for evaluation of corneal epithelial and stromal thickness. Corneal sections from these
same rats were used for immunostaining. Three additional rats from each group (n=9)
were used for Western Blot analysis. Five rats with only corneal denervation, and 10 that
received corneal neurotization were assessed for corneal epithelial ulceration using
fluorescein staining.
Normally innervated
cornea (control)
Corneal denervation (NK model,
negative control)
Corneal denervation +
corneal neurotization
n/group
V – Evaluation of
corneal epithelial and
stromal thickness
7 7 7 21
VI – Evaluation of
corneal ulceration,
scarring and
perforation
0 5 10 15
VII –
Immunohistochemistry
1 1 1 (biological
replicates of
IV)
VIII – Western blot 3 3 3 9
Total n 45
Table 2-1. Sample size & group distribution
50
III – Sterile surgeries
Surgical procedures were performed in an aseptic manner using an operating microscope
(Leitz, Willowdale, ON, Canada). Rats were operated on under inhalational anesthetic
(2% isoflurane in 98% oxygen; Halocarbon Laboratories, River Edge, NJ, USA) and given
metacam (2 mg/kg; Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO, USA) for pain
relief. At the final outcomes assessment, rats were euthanized under deep anesthesia
using intraperitoneal Euthanyl (sodium pentobarbital, 240 mg/mL concentration, 1 mL/kg;
Bimeda-MTC, Cambridge, ON, Canada).
Figure 2-1 Rat models. In our rat model, A. denervation of the left cornea was achieved
with stereotactic ablation of the left ophthalmomaxillary nerve (V1), and B. corneal
neurotization was achieved by coapting common peroneal (CP) and sural nerve
autografts to the right infraorbital nerve and implanting the grafts into the left cornea
(illustrated by Kasra Tajdaran).
Corneal denervation and corneal neurotization in a rat model of NK was described in
detail previously (Joseph Catapano et al., 2018). Briefly, denervation of the left cornea
was achieved by stereotactic ablation of the left ophthalmomaxillary nerve (V1) (Figure 2-
1A). This ablation was performed using a modified insulated 22-G monopolar electrode
(UP 3/50; Pajunk GmbH, Geisingen, Germany), in which 1mm of the insulation at the
Ophthalmomaxillary Nerve (V1)
Trigeminal Ganglion
Trigeminal ganglion
A B
51
electrode tip was removed with a scalpel prior to the ablation surgery. In surgery, rats
were mounted onto a stereotactic frame (Harvard Apparatus, Hollingston, MA, USA). A
midline cranial incision was made to expose bregma. Bregma is the location where the
sagittal and coronal sutures of the skull cross, from which the coordinates of V1 were
calculated. A 1 mm burr-hole was made through the skull with a drill 2.0 mm anterior and
1.5 mm lateral (left) to bregma. The modified electrode was then lowered to a depth of 10
mm through the burr hole, such that the non-insulated portion of the electrode pierced
through V1. Subsequently, the ophthalmomaxillary nerve was ablated (10 W for 60
seconds) using an electrosurgical generator (Force FC-8C; Medtronic, Fridley, MN, USA).
The electrode was then removed, and the skin incision sutured. Ablation was confirmed
with observation of a blown pupil, absent blink reflex and absent response to suturing of
the eyelid. The coordinates of V1 were confirmed after sacrifice.
Corneal neurotization was performed under an operating microscope (Figure 2-1B). A
skin incision made connecting the greater trochanter and knee joint, as well as the knee
and ankle joint. Thereafter the biceps femoris muscle was separated from the vastus
lateralis muscle to expose, dissect, and harvest ~30 mm of the common peroneal (CP)
nerve. The gastrocnemius muscle was freed from the tibialis anterior muscle to expose,
dissect and harvest the same length of the sural nerve. The nerves were each placed on
a saline soaked gauze until the coaptation of the two nerves to the proximal stump of the
transected right infraorbital nerve and tunneled subcutaneously to the left cornea (Fig. 2-
1B). The latter nerve was exposed an incision that was made parallel to the right
contralateral whisker pad and the nerve branches to the whisker pad were identified. Two
branches were transected and the proximal stump of each cross-sutured to the CP and
to the sural autografts. The autografts were tunneled under the conjunctiva and loosely
sutured onto the corneoscleral junction at the 12 and 6 o’clock position with 9-0 vicryl.
Finally, the conjunctiva was closed over the grafts to promote revascularization.
A protective tarsorrhaphy, suture of the eyelids, was applied after each corneal
neurotization and denervation surgery to prevent damage to the cornea after corneal
denervation and to protect the autografts following corneal neurotization.
52
Timeline. In the treatment group, corneal neurotization was performed six weeks prior to
the first corneal denervation to allow sufficient time for the axons to grow through the
grafts. In the neurotized and NK groups, the second denervation was performed three
weeks after the first to prevent native corneal nerves from re-innervating the cornea.
Subsequent assessments were performed beginning four weeks after initial corneal
denervation, (Figure 2-2).
Figure 2-2 Experiment timeline. Timing of corneal neurotization, denervation,
tarsorrhaphy removal and corneal harvest represented on timeline. Experiments to which
these procedures belong are represented by the assigned roman numerals within the
Material and Methods section (V – epithelial and stromal thickness; VI – ulceration,
scarring and perforation; VII – immunohistochemistry; VIII – western blot). Rat groups are
denoted by colour and number (figure legend in top right corner). In group 1, corneal
neurotization is performed 6 weeks prior to corneal denervation. In groups 1 and 2,
corneal denervation is performed twice, 3 weeks apart. One week after the second
denervation the affected corneas are harvested with the exception of the rats undergoing
observation of the ocular surface for ulceration (VI) in which corneas are harvested one
week later.
I
I
53
IV – Corneal harvest
At the time of harvest, eyes were enucleated and whole-globes were fixed in 4%
paraformaldehyde (PFA) and 0.2% saturated picric acid in 1X Phosphate Buffer Solution
(PBS) for 15 minutes over ice. The cornea and limbus were then dissected and fixed for
another 45 minutes over ice. Following fixation, corneas were placed in 30% sucrose, 1X
PBS overnight in 4 C. Corneas were embedded in OCT, kept in a -80 C freezer, and
cut in 10 μm cross-sections onto gel-coated slides (Fisherbrand Superfrost Plus
Microscope slides) using a cryostat (Lecia CM3050).
V – Evaluation of corneal epithelial and stromal thickness
Denervated corneas and those denervated corneas which were neurotized (n=7 per
group) were harvested four weeks after the initial V1 ablation (Figure 2-2 A). Normal
unoperated and innervated corneas (n=7) were harvested at the same timepoint. Three
10 μm cross-sections from each cornea were sampled from representative areas of the
cornea and stained with Hematoxylin and Eosin. Sections were subsequently imaged with
bright-field microscopy under a 20X objective (overall 200X magnification; Leica). The
central and peripheral epithelial and central stromal thicknesses of each section were
measured using the computer program ImagePro.
VI – Evaluation of corneal ulceration, scarring and perforation
Four weeks after the initial V1 ablation, the tarsorrhaphy was removed in all rats exposing
the denervated cornea (Figure 2-2 B, D). The denervated corneas in the rats that did not
receive treatment (negative control, n = 5) were compared to those in which corneal
neurotization was performed (treatment group, n = 10). Standardized digital photographs
(Nikon D 5100; Nikon, Tokyo, Japan) of the corneas were taken daily for one week. To
keep the camera at a fixed distance from the cornea, imaging was performed using a
standardized frame. The photographs were obtained under i) normal light and ii) with a
Wood’s lamp/fluorescein staining (DioFluor Strips; Innova Medical Opthalmics, Inc.,
Toronto, Canada) to assess corneal scarring and epithelial breakdown, respectively.
Perforation was assessed by in vivo observation of a hole in the cornea. Incidence of
ulceration and perforation was considered a binary outcome that was compared between
54
experimental groups using a Fisher’s exact test. The area of de-epithelialization was
calculated as a percentage of the entire cornea using the computer program ImageJ.
VII - Immunohistochemistry
Slides were permeabilized with methanol for 10 minutes at -20 C. Slides were
subsequently washed with 1X PBS at room temperature (RT). Antigen retrieval was
performed using a citrate buffer solution (Sigma Aldrich) that was boiled in a microwave.
Slides were put into solution and brought back to a boil before cooling over ice to RT. The
boil and re-cooling were repeated. Slides were then washed in 1X PBS and subsequently
incubated for two nights with primary antibody (See Table 1) diluted with 1X PBS with
0.1% Triton X and 5% serum in 4 C. Control corneal sections were incubated with the
aforementioned diluting solution without primary antibodies. Slides were washed with 1X
PBS, incubated with secondary antibodies (See Table 1) in 1X PBS with 0.1% Triton X
and 5% serum in RT for 30 min. Subsequently slides were washed at RT and mounted.
Slides were imaged using Zeiss AxioVision 4.8.2 software under a fluorescent Zeiss
Axioplan 2 upright microscope (Toronto, ON) with a Hamamatsu ORCA-R2 C10600
camera (Bridgewater, NJ) at 20X magnification.
Primary antibody
Dilution Host Company Secondary antibody
Dilution Company
Substance P (SP)
1:50 Mouse Santa-Cruz Biotechnology
Cy3 1:1000 Jackson Immuno
Calcitonin gene-related peptide (CGRP)
1:50 Rabbit Sigma-Aldrich anti-biotin
1:1000 Jackson Immuno
Streptavidin-Cy5
1:500 Jackson Immuno
ß-iii Tubulin
1:300 Chicken Abcam 488 1:1000 Jackson Immuno
Table 2-2. Antibody solutions and dilutions used in thin-section and whole-mount
immunohistochemistry
55
VIII - Western blot
For immunoblotting analysis of Substance P, three corneas were harvested from each
group four weeks after initial denervation (Figure 2-2 A). After washing twice with 1X PBS,
the corneas were homogenized with 300 µl of lysis buffer (150 mM Tris-HCl, pH 8.0, 150
mM NaCl, 2 mM EDTA, 0.1% Sodium dodecyl sulphate, 1% Nonide P-40, 0.5%
Sodium Deoxycholate, 10% protease inhibitors) on ice. After centrifuging at 10,000 rpm
for 30 min at 4 °C, the supernatant was collected.
Protein concentration was measured using a BCA protein assay kit [Bovine Serum
Albumin (BSA) set, Thermo Fisher Scientific, cat. no. 23208] with BSA as the standard.
Tissue lysates (40 µg for each sample) were resolved on 10% SDS-PAGE mini
electrophoretic gels. Following SDS-PAGE, the protein was transferred onto PVDF
membrane (Immuno-Blot PVDF membranes, Bio-Rad Laboratory, cat. No. 162-0177) and
subjected to electrophoresis for 1.5 hours at RT. The proteins were blocked with a 3% of
BSA in Tris-buffered saline with Tween 20 (TBST), incubated with the primary antibodies
(anti-Substance P antibody, Santa Cruz, cat. no. sc-9758, 1:100; anti-GAPDH, Sigma,
cat. no. G9545, 1:3000) at 4 °C overnight, and incubated with a secondary antibody that
was conjugated to horseradish peroxide for 1 hour at RT.
For semi-quantitation of protein, the VDF membranes were incubated in an enhanced
chemiluminescence-detection reagent (Pierce ECL Western blotting substrate, Thermo
Fisher Scientific, cat. no. 32209) and the chemiluminescent signals were captured using
a ChemiDoc™ MP Imaging System (Bio-Rad Laboratories, cat. no. 1708290).
IX – Statistics
Statistical analysis was performed using GraphPad Prism version 8.0 for Mac (GraphPad
Software, Inc., San Diego, CA, USA). One Way ANOVAs were used to compare the three
groups with regard to epithelial and stromal thickness. The mean ± standard deviation
(SD) area of de-epithelialization in each group was calculated daily, and means were
compared at each time point using a Student’s t-test. Fisher’s exact tests and unpaired t-
56
tests were used to compare the denervated and neurotized corneas with respect to
ulceration and perforation. Statistical significance was accepted as p<0.05, and all data
were expressed using mean ± SD.
2.3 RESULTS I - Corneal neurotization prevents central epithelial thinning in the denervated corneal
model of NK
Four weeks after the initial denervation of the cornea and just prior to globe enucleation,
corneal dissection and fixation, the protective tarsorrhaphy was removed (Figure 2-2 A).
Thinning of the central epithelium of the denervated cornea as compared to the normally
innervated cornea and the recovery of thickness after neurotization are apparent from
representative images shown in Figure 2-3 A. In contrast, this thinning and its recovery
are not apparent in the epithelium of the peripheral cornea (Figure 2-3 A). Measurements
of thickness revealed significant thinning after corneal denervation, the mean (± SD) of
the central corneal epithelium in the denervated cornea (negative control) being
significantly thinner than that of the uninjured group (positive control, 10.84 ± 1.37 μm vs.
16.19 ± 1.34 μm, p < 0.01) and thinner than the central cornea in the neurotized treatment
group of rats (10.84 ± 1.37 μm vs. 15.08 ± 0.42 μm, p < 0.01). The corneal reinnervation
by implanted sural and CP nerve autografts in the neurotized treatment group resulted in
the corneal epithelial thickness equaling that in the uninjured control group, there being
no significant difference between the thicknesses of 15.08 ± 0.42 μm and 16.19 ± 1.34
μm (p>0.05), in the treatment and uninjured groups, respectively. Hence, the corneal
neurotization procedure either prevents central epithelial thinning and/or restores
thickness in the reinnervated cornea (Figure 2-3 B).
The central epithelium in the normally innervated cornea of the rat is significantly thicker
than the peripheral corneal epithelium (16.19 ± 1.34 μm vs 12.11 ± 0.49 μm, p<0.01).
After denervation, there was no significant difference between the central and peripheral
cornea due to the central epithelial thinning in the denervated cornea (10.84 ± 1.37 μm
vs 11.26 ± 0.93 μm, p>0.05). The relationship between the healthy central and peripheral
57
epithelial thickness is restored in the reinnervated cornea after corneal neurotization
(15.08 ± 0.42 μm vs 12.25 ± 1.48 μm, p<0.01) (Figure 2-3 B). There was no difference
(p>0.05) in the mean (± SD) central stromal thickness between the neurotized and
normally innervated (38.66 ± 2.29 μm) nor denervated (39.37 ± 10.10 μm) corneas. The
central stroma of the denervated cornea was thinner than that of the normally innervated
cornea (p<0.05) (Figure 2-3 C). Peripheral stromal thickness could not be accurately
measured due to fragility of corneal sections and consequently tissue sample loss.
0
5
10
15
20Ep
ithel
ial T
hick
ness
(mm
)
Normal innervation (n=7)
Corneal neurotization (n=7) Corneal denervation (n=7)
Normal Denervation Neurotization0
5
10
15
20
Thic
knes
s (m
m)
Central cornea
Peripheral cornea
Normal Denervation Neurotization0
5
10
15
20
Thic
knes
s (m
m)
Central cornea
Peripheral cornea
Central cornea (C) Peripheral cornea (P)
*** p < 0.0001 • p < 0.05
Central cornea
Peripheral cornea
Nor
mal
Co
rnea
l de
nerv
atio
n Co
rnea
l ne
urot
izatio
n
***
A
C
*** *** ***
B
0
20
40
60
Stro
mal
thic
knes
s (m
m)
*
58
Figure 2-3 Corneal epithelial and stromal thickness after corneal neurotization. A. Representative 1X1 images of the central and peripheral cornea stained with Hematoxylin
& Eosin in the three groups of rats in which the corneas were normally innervated,
denervated and neurotized corneas. Scale bars 20 μm B. The central epithelial thickness
in the denervated cornea (black C) was significantly thinner than that of the normally
innervated cornea (clear C; p<0.01) and neurotized cornea (blue C; p<0.01). There were
no differences between peripheral (P) epithelial thicknesses in any of the three groups.
C. There was a difference between the central stromal thickness of the normally
innervated and denervated corneas. There were no differences in the central stromal
thickness between the neurotized corneas (blue) and the normally innervated corneas
(clear) or the denervated corneas (black). In B and C, the mean values + SD are shown
with individual data points.
II - Corneal neurotization protects the denervated cornea from ulceration, scarring and
perforation
Seven days after removal of the protective tarsorrhaphy (Figure 2-2), the cornea after
corneal neurotization had significantly less corneal epithelial breakdown than those of the
denervated negative control cornea (0.0 ± 0.0 vs. 30.1% ± 12.7, p < 0.01) (Figure 2-4 A,
B). All denervated rat corneas developed progressive epithelial breakdown, while the
denervated corneas that received neurotization in only two rats developed an ulcer that
healed within one week (p < 0.01) (Figure 2-4 C). Hence, the neurotization protects the
denervated cornea from epithelial breakdown. Corneal neurotization reduced corneal
scarring (Figure 2-5 A), and no corneas developed a perforation after neurotization as
compared to the 80% of corneas that did in the untreated rat group (p < 0.05) (Figure 2-
5 B). The corneal neurotization procedure thus prevents scarring and perforation in the
rat model of corneal NK.
59
Denerv
ation
(n=5
)
Neurot
izatio
n (n=
10)
0
50
100
% C
orne
as th
at fo
rmed
ulc
ers
24 48 72 96 120 144 168-10
0
10
20
30
40
50
Hours after tarsorrhaphy removal
% U
lcer a
rea
Denervation (n=5)Neurotization (n=10)
*
***
*
24 48 72 96 120 144 168-10
0
10
20
30
40
50
Hours after tarsorrhaphy removal
% U
lcer a
rea
Denervation (n=5)Neurotization (n=10)
*
***
*
Corn
eal
dene
rvat
ion
Corn
eal
neur
otiza
tion
Mea
n %
ulc
erat
ion
area
Day 0 Day 2 Day 4 Day 7 A
B
C * p < 0.05 *
Figure 2-4 Corneal ulceration after corneal neurotization. A.
Fluorescein staining (green) of
the cornea between 0 and 7 days
shows corneal epithelial
ulceration in the denervated and
neurotized groups. B. There was
a significant time-dependent
increase in the % of the cornea
that was ulcerated in the
denervated (black) group
compared to the neurotized
(blue) group between 2 to 7 days.
C. Corneal neurotization
significantly reduced incidence of
corneal ulceration in the
denervated cornea.
Days after tarsorrhaphy removal
1 2 3 4 5 6 7
60
III - Corneal neurotization restores SP and CGRP in the denervated cornea
Immunohistochemical staining of corneal thin-sections showed co-localization of SP and
CGRP with the axons innervating the cornea, both in the normally innervated cornea (Fig
2-6 A) and the denervated cornea that were reinnervated by corneal neurotization (Fig 2-
6 C). No staining was observed in the untreated denervated cornea (Figure 2-6 B). There
was no staining of SP or CGRP under the control condition of incubation with only the
secondary and no primary antibody incubation (Figure 2-7). Western blot analysis
confirmed the absence of SP after denervation and presence in the neurotized cornea
(Figure 2-8). These findings indicate that SP and CGRP from reinnervating infraorbital
nerves may be responsible, at least in part, for the proliferation, migration, and adhesion
of epithelial cells in the cornea.
Denervation (n=5) Neurotization (n=10)0
20
40
60
80
100
% c
orne
as th
at p
erfo
rate
dFigure 2-5 Corneal scarring and perforation after corneal neurotization. A.
Representative images of the
same cornea in each group
over seven days after
tarsorrhaphy removal.
Corneal neurotization
protected the denervated
cornea from scarring (4 days)
and perforation (7 days). B.
Corneal neurotization
completely prevented the
denervated cornea from
perforating.
Corn
eal
dene
rvat
ion
Corn
eal
neur
otiza
tion
A
B * p < 0.05
Day 0 Day 2 Day 4 Day 7
61
A
B
C
Figure 2-6 SP and CGRP immunostaining.
Immunohistochemical
staining of i. ß-3
tubulin (green), ii. Substance P (SP)
(red), iii. Calcitonin
gene-related peptide
(CGRP) (purple), iv.
merged in the A
normally innervated
(n=1), B denervated
(n=1) and C. neurotized (n=1)
corneas. White arrows
point to true staining,
not artifact. A and C
demonstrate co-
localization of SP and
CGRP with axon fibers
in the normally
innervated and
neurotized corneas,
whereas in B there is
an absence of staining
in the denervated
cornea.
Do 1, 2, 3, 4 instead of
62
Figure 2-8 SP western blot analysis.
Corneal neurotization restores
Substance P (SP) in the denervated
cornea. There is no SP detected in the
untreated denervated (NK) cornea. (n=3
per group)
Figure 2-7 Control
immunostaining. No
primary antibody
incubation resulted
in no staining of ß-3
tubulin (green), SP
(red), or CGRP
(purple) in the A.
normally innervated
(n=1), B denervated
(n=1), and C
denervated +
neurotized (n=1)
corneas at 20X
magnification.
A
B
C
63
2.4 DISCUSSION
In this study, we demonstrated for the first time that corneal neurotization in an animal
model improves the epithelial integrity of the denervated cornea, possibly by restoring
trophic support of nerve-derived neuropeptides. Specifically, we showed that 1) corneal
neurotization protects the denervated cornea from epithelial thinning, ulceration, scarring
and perforation, and 2) axons from the common peroneal (CP) and sural nerve autografts
reinnervate the denervated cornea with nerve-derived trophic factors, substance P (SP)
and calcitonin-gene related peptide (CGRP).
The animal model that was used in these experiments paralleled the corneal neurotization
procedure that has been used successfully in human neurotrophic keratopathy (NK).
Unlike the human surgery, we used two branches of the donor infraorbital nerve to cross-
suture to the CP and sural nerve autografts that were tunneled and sutured to the
denervated cornea. The rat surgical procedure mimicked quite closely the human surgery
because the two fascicles of the infraorbital nerves that were used to reinnervate the rat
cornea that is smaller than the human cornea, likely were sufficient to mimic the splaying
of several fascicles of the dissected sural nerve autograft in the human cornea,
Prior to discussing our findings, It must be recognized that our experimental conditions of
denervation and reinnervation in the rat are somewhat artificial. They were used because
of the rapid spontaneous reinnervation of the denervated cornea (within 3 weeks) by
nerve fibers from the ablated ophthalmomaxillary nerve (Catapano et al., 2018) whilst
infraorbital nerve fibers were regenerating from the contralateral side to the denervated
cornea. The ophthalmomaxillary nerve was ablated twice (Figure 2.1), three weeks apart
before harvesting a denervated cornea seven days later to assess epithelial and stromal
thinning and immunohistochemical detection of neuropeptides (Figure 2.6). For
assessment of corneal ulceration, scarring, and perforation, the tarsorrhaphy was
removed to open the eyelid at the same seven-day timepoint after the second ablation
and the assessment performed daily for zero to seven days. For observations and
measurements from reinnervated cornea, the cross-suture of two branches of the donor
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infraorbital nerve to reinnervate the contralateral cornea via the CP and sural nerve
autografts, was performed 6 weeks prior to the first and second ophthalmomaxillary nerve
ablations described for the corneal denervation. Thus, this model is most representative
of patients receiving corneal neurotization who are diagnosed before extensive epithelial
ulceration and scarring, or patients who can expect corneal anesthesia secondary to an
intracranial surgery, for example.
Epithelial thinning was demonstrated in the denervated cornea with Hematoxylin and
Eosin staining (Figure 2-3). This finding concurred with similar findings in several animal
models of corneal denervation (Reid et al. 1993; Garcia-Hirschfeld et al., 1994; Ko et al.,
2009) as well as in the insensate cornea in human NK patients (Sigelman and
Friedenwald, 1954). In animal studies, Hematoxylin and Eosin was similarly used to
assess thinning, and in human studies, the thinning was detected using in vivo confocal
microscopy and optical coherence tomography (Villani et al. 2014; Mastropasqua et al.
2018). It is suggested that the thinning is due to impaired adhesion and proliferation of
corneal epithelial cells. This conjecture was based on the coincidence of the impairment
with epithelial thinning (Reid et al. 1993; Garcia-Hirschfeld et al., 1994; Ko et al., 2009).
Whatever the basis for the thinning, our findings of normal epithelial thickness in
longitudinal sections of reinnervated rat cornea (Figure 2-3) demonstrate unequivocally
the maintenance of epithelial thickness and/or the reversal of denervation-induced
thinning by peripheral nerve reinnervation. Though epithelial thickness was not assessed
in human patients with and without corneal neurotization, reduced persistent epithelial
defects in the majority of NK patients treated with corneal reinnervation (Catapano et al.,
2019) support this finding.
A significant reduction in stromal thickness was detected in the denervated cornea for the
first time, using Hematoxylin and Eosin staining, indicating that corneal innervation is
essential to maintain the stroma. This thinning occurred despite protection from external
abrasion by a protective tarsorrhaphy. However, the stromal thickness in the reinnervated
cornea, was not different from either the intact or denervated cornea, possibly due to the
scatter in the measurements (Figure. 2.3C). This may be reduced by increasing the
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number of observations. Additional explanations for the findings include the unintentional
stromal trauma of the rat neurotization surgery and varied orientation of the corneal cross-
sections relative to the autograft insertion onto the corneal surface. The latter may be
addressed in future studies by measuring innervation density (Cruzat et al., 2017)
concurrent with stromal thickness using confocal microscopy (Reinstein et al., 2009).
The denervated cornea that undergoes corneal ulceration, scarring and perforation, also
seen in previous studies (Huang and Tseng, 1991; Araki et al., 1993; Rao et al., 2010;
Blanco-Mezquita et al., 2013), was fully protected by corneal neurotization (Figures 2-4
and 2-5). As in the previous studies, the area of epithelial ulceration was measured with
in vivo fluorescein staining (Huang and Tseng, 1991; Araki et al., 1993; Rao et al., 2010;
Blanco-Mezquita et al., 2013). Normally fluorescein staining is not seen because the
epithelial cells form a tight barrier (Lambiase et al., 1998; Matsumoto et al., 2004) which
is broken down following corneal denervation. The fluorescein staining became evident
as the cornea ulcerated (Figure 2-4A) and without innervation, proceeded to corneal
scarring at day 4 and perforation at day 7 that was seen under the microscope with normal
light (Figure 2-5A). Hence, reinnervation is essential for the preservation of normal
corneal epithelium.
A proviso of our experimental method was the eyelid closure with a stitch (tarsorrhaphy)
immediately after all the surgeries. The tarsorrhaphy was removed just prior to the corneal
observations from zero to seven days, a week after the second denervation (Figure 2-2).
The findings of the progression of ulceration, scarring and perforation are attributed to the
removal of the tarsorrhaphy itself as well as the corneal denervation in contrast to the
epithelial thinning (Fig. 2-3) that occurred whilst the eyelids were closed. The tarsorrhaphy
was removed at day zero because vascularization in the conjunctival tissue of the eyelids
may otherwise have provided the ulcerated cornea with factors to promote healing (Müller
et al., 2003) and would thereby confound the results. However, the lack of protection after
the first day may have hastened corneal abrasions in our rat model of corneal
denervation, unlike the protection offered by lenses or protective glasses in NK patients
(Donnenfeld et al., 1995; Ramaesh et al., 2007). Nonetheless, despite rapid symptom
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progression in the rat untreated denervated cornea, we demonstrated that corneal
neurotization prevented ulceration, scarring, and perforation. A protective cone would be
efficacious in protecting the rat cornea from external abrasions in future experiments. In
addition, the same ophthalmic slit lamp used by ophthalmologists in the clinic would allow
assessment of the depth of the ulceration.
Our immunostaining of the reinnervating infraorbital nerve axons via the peripheral nerve
autografts used in the corneal neurotization procedure demonstrated their expression of
the SP and CGRP neuropeptides. These peptides were co-localized with the re-
innervating axons and were absent in the untreated denervated cornea (Figure 2-6 and
7). A “no primary antibody” control was used to confirm staining, in which the tissues of
interest were only incubated with the secondary antibodies, but there are several other
controls that could have been used. A similar method would be to incubate the tissues of
interest with only the primary and no secondary antibodies. Alternatively, a tissue that is
known to express or not express the target antigen can be used as a positive or negative
tissue control. For example, a positive control for the SP antibody could be the trigeminal
ganglion, in which SP is produced (Lehtosalo, 1984), where we would expect true staining
to appear. Conversely, any staining observed in tissue of a SP knock-out model can be
would be a false-positive or non-specific. An isotype control, an antibody with the same
isotype, clonality, and host-species as the primary antibody, can be used in lieu of the
primary antibody to ensure the observed staining is not due to non-specific interactions
of the antibody and tissue. Lastly, any staining observed in the tissue incubated with a
primary antibody pre-absorbed with the immunogen of interest, SP or CGRP, would also
indicate non-specific staining.
Our western blot analysis confirms that SP is restored in the denervated cornea by
corneal reinnervation by neurotization. Whilst the calculated molecular weight for SP is
~13kDa, the observed molecular weight with Western blot can vary due to post-
translational modifications, cleavages, charges and so forth which explains our
observation of the SP band at 18kDa (Figure 2-8). To validate the band in Western blot
is in fact SP, a positive and negative tissue control, such as those mentioned above for
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immunohistochemistry controls, could be used. A band at the same observed molecular
weight for a positive tissue control, and an absence of a band for a negative tissue control
would confirm the band is indeed SP.
In the normally innervated cornea, neuropeptides, such as SP and CGRP, function to
maintain epithelial cell adhesion and promote their epithelial cell proliferation and
migration to the central cornea in response to the ongoing epithelial cell turnover and
healing after injury (Reid et al. 1993; Garcia-Hirschfeld, Lopez-Briones, and Belmonte
1994; Mikulec and Tanelian 1996; Nakamura et al. 1997; Chikama et al. 1998; Araki et
al. 1993; Ko, Yanai, and Nishida 2009). In human NK, topical applications of SP and
CGRP promote healing of epithelial defects (Brown et al., 1997; Chikama et al., 1998;
Yamada et al., 2008). The observed improvement in the restoration and/or maintenance
of the corneal epithelium after reinnervation of the denervated cornea in our rat model of
NK (Figures 2-3 to 2-5), suggests that the peptides may function similarly in neurotized
NK corneas and normally innervated corneas to promote healing of epithelial defects.
Future studies need to be performed to elucidate the specific mechanism(s) by which SP
and CGRP promote epithelium maintenance in the neurotized cornea.
In summary, this study demonstrated that corneal neurotization restores and/or maintains
epithelial integrity in the denervated rat cornea by preventing epithelial thinning,
ulceration, scarring, and perforation. We hypothesize that this may be due, at least in
part, to the restoration of the nerve-derived peptides, SP and CGRP, in the neurotized
cornea. Our study provides further insight into why corneal neurotization is effective in
preventing disease progression and maintaining corneal epithelial integrity in the majority
of NK patients.
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CHAPTER 3 Conclusions and
Future Directions
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3.1 – Summary of results
In this thesis, we have demonstrated that corneal neurotization maintains epithelial
integrity of the denervated cornea in our rat model of Neurotrophic Keratopathy (NK). We
have shown corneal neurotization protects the cornea from the epithelial thinning,
ulceration, scarring and perforation that occurs in the denervated cornea. In denervated
corneas that received corneal neurotization, nerve-derived peptides Substance P (SP)
and calcitonin gene-related peptide (CGRP) were restored by the re-innervating axons
from the nerve autografts, which offers a potential mechanism of improved ocular surface
health.
3.2 – Future Directions
Our future directions and hypotheses derive from the results of this thesis and published
literature. Due to the severity of NK and the difficulty of managing symptoms in patients,
an extensive amount of research has focused on normal and impaired corneal
innervation. While some groups have investigated nerve regeneration in corneal diseases
associated with corneal hypoesthesia (Martin, 1996; Lambiase et al., 2008; Chikamoto et
al., 2009; Yin et al., 2011), only one paper, published by our group, has evaluated the
effects of re-innervating the cornea with donor nerves (Catapano et al., 2018). Previously
published literature about corneal deficits following denervation in models of NK inform
our future directions in investigating corneal reinnervation by nerve autografts (future
directions 1, 2, 5). Little is known about the relationship between innervation, Schwann
cells (SCs), and limbal stem cells (LSCs). Published literature about innervation, SCs and
stem cells in other tissues as well as preliminary investigations by our laboratory inform
future directions 3 and 4. These investigations focus on understanding the relationship
between these cells and innervation in the normal and denervated cornea to inform
investigations into neurotization’s effect on this relationship.
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FUTURE DIRECTION 1 – CORNEAL EPITHELIUM
Evaluating ocular surface health is the first step to understand how corneal re-innervation
influences the denervated corneal epithelium. In our rat model, corneal neurotization
prevents the central corneal epithelium from the thinning, ulceration, perforation and
scarring that follows corneal denervation (Ch. 2). Corneal epithelial thinning in NK is
associated with changes in epithelial cell morphology. In response to corneal denervation
epithelial cells demonstrate swelling in rabbits (Gilbard and Rossi, 1990) and, loss of
surface cell microvilli in rats (Alper, 1975; Hosotani et al., 2006). A future direction is to
investigate whether the morphological changes of the epithelial cell that typically
accompany corneal denervation, remain in the reinnervated cornea. Due to the
similarities between the ocular surface health in the normally innervated and denervated
corneas treated with corneal neurotization, we hypothesize the neurotization procedure
will preserve the normal healthy epithelial cell morphology. Immunohistochemical staining
of Keratin 3/12 (K3 and K12) and CD44-postive epithelial cells can be used (Table 3-1).
Alternatively, epithelial cell morphology may be assessed with electron microscopy as in
other studies (Hosotani et al., 2006; Chalvatzis et al., 2012)
FUTURE DIRECTION 2 – TROPHIC SUPPORT
In this thesis, we confirmed the absence of nerve-derived peptides, SP and CGRP, in the
denervated cornea and demonstrated their co-localization with the re-innervating axons
of autografts used in the corneal neurotization surgical procedure. To do this we used
thin-section immunohistochemistry to permit the visualization of smaller nerve segments
in the cornea. We are currently performing whole-mount SP and CGRP
immunohistochemical staining in the rat cornea which allows a larger number of nerve
fibers to be visualized in several layers of the cornea and in continuity. However, this
technique has proven to be very difficult for staining the weaker antibodies, and
optimization of this technique in our laboratory is ongoing. Whilst we have characterized
these nerves as containing SP and CGRP, we have not yet evaluated whether the trophic
support, normally provided by these nerve-derived peptides, is restored in the neurotized
cornea.
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Primary antibody Indication of K3 (cyto-)keratin 3 Epithelial cell marker
- With K12, specific for corneal epithelium K12 (cyto-)keratin 12 Epithelial cell marker
- With K3, specific for corneal epithelium CD44 Cell surface adhesion
receptor Corneal epithelial cell marker
- Receptor on corneal epithelial cell membrane
pEGFR Phosphorylated epidermal growth factor receptor
Epithelial cell proliferation - Trophic influence of SP in cornea
ZO-1 Adhesion marker - Tight junction protein - Trophic influence of SP in cornea
E-cadherin Adhesion marker - Tight junction protein, anchoring complex
formation - Trophic influence of SP in cornea
ß-4 integrin Adhesion marker Necl4 A.K.A. SynCAM4 = Cell
Adhesion Molecule 4 Schwann cell marker
- Especially of those interacting with axons GFAP Glial fibrillary acidic protein Schwann cell marker
- Non-myelinating MAG Myelin-associated
glycoprotein Schwann cell marker
- Pre-myelinating, where Schwann cells engulf axons destined to be myelinated
MBP Myelin basic protein Schwann cell marker - Myelinating
NGF Nerve growth factor Neurotrophin trkA trkA receptor
K14 Keratin 14 Limbal stem cell marker
- Lineage tracing of epithelial cells C/EBPδ, Limbal stem cell marker
- Quiescent Bmi1 Limbal stem cell marker
- Quiescent SOX9 Limbal stem cell marker
- Quiescent ΔNp63α Limbal stem cell marker
- Quiescent, early activated, ΔNp63β Limbal stem cell marker
- Activated, transient amplifying cells ΔNp63γ Limbal stem cell marker
- Activated, transient amplifying cells Wnt/β-catenin Limbal stem cell marker
- Activated, transient amplifying cells ß-iii tubulin
Axonal marker - Microtubule element of tubulin family found
in neurons Ki67 Cell proliferation marker
Table 3-1. Antibodies for future directions. A description of antibodies that can be used
for future investigations and what their indication is in the cornea
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Corneal sensory nerves contain several nerve-derived peptides that play a role in
epithelial maintenance and healing in the cornea. The majority of corneal nerves contain
either SP or CGRP, both of which were evaluated in the present thesis. Some nerves in
the cornea also contain pituitary adenylate cyclase-activating peptide, galanin, vasoactive
intestinal polypeptide, and met-enkephalin (Müller et al., 2003).
CGRP facilitates epithelial cell migration, and is involved in epithelial wound healing (Reid
et al., 1993; Mikulec and Tanelian, 1996). SP stimulates corneal epithelial cell proliferation
(Reid et al., 1993; Garcia-Hirschfeld, Lopez-Briones and Belmonte, 1994), upregulates
corneal epithelial cell migration, in conjunction with insulin-growth factor 1 (IGF-1)
(Nakamura, Nishida, et al., 1997; Chikama, Nakamura and Nishida, 1999) and increases
the expression of tight-junction proteins in the corneal epithelium (Araki‐Sasaki et al.,
2000; Ko, Yanai and Nishida, 2009). SP is the most extensively studied neuropeptide in
the literature, and has been shown to decrease in patients with corneal hypoesthesia
(Stone and Kuwayama, 1985; Yamada et al., 2000). Capsaicin administration, which
depletes SP and prevents the peptide from binding to its receptors, results in corneal
changes consistent with NK including corneal opacity and corneal axonal loss (Fujita et
al., 1984; Shimizu et al., 1987). Further, topical SP in conjunction with IGF-1 has been
shown to heal persistent epithelial defects (PEDs) in animals (Murphy et al., 2001;
Nagano et al., 2003) and patients (Brown et al., 1997; Chikama et al., 1998; Yamada et
al., 2008). Presumably the presence of SP, and CGRP, in the neurotized cornea suggests
the trophic influence of the nerve-derived peptides is restored, but this hypothesis needs
to be confirmed with further studies.
SP upregulates tight-junction proteins, E-cadherin (Araki‐Sasaki et al., 2000) and ZO-1
(Ko, Yanai and Nishida, 2009) in the corneal epithelium. SP is also implicated in
facilitating the adhesion of epithelial cells to the underlying basement membrane and
extracellular matrix through the upregulation of the aforementioned adhesion proteins as
well as integrins. Further, trigeminal ganglions, containing SP (Lehtosalo, 1984), influence
the expression of collagen VII on rabbit corneal epithelial cells in vitro (Baker et al., 1993),
a key component of the hemidesmosome anchoring complex. Innervation and SP are
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hence involved in formation of the hemidesmosome-anchoring complex. We hypothesize
that corneal neurotization increases the expression of tight junction proteins, ZO-1 and
E-cadherin, as well as of proteins associated with the anchoring complex, including ß-4
integrin (Future direction 1). Immunohistochemistry will be used to localize expression of
these proteins (antibodies described in Table 3-1), as well as for semi-quantitation.
Protein and mRNA semi-quantification can be performed using Western blot and Real
Time -Polymerase Chain Reaction (RT-PCR) respectively. Capsaicin can be used to
evaluate if the hypothesized upregulation of adhesion proteins in the neurotized cornea
is reversed by prohibiting SP function, thus confirming the neuropeptide’s trophic
influence on the reinnervated corneal epithelial integrity.
SP stimulates epithelial cell proliferation in vitro (Reid et al., 1993; Garcia-Hirschfeld et
al., 1994), and wound healing in mouse models of diabetic keratopathy (Yang et al.,
2014). In the hypo-innervated diabetic rabbit cornea, the epidermal growth factor (EGF)
receptor does not phosphorylate, thus is not activated (Xu nd Yu, 2011). This
phosphorylation is necessary in corneal epithelial cell turnover and wound healing which
is mediated by the EGF-signalling pathway (Nakamura, Nishida, et al., 1997; Peterson et
al., 2014; Rush et al., 2014). SP re-activates this signalling pathway, thereby promotes
wound healing in diabetic mice (Yang et al., 2014). Thus, to determine whether the trophic
influence of SP is restored in the denervated cornea treated with corneal neurotization,
as we would expect, we will evaluate the presence and protein levels of phosphorylated
EGF receptors (pEGFR) using immunohistochemistry and Western blot analysis
respectively (antibody description in Table 3-1). We can confirm these results by
evaluating changes in ocular surface health and pEGFR levels in the neurotized cornea
whilst using capsaicin to block SP binding to its receptors. We hypothesize pEGFR levels
will be restored in the neurotized cornea. This would suggest that SP, co-localized with
the reinnervating axons in the treated denervated cornea, restores trophic support by
influencing epithelial proliferation through the EGF signalling pathway.
In this thesis, our observations of the denervated corneal epithelium, including thinning
and ulceration, suggest an impairment in epithelial adhesion and proliferation in our model
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of NK. Our observed improvements in epithelial integrity of the denervated cornea treated
with corneal neurotization suggest there is an improvement in these SP and CGRP-
mediated molecular processes. Presence of nerve-derived peptides in the neurotized
cornea suggests the trophic influence of these peptides may be restored. We hypothesize
the pEGFR and adhesion protein levels will be restored in the neurotized cornea, and that
this restoration and the improvement in ocular surface health observed in the neurotized
cornea (Chapter 2), will be at least partially reversed by capsaicin administration.
FUTURE DIRECTION 3 – SCHWANN CELLS
In chapter 1.3 of this thesis, we briefly discussed the understudied Schwann cell (SC)
presence in the cornea. The SCs are present in both the cornea and limbus (Figure 3-1).
However, the function these SCs in the cornea and/or limbus is unknown, and no literature
has been published regarding SC response to corneal denervation or hypoinnervation.
Figure 3-1 Schwann cells ensheath axons in the cornea and limbus. Left. Early cross-section of
rat cornea and limbus (overview image), demonstrating ßIII tubulin–positive axons (green) ensheathed
by Necl-4 (white) and GFAP (red)–positive Schwann cells. Scale bars: 200 μm and 20 μm, respectively.
Right. Cross-section of the rat cornea at limbal region (overview image), demonstrating a cross-section
of single ßIII tubulin–positive axon ensheathed by MAG–positive non-myelinating Schwann cells. Scale
bars: 50μm and 2.5 μm in the, respectively.
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In response to peripheral nerve injury SCs de-differentiate and proliferate to participate in
axonal regeneration (Stoll and Müller, 1999). Long-term denervation of tissues results in
a reduced ability of these SCs to support nerve regeneration and a decline in SC gene
expression (Fu and Gordon, 1997; You et al., 1997; Höke et al., 2002; Höke, 2006;
Brushart et al., 2013). We hypothesize that corneal SC numbers will thus decrease in
response to long-term corneal denervation. Investigating presence and localization of
SCs can be performed with immunohistochemical staining for SC antibodies (Necl4,
GFAP, MAG; Table 3-1) in the normally innervated cornea and denervated cornea of our
NK rat model, and SC levels can be confirmed with Western blot analysis both corneal
groups.
Figure 3-2 Schwann cells are located in close proximity to Sox9 positive cells. Extended depth focus immunofluorescent image of limbal area of whole mount rat cornea.
Sox9-positive nuclei, possibly LSC (red), are in close vicinity to Necl-4–positive Schwann
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cells (green) ensheathing axons in the limbus. The border between the limbus and cornea
is annotated by a dashed line. Scale bars: 50 μm.
In our preliminary staining of the whole-mount limbal epithelium, we observed Sox9
positive cells, possibly indicating LSCs, located in close proximity to axon-ensheathing
Necl4 positive SCs in the limbus (Figure 3-2). Although the relationship between SCs and
LSCs has not yet been investigated, the close proximity of these cells to each other
indicate they may influence one another. In fact, SCs in the bone marrow have been found
to support homeostasis of hematopoietic stem cells (Yamazaki et al., 2011; Yamazaki
and Nakauchi, 2014) and release neurotrophins, including NGF, brain derived
neurotrophic factor (BDNF), and others, in response to axonal injury (Meier et al., 1999;
Fontana et al., 2012; Brushart et al., 2013). Neurotrophins including NGF, neurotrophin-
3 and 4/5, and BDNF, and their receptors are present in the cornea. Further NGF’s trkA
receptors are specifically located on the limbal epithelium, where the LSCs are found.
This suggests that if SCs are a source of NGF and other neurotrophins, these cells may
potentiate LSC function through the release of the neurotrophins. Perhaps corneal
epithelial wounding results in intraepithelial axonal injury, stimulating the release of NGF
from SCs to act on its receptors on LSCs (Figure 3-3). This hypothesis was formed by
applying knowledge about SC function(s) in other tissues in the context of the cornea.
Therefore, many techniques and studies need to be performed to test this hypothesis and
to elucidate the function of SCs and the underlying mechanism(s) in the cornea
specifically. In our current rat model of NK, immunohistochemical staining and Western
blot can be used to first evaluate neurotrophin (NGF) and neurotrophin receptor (trkA)
presence in the normally innervated cornea, and subsequently in the denervated cornea
(Table 3-1). However, even if the SC phenotype changes, for example these SCs de-
differentiate, or SCs are lost in response to corneal denervation, the changes we expect
to see in neurotrophin and neurotrophin receptor levels could not be exclusively attributed
to loss of innervation nor loss of normal SC phenotype. Cell-culture experiments can be
used to evaluate if isolated corneal SCs, on pre-conditioned media, release growth factors
including NGF.
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Figure 3-3 Proposed mechanism of action of corneal SCs in response to axonal injury. Schematic diagram – we propose that following axonal injury SCs release NGF
which acts on its trkA receptors in the limbal epithelium to stimulate LSC proliferation and
differentiation.
Several transgenic mice exist and thus, translating our rat NK and corneal neurotization
models into mice offers greater research potential than our current rat model. The
relationship between NGF’s receptor trkA and innervation has previously been
investigated in a transgenic trkA knock-out mouse model, in which trkA (-/-) mice had
reduced corneal sensation and nerve terminals (de Castro et al., 1998). Further, trkA is
co-localized with several LSC-associated markers (Touhami et al., 2002; Qi et al., 2008).
Tamoxifen application in the trkACreERT2/+;R26-LSL-TdT;R26-LSL-DTA mouse will
induce apoptosis of trkA positive cells exclusively in the cornea by the activation of the
diphtheria toxin. Immunolabelling of NGF, ß-III tubulin, SC and LSC-associated markers
Legend
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in the cornea of this mouse model may help elucidate the proposed mechanism in Figure
3-3.
Stereotactic ablation of the ophthalmomaxillary nerve to achieve corneal denervation has
previously been performed in mice (Ferrari et al., 2011) and can be used in transgenic
mouse models to study the relationship between corneal SCs and innervation. In the
Sox2CreERT2/+;R26-LSL-TdT mouse, topical tamoxifen application induces TdTomato
expression on Sox2-positive cells, SCs. Co-localization of TdTomato and other SC
markers (Table 3-1) in the transgenic mouse cornea will confirm this model as suitable
for use in further corneal SC investigations. In vivo fluorescent confocal microscopy can
then be used, in this mouse model, to track what happens to SCs in the days and weeks
following corneal denervation. Further, we can use transgenic mouse models to
selectively ablate SCs in the mouse cornea, without damaging axons. Topical tamoxifen
application in the Sox2CreERT2/+;R26-LSL-TdT;R26-LSL-DTA mouse will induce
apoptosis of SC (Sox-2 positive) cells by activation of the diphtheria toxin. This ablation
will be confirmed by disappearance of TdTomato labelled cells and of SC marker
expression. This model can be used to observe possible changes to corneal innervation,
through ß-III tubulin immunostaining, and observations of changes in ocular surface
health and healing. The effect of SC ablation on the presence of NGF, other neurotrophins
and their receptors can also be assessed with immunostaining (Table 3-1).
Once SC response to corneal denervation is established, we can investigate SC presence
and function in the denervated cornea after treatment with corneal neurotization. Using
immunohistochemical staining in our current rat model, we can investigate whether there
are SCs in the re-innervated cornea, where they are expressed and what their function
is, if any. While the majority of native corneal nerves are unmyelinated, the nerve
autografts used in corneal neurotization contain a diverse population of both myelinated
and unmyelinated nerves. Thus, in our established rat model, immunostaining with a
myelinating SC marker, such as MBP, and non-myelinating SC marker, such as GFAP,
can be used to confirm whether the reinnervating axons from the autografts are
myelinated or unmyelinated (Table 3-1).
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Development of a mouse model is necessary for the following proposed investigations.
The experiments in transgenic mouse models described above can be repeated in the
neurotized mouse cornea. Catapano et al (2018) demonstrated that only a small
proportion of the axons regenerating through the autografts reinnervated the denervated
cornea in our rat model of NK. In vitro, long-term denervated SCs retain the capacity to
re-activate to support axons (Gordon et al., 2019). Thus, it is possible that the
regenerating axons are guided to the cornea by remaining non-myelinating SCs native to
the denervated cornea which are re-activated by close proximity to the nerve grafts.
Alternatively, it is possible the SCs of the nerve autograft may in some way migrate into
the cornea, and perhaps aid corneal reinnervation. By using transgenic mouse models,
we can investigate whether nerve isograft derived fluorescently-labelled SCs migrate to
the cornea to support axon regeneration and corneal reinnervation, or if the native SC
remain or re-activate to proliferate and support re-innervation following corneal
neurotization. The aforementioned Sox2CreERT2/+;R26-LSL-TdT mouse can be used to
selectively induce TdTomato expression of the SCs in the harvested sural and common
peroneal isografts in the corneal neurotization procedure. These isografts can then be
used to reinnervate the cornea of a genetically identical mouse lacking SC activation by
tamoxifen and, at the end-point, harvested corneas can be immunolabelled for SC
markers (Table 3-1). Co-localization of TdTomato expression with SC markers would
suggest the SC in the re-innervated cornea are derived from the isograft. Conversely,
absent TdTomato expression or absent co-localization would suggest the native corneal
SCs remain in the denervated cornea.
FUTURE DIRECTION 4 – LIMBAL STEM CELLS
There is an ill-defined relationship between limbal epithelial stem cells (LSCs) and
corneal/limbal sensory innervation, but LSCs and corneal innervation are critical for
corneal epithelial wound healing. Because of differences in symptoms between LSC
deficiency and NK patients, the absence of corneal innervation likely does not result in a
reduced LSC quantity. However, perhaps innervation influences LSC differentiation and
proliferation in response to epithelial cell turnover and wound healing. Although a
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definitive LSC marker does not exist, several have been suggested in the literature and it
is suggested that LSC marker expression changes in response to LSC activation. K14 is
an accepted LSC marker which has been used for epithelial cell lineage tracing in mouse
models (Figueira et al., 2007; Amitai-Lange et al., 2015; Nasser et al., 2018). Quiescent
LSCs express C/EBPδ, Bmi1, SOX9, ΔNp63α, and activated LSCs/transient amplifying
cells express ΔNp63β, ΔNp63γ and Wnt/β-catenin (Di Iorio et al., 2005; Barbaro et al.,
2007; Menzel-Severing et al., 2018). We hypothesize that in the absence of innervation,
LSCs will remain in a quiescent state and fail to activate to become transient amplifying
cells.
We performed a preliminary microarray analysis of the RNA of the limbal areas dissected
from the normally innervated and denervated rat corneas 2 days after corneal epithelial
wounding. There were several significant differences in gene expression between the two
groups (Figure 3-4). However, the limitation of this method is that we were not able to
specifically isolate the LSCs. Single cell-sequencing would allow us to evaluate specific
differences in LSC gene expression between the normally innervated and denervated rat
corneas and target these differences in gene expression for futures research. We are
currently optimizing the techniques used to isolate LSCs in culture, to be used for single-
cell sequencing analysis.
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Figure 3-4 Preliminary microarray analysis. A. Microarray analysis of limbus of
normally innervated and 7-day denervated rat corneas 2 days after corneal epithelial
injury. B. Quantification demonstrates significant changes (downregulation in green and
upregulation in red) in gene expression between the denervated and normally innervated
groups.
In our existing NK rat model, we will use immunohistochemical staining to evaluate
differences in LSC marker (Table 3-1) expression with and without epithelial wounding in
the normally innervated and denervated corneas. Topical BrdU application after epithelial
wounding can identify LSCs as slow-cycling label-retaining cells (Figueira et al., 2007).
Ki67, a proliferation marker (Table 3-1), can be stained for in conjunction with the LSC
markers to specifically look at the proliferation of these cells. If a difference in marker
expression is found, this can be repeated in the neurotized cornea to evaluate whether
the re-innervating nerves influence the LSC activation in the same way as the native
corneal sensory nerves.
In the Sox9CreERT2/+;R26-LSL-TdT mouse, tamoxifen application induces TdTomato
expression of Sox9-positive cells. In the normally innervated cornea, the LSC phenotype
of these cells will be confirmed by immunolabelling with other LSC markers (Table 3-1)
and assessing co-localization of marker expression with TdTomato expression. Corneal
denervation in this model can be used to evaluate changes in LSC expression in the
absence of innervation. The R26-confetti/K14-Cre mouse model allows lineage tracing of
the corneal epithelium (Nasser et al., 2018). Topical application of tamoxifen in these
mouse corneas induces K14-positive cells (LSCs) to express a fluorescent protein (green,
red, cyan, yellow) and allows for lineage tracing of terminally differentiated epithelial cells
(Amitai-Lange et al., 2015; Nasser et al., 2018) (Figure 3-5). Thus, reduced or absent K14
lineage tracing would be observed if LSC function is impaired. Corneal denervation in this
mouse model can be used to test the hypothesis that LSC function is impaired in the
absence of innervation.
82
The ability to cross transgenic mice (R26-confetti/K14-Cre and Sox2CreERT2/+;R26-
LSL-TdT;R26-LSL-DTA) may allow for investigation of LSC response to SC-induced
death in the normally innervated cornea. This would further confirm or deny SC influence
on LSCs, as discussed in “future direction 3,” and help elucidate whether the
hypothesized impairment in LSC function is due to loss of corneal innervation or SCs.
Lastly, the above LSC investigations using mouse models can be repeated in the
neurotized mouse cornea to assess the effect of re-innervation on the denervated
cornea’s LSCs.
The use of transgenic mouse models and in vivo confocal microscopy allows for countless
manipulations to corneal innervation and cells to be studied in live animals and in real-
time. Because of its transparency, the cornea is an ideal tissue in which to study the
relationship between innervation, stem cells and SCs, and could have implications in a
broader context beyond preservation of corneal health in NK.
Figure 3-5 Transgenic confetti mouse. Lineage tracing from a R26-confetti/K14-Cre
mouse. K15 positive cells induced to express
Green (GFP) Red (RFP) Cyan (CFP) and Yellow
(YFP) fluorescent proteins as they differentiate
from the LSCs in the limbus (indicated by dotted
line) to terminate as epithelial cells in the central
cornea. (Modified from Stem Cells, Amitai-
Lange et al, Lineage tracing of stem and
progenitor cells of the murine corneal epithelium
pages 230-239, copyright (2014) with
permission from Elsevier: License #
4595700226918
83
FUTURE DIRECTION 5 – TOPICAL AGENTS
In the majority of patients, the corneal neurotization procedure is effective in treating NK.
However, some patients do not benefit from the procedure (Catapano et al., 2019).
Identifying an underlying mechanism involved in corneal epithelial maintenance or healing
that is not restored following corneal reinnervation may help elucidate a target for future
interventions. A topical agent could then perhaps be used to supplement the current
neurotization surgical procedure to improve outcomes. Below I outline some possible
topical agents, based on our current knowledge, and propose experiments to test their
efficacy.
Corneal neurotization treats the underlying condition of NK by restoring innervation in the
affected cornea. In the denervated rat cornea, we have previously shown corneal
neurotization improves healing over time (Catapano et al. 2018). It is likely that just as
numerous factors contribute to the impairment in healing in NK, several factors may
contribute to the observed improvement in healing following corneal neurotization.
Although not yet confirmed, we hypothesize the improvement in healing observed
(Catapano et al. 2018) is due to the restoration of nerve-derived peptides, such as SP
and CGRP. As previously discussed in Chapter 1.3 and 1.4 of this thesis, in the normally
innervated cornea these peptides provide trophic support to the cornea by promoting
adhesion (Araki‐Sasaki et al., 2000; Ko, Yanai and Nishida, 2009), proliferation (Reid et
al., 1993; Garcia-Hirschfeld, Lopez-Briones and Belmonte, 1994), and migration
(Nakamura, Nishida, et al., 1997; Chikama, Nakamura and Nishida, 1999) of epithelial
cells. Previous groups have demonstrated that topical agents containing these nerve-
derived peptides are most effective in healing epithelial wounds when combined with
other trophic factors, such as SP with IGF-1 or CGRP with SP (Nishida et al. 1996;
Nakamura et al., 1998; Chikama et al., 1999; Ko et al. 2013). Although, in Chapter 2 of
this thesis we have shown that corneal neurotization restores the presence of the nerve-
derived peptides SP and CGRP, the trophic activity of these peptides in the neurotized
denervated cornea needs to be confirmed. To test whether neurotization restores trophic
support, we will repeat the previously published corneal healing experiment (Catapano et
al. 2018) in the denervated + neurotized rat cornea with topically applied capsaicin. This
84
topical agent will ablate the effect of SP, one of the nerve-derived mediators that provides
the cornea with trophic support. An observed impairment in healing in this model, would
confirm the previously documented improvement (Catapano et al., 2018) was, at least in
part, due to the restoration of SP. Further, perhaps topical agents containing SP and/or
CGRP will facilitate corneal neurotization’s improvement in corneal epithelial healing and
health in the denervated cornea.
Topical agents containing NGF promote corneal epithelial cell differentiation, proliferation
and migration in a corneal injury model in hens (Blanco-Mezquita et al., 2013) and rodents
(Lambiase et al., 2000). In patients, topical NGF effectively heals corneal PEDs of a
variety of etiologies (Bonini et al., 2000a, 2000b; Tan, Bryars and Moore, 2006; Lambiase,
Sacchetti and Bonini, 2012; Sacchetti, Lambiase and Ph, 2017), despite the lack of
knowledge of the source and mechanism of action of the neurotrophin in the normal
healthy cornea (Bonini et al., 2000a; Lambiase et al., 2000; Vesaluoma et al., 2000; Müller
et al., 2003). However, success in NGF treatment is primarily documented in cases of
corneal hypoinnervation, not complete denervation (Lambiase, Se, et al., 1998; Bonini et
al., 2000; Tan et al., 2006; Lambiase et al., 2012; Sacchetti et al., 2017). Because corneal
neurotization treats the underlying cause of NK, it is likely that NGF is not as effective in
improving corneal epithelial healing and health of the completely denervated cornea.
However, it is possible that in combination with the procedure, topical NGF would
advance the previously observed improvement.
As discussed above, SCs that are present and ensheath axons in the cornea and limbus
may be a source of growth factors, including the neurotrophin NGF. In response to
peripheral nerve axonal injury, SCs are known to release growth factors such as NGF
(Meier et al., 1999; Fontana et al., 2012; Brushart et al., 2013), as well as support axon
and digit-tip regeneration, epithelial wound healing and stem cell function (Jessen and
Mirsky, 2016; Carr and Johnston 2017). Schwann cell conditioned media (SCcm) consists
not only of NGF (Brushart et al., 2013), but also of other factors SCs release such as NT-
3, and PDGF (Meier et al., 1999; Brushart et al., 2013). Thus, if in patients, topical
85
application of NGF is effective in healing PEDs, presumably SCcm would be at least as
effective.
FK506 (tacrolimus) is commonly used as an immunosuppressant agent in corneal
applications such as preventing rejection of corneal grafts (Wu et al., 2019) and LSC
transplants (Ballios et al., 2018). Our group has shown local FK506 enhances peripheral
nerve regeneration (Tajdaran et al., 2015, 2016b) by promoting proliferation of SCs
(Tajdaran et al., 2016a). However, the nerve regeneration capabilities of this drug have
not been studied in the cornea. This drug is FDA and Health Canada-approved and is
available as a topical ophthalmologic agent. Thus, as with topical applications of SP,
CGRP, NGF, and SCcm, this drug may aid the advancement of epithelial healing and
health in the neurotized denervated cornea.
We have previously published an experiment in which we observed corneal neurotization
improves corneal healing over time as compared to the denervated cornea that received
no treatment (Catapano et al., 2018). In future experiments, we will use the same
experiment paradigm and experimental groups to test the hypotheses that in the
denervated cornea 1) corneal neurotization is more effective in healing epithelial defects
than the aforementioned topical agents alone, and 2) corneal neurotization is more
effective in healing corneal epithelial defects in combination with the topical agents than
alone. To allow for comparisons to our previously published work, the groups used will be
as they were previously: (A) normally innervated corneas (control), (B) denervated
corneas (negative control), and (C) denervated + neurotized corneas. The topical agents
used will be i) SP + IGF-1 dissolved in Hanks Balanced Salt Solution (HBSS), ii) CGRP
+ SP in HBSS, iii) NGF in HBSS, iv) SCcm, v) FK506 vi) HBSS (negative control) and vii)
SCpm (SC pre-conditioned media, negative control for SCcm).
Prior to use of topical agents in vivo, the activity of the agents will be tested in vitro. The
concentrations used will be based on previously published literature (Nishida et al. 1996;
Nakamura et al., 1998; Chikama et al., 1999; Lambiase et al. 2000; Bonini et al., 2000a,
2000b; Tan, Bryars and Moore, 2006; Lambiase, Sacchetti and Bonini, 2012; Ko et al.
86
2013; Sacchetti, Lambiase and Ph, 2017). SP and CGRP have been shown to promote
the stratification of corneal epithelial cells (Ko et al., 2013), thus the efficacy of i), ii) and
vi) will be tested in cultures of human corneal epithelial cells. FK506, NGF, and SCcm all
promote neurite outgrowth, and thus the efficacy of iii) iv), v), vi) and vii) will be tested on
DRG cultures. Once concentrations are confirmed to ensure maximum effect in vitro, the
topical agents will be used to promote healing in the three groups in vivo. Further, to
ensure the topical agents are acting on-target, they can be applied with receptor-
antagonists.
The protective tarsorrhaphy will be removed, four weeks after the initial denervation, and
the corneal epithelium will be carefully debrided using an Algerbrush II (Alger Company,
Inc., Lago Vista, TX, USA). Fluorescein (DioFluor Strips, Innova Medical Opthalmics, Inc)
will be applied to the cornea and the staining imaged digitally (Nikon D 5100; Nikon) under
a ultraviolet lamp each day prior to application of topical drops applied to the denervated
cornea. Topical drops will be applied 4X per day between 8 AM and 8 PM. Imaging will
be performed using a standardized frame to keep the camera a fixed distance from the
corneal surface. The area of epithelial injury will be measured using ImageJ. The percent
of the corneal area healed over time will be calculated by standardizing the de-
epithelialized area at each timepoint to the initial wound size. The mean (± SD) percent
of corneal area healed in each group at each time point will be calculated and compared
using unpaired t-tests. In contrast to the previously published experiment, we will increase
the time of observation to one week. Though, with the addition of topical agents, we
hypothesize healing to improve within the four days it took for an epithelial wound to heal
in the denervated + neurotized cornea, we expect it will take longer for a wound to heal,
if at all, with applications of topical agents on the completely denervated cornea.
The experiments performed in Chapter 2 in this thesis, evaluating corneal epithelial and
stromal thicknesses, and evaluating corneal epithelial ulceration, scarring and
perforation, can similarly be repeated with the aforementioned topical agents and groups.
87
Of all the previously discussed topical agents, NGF is the treatment that has the largest
documented success in healing epithelial defects in patients. Even so, the documented
successes have been primarily in patients with corneal hypoesthesia, decreased
sensation, rather than complete anesthesia, absent sensation (Lambiase et al., 1998;
Bonini et al., 2000; Tan et al., 2006; Lambiase et al., 2012; Sacchetti et al., 2017). It is
therefore reasonable to hypothesize the other proposed topical agents would similarly be
more efficacious in cases of hypoinnervation. In our current model of NK, to achieve
complete denervation of the cornea for four weeks, we perform the ophthalmomaxillary
ablation twice. In the absence of the second ablation we have observed the early stages
of corneal reinnervation (Catapano et al., 2018) that resembles corneal hypoinnervation.
Thus, in the future we will re-evaluate the effect of topical SCcm and NGF application on
healing in the completely denervated (two ablations) and hypoinnervated (one ablation)
cornea over a longer period of time. This can also be done using corneal ablation in
healthy mice and mouse models of hypoinnervation such as herpetic keratitis in which
NGF topical treatment was effective in healing epithelial injury (Lambiase et al., 2008).
Repeating the healing experiment with the addition of topical agents in a corneal
hypoinnervation model (group D) might be extremely informative and applicable for
patients.
In cases of corneal hypoesthesia due to metabolic diseases, such as in diabetic or
herpetic keratopathy, corneal neurotization may not be a suitable option because of a
lack of viable donor nerves for coaptation of the nerve autograft. In cases where
hypoesthetic NK develops as a result of lamellar flap surgery, corneal abrasions, corneal
transplants, and LASIK, for example, less severe symptoms may not necessitate the
invasive, relative to topical drops, neurotization procedure. If corneal SCs are in fact the
source of NGF and other growth factors or neurotrophins, SCcm could be more effective
in treating PEDs than NGF alone. This is a similar, but more specific approach to
treatment of NK with autologous serum (Matsumoto et al., 2004). Contrary to the
autologous serum, the corneal SCcm would solely consist of the growth factors
specifically released by SCs in the cornea, and no other factors or molecules beyond
what is normally present in the healthy cornea. Further, FK506 is a promising drug for
88
topical use to enhance corneal nerve regeneration in hypoinnervated corneas because
of its effectiveness in enhancing nerve regeneration in other tissues, and its safety for
ophthalmic use in patients.
COLLABORATIONS FOR FUTURE DIRECTIONS
Our laboratory has developed the only animal model of corneal neurotization in rats, as
well as an animal model of NK with the longest-documented period of corneal denervation
(4 weeks). We have, in this thesis, established protocols for immunohistochemistry and
Western blot analysis of rat corneas and have experience with these techniques in
peripheral nerve analysis. Many of the methods proposed for future directions, such as
use of transgenic mouse models, necessitates collaboration with experts in other fields.
Collaboration with the Kaplan-Miller laboratory (The Hospital for Sick Children, Toronto)
will greatly benefit our investigations into corneal SCs. Expertise in the area of LSCs will
be provided through collaboration with the Shalom-Feuerstein laboratory (Technion,
Israel). Collaboration with both of these laboratories will be instrumental in understanding
the underlying mechanisms involved in corneal epithelial maintenance in the normally
innervated, denervated and neurotized corneas and how this relates to SCs and LSCs.
3.3 – Concluding remarks
This thesis demonstrates corneal neurotization is effective in maintaining corneal
epithelial integrity and health in the denervated rat cornea of our NK model. Corneal
neurotization is more effective than topical applications of NGF in treating epithelial injury
in the completely denervated cornea. Presence of nerve-derived peptides, SP and CGRP,
suggests a possible mechanism by which epithelial integrity is maintained, but further
studies need to be done to support this claim.
Despite the procedure’s demonstrated effectiveness in maintaining and healing the
corneal epithelium in both our NK rat model (Chapter 2; Catapano et al., 2018) and
patients (Catapano et al., 2019), about 15% of patients fail to reach levels of protective
89
corneal sensation post-operatively (Catapano et al., 2019). Additionally, once NK
symptoms progress to affect corneal layers deeper than the superficial epithelium, re-
innervating the cornea will not reverse the existing scarring or opacification (Catapano et
al., 2019) and these patients may still require corneal transplants/keratoplasties. In most
clinical cases patients are seen in late-stage NK, already presenting with moderate to
severe complications. The most direct application of this model and this study is thus for
patients with early stage NK, without severe ulceration or scarring. This emphasizes the
importance of identifying and treating these patients as early as possible.
This thesis work and our outlined future directions (Chapter 3.2) enhances and will add
to the understanding of 1) the relationship between innervation, SCs and LSCs, 2) the
molecular basis of NK, and 3) the mechanisms underlying the improvement in ocular
surface health following neurotization of the denervated cornea. The ultimate goal of
research into corneal innervation is to benefit the NK patients that do not currently benefit
from the corneal neurotization procedure. We believe our investigations into this area will
benefit patients that suffer from other ocular surface diseases, and perhaps have
implications in the broader context of peripheral nerves, SCs and stem cells.
90
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