Immunohistochemical Mapping of Corneal Sensory Innervation in Mice: Effects of Sjögren`S Syndrome Associated Dry Eye

IMMUNOHISTOCHEMICAL MAPPING OF CORNEAL SENSORY INNERVATION IN MICE: EFFECTS OF SJÖGREN`S SYNDROME ASSOCIATED DRY EYE

RENATA VELLOSO RAMOS

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2016

To Viviane and Otto

ACKNOWLEDGMENTS

I would like to recognize and express my profound gratitude to my advisor Dr.

Caryn Plummer for believing in me and giving me the opportunity of pursing my Master of Science degree and residency under her mentorship. I am greatly thankful for her guidance, encouragement, and friendship. I admire her remarkable ability to mentor, lead and teach while also being a caring friend; she will always be a professional model for me. I would like to extend my sincere gratitude to my co-advisor Dr. Rick Johnson for helping me in the development of critical thinking and commitment to high quality research. I am very honored to be able to benefit from his guidance, experience and friendship.

I would like to thank Dr. Whitley, Dr. Brooks and Dr. Hamor for their intellectual guidance, support and enthusiasm in both research and clinical settings.

I would like to express my gratitude to Dr. Cuong Nguyen, for the use of his mouse colony also offering his knowledge and support. I extend my appreciation to Dr.

Dan Gibson and Dr. Jasenka Zubcevic for their continued input, interest and brilliant solutions along the way. Their encouragement helped me to transform moments worth of frustration into a successful endeavor.

I would like to thank Victoria Dugan, Huy Nguyen, Alexandria Voigt and Dr. Arun

Wanchoo for their patience and support in the lab.

I extend my sincere appreciation to my dearest resident-mates, past and present,

Dr. Bianca Martins, Dr. Caroline Monk, Dr. Sarah Czerwinski, Dr. Proietto and Dr.

William Berkowski and to our remarkable technicians Holly Kitchen, Michelle Wilhelmy and Katherine Devine for their guidance, support and camaraderie over this journey.

Each of them has taught me something that I will take for life. I am forever grateful for

the opportunity to train in the country’s premier ophthalmology residency and become part of the ophthalmology family at UF.

I would like to thank UF College of Veterinary Medicine Graduate Office for their financial support, and Sally O’connell for her unrelenting help and positive attitudes.

I would also like to express my gratitude to my family and friends. The distance that separated us in the last years has not prevented them to continue nurturing our love. I specially thank my parents, Gastão and Isabella Ramos, for supporting me not only during this time but also throughout all my life.

Lastly, I would like to thank my partners in crime, Fernanda Ferreira and Achilles

Vieira Neto, without their love, strength and unending support this thesis would not have been possible.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF FIGURES ...... 8

LIST OF ABBREVIATIONS ...... 9

ABSTRACT ...... 11

1 INTRODUCTION ...... 13

2 LITERATURE REVIEW ...... 15

The Cornea ...... 16 Anatomy and Physiology ...... 17 Innervation ...... 19 Neuropeptides ...... 21 CGRP ...... 21 Substance P ...... 22 Protein Gene Product 9.5 – PGP 9.5 ...... 23 Dry Eye Syndrome ...... 24 Clinical Significance of Sjögren’s Syndrome ...... 26 Immunopathology ...... 27 Epidemiology ...... 28 Diagnostic, Treatment and Prognosis ...... 29 C57BL/6.NOD-Aec1Aec2 Mouse Model ...... 29

3 CHARACTERIZATION OF THE ARCHITECTURAL PATTERN OF CORNEAL INNERVATION IN C57BL/6.NOD-AEC1AEC2 MICE WITH SJÖGREN`S SYNDROME COMPARED TO WILD TYPE ...... 32

Background ...... 32 Materials and Methods...... 35 Animals...... 35 Antibodies ...... 35 Tissue Preparation and Immunofluorescence Staining ...... 35 Imaging...... 36 Data Analysis ...... 37 Results ...... 38 Stromal Nerve Architecture ...... 38 Epithelial Nerve Architecture ...... 39 PGP 9.5 and SP ...... 39 Co-Localization of SP and CGRP ...... 40 Sjögren’s Syndrome-Related Changes in Nerve Density ...... 40 Discussion ...... 41

Corneal Nerve Anatomy ...... 41 Methodological Considerations ...... 43 CGRP and SP ...... 45 Disease-Related Changes in Corneal Nerve Density ...... 46

4 CONCLUSIONS ...... 55

LIST OF REFERENCES ...... 57

BIOGRAPHICAL SKETCH ...... 70

LIST OF FIGURES

Figure page

3-1 Immunofluorescence imaging of wild type mouse cornea stained with PGP 9.5 ...... 50

3-2 Subbasal nerves in a wild type mouse cornea with whorl-like structure or vortex, labeled with PGP 9.5 antibody ...... 50

3-3 Representative images showing the expression of PGP 9.5-and-SubP positive nerves in the central and peripheral cornea ...... 51

3-4 Difference of corneal nerve density between the central and the peripheral zones ...... 52

3-5 Difference of corneal nerve density between the central and the peripheral zones ...... 52

3-6 Representative images showing the expression of CGRP-and-SubP positive nerves in the central and peripheral cornea ...... 53

3-7 Difference of corneal nerve density between the C57BL/6.NOD-Aec1Aec2 and wild type mouse ...... 53

3-8 Representative images showing decrease in the expression of PGP 9.5 positive nerves in normal mice and C57BL/6.NOD-Aec1Aec2 mice at the central and peripheral cornea ...... 54

LIST OF ABBREVIATIONS

5-HTT Serotonin-transported gene

Aec Autoimmune exocrinopathy

ANA Antinuclear antibody

AOI Area of interest

ARVO Association for Research in Vision and Ophthalmology

CGRP Calcitonin gene-related peptide

CO2 Carbon dioxide

DSU Disk scanning unit

GAL Galanin

IACUC Institutional Animal Care and Use Committee

Idd Insulin dependent diabetes

IFN Interferon

IL Interleukin

IP3 Inositol 1,4,5-triphosphate

IVCM In vivo confocal microscopy

KCS Keratoconjuctivitis sicca

M-ENK Methionine-enkephalin

MGV Mean gray value

NK Neurokinins

NK-1 Neurokinin type 1

NKA Neurokinin 

NKB Neurokinin 

NOD Nonobese diabetic mice

NPY Neuropeptide Y

PFA Paraformaldehyde

PGP 9.5 Protein gene product 9.5

PLC Phospholipase C pSS Primary Sjögren’s syndrome

RA Rheumatoid Arthritis

RF Rheumatoid factor

SDCM Spinning disk confocal microscopy

SLE Systemic Lupus Erythematous

SP Substance P

SS Sjögren’s syndrome sSS Secondary Sjögren’s syndrome

T1D Type 1 Diabetes

TG Trigeminal ganglion

UCH-L1 Ubiquitin carboxyl-terminal hydrolase-1

VIP Vasoactive intestinal polypeptide

Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

IMMUNOHISTOCHEMICAL MAPPING OF CORNEAL SENSORY INNERVATION IN MICE: EFFECTS OF SJÖGREN`S SYNDROME ASSOCIATED DRY EYE

Renata Velloso Ramos

August 2016

Chair: Caryn Plummer Major: Veterinary Medical Sciences

Purpose: The anatomy of the corneal innervation in a variety of species has been subject of much investigation; however a comprehensive description of mice corneal nerves architecture changes after exposure to a desiccating stress remains elusive. The purpose of the present study was to provide a detailed description of the mice corneal innervation involving immunohistochemically stained anterior-cornea whole mounts in a rodent model of dry eye and compare to that of the wild type. Methods: six

C57BL/6.NOD-Aec1Aec2 mice and fourteen wild type controls were enucleated after euthanasia via CO2 inhalation in a closed chamber. All corneas were harvested with narrow corneoscleral rims and incisions were made to produce a cloverleaf-shaped flat mount. The corneal sensory axons were labeled with immunofluorescence using primary antibodies against protein gene product (PGP) 9.5, substance P (SP), calcitonin gene-related peptide (CGRP); and subjected to quantitative and qualitative analyses using a spinning disk confocal microscope. Results: The results showed that main stromal nerve bundles entered the cornea at the cornealscleral limbus. Each stromal bundle gave rise through repetitive branching to a mildly dense midstromal plexus and

dense subepithelial plexus. Sub-basal density and anatomical complexity varied considerably among corneas and was less dense in the central cornea especially on dry eye syndrome corneas. Corneas from the C57BL/6.NOD-Aec1Aec2 mouse model have significantly decreased nerve fiber density of axons labeled for PGP 9.5 in the peripheral cornea compared to wild type. Similar findings were observed in the central cornea, however with no statistical difference. Conclusion: This study provide comprehensive descriptions of the architecture of mouse corneal sensory innervation including the distribution of the two major neuropeptides, as well as, the innervation of the mouse cornea under desiccant conditions, such those associated with Sjögren’s syndrome disease. Therefore this study provides an opportunity to gain important information that could lead to new treatments for dry eye in both humans and veterinarian patients.

CHAPTER 1 INTRODUCTION

Because vision is a fundamental phenomenon that affords distinct survival advantage, evolutionary development has provided the structures that sustain this function with special protection against injury. The cornea is the most powerful refractive surface of the eye, and must maintain optical transparency and structural integrity to best achieve its functions of transmitting incident light and protecting intraocular structures from traumas and pathogens.1,2 Thus, the cornea is the most densely innervated peripheral tissue of the body with respect to detecting noxious stimuli.3

Corneal nerves vary in their chemical composition and electrophysiological characteristics and can be classified according to the stimuli that foremost activate them: mechanical forces, thermal energy, or irritant chemicals. In addition to their protective function, these neurons play critical role in the blink reflex, pupil constriction, globe retraction and tear production in order to maintain the healthy of the ocular surface.4,5

The normal neuroanatomy and neurochemistry of the cornea has been studied for many years by a variety of methods, including light and electron microscopy, immunohistochemistry and in vivo confocal microscopy (IVCM). Several studies have used different immunochemical techniques to assess the quantities of the most abundant neuropeptides in the cornea, calcitonin gene-related peptide (CGRP), and substance P (SP). Despite several different approaches to the description of the anatomy and physiology of the corneal innervation, using (i) numerous neuronal markers, (ii) staining methodologies and (iii) image acquisition techniques, there are many aspects of corneal nerve function and architecture that are still poorly

understood.5-8 Therefore, there has been a renewed interest in studying corneal nerves.

Studies have established that nerve dysfunction is a frequent pathobiological feature of corneal diseases that can cause opacities and result in discomfort and blindness.9

Several infectious and noninfectious inflammatory corneal injuries including surgical manipulation or trauma, can lead to lost or compromised innervation to varying degrees.

As a consequence, the functional and morphologic properties of corneal nerves can change substantially leading to discomfort and pain.9-11

Dry eye syndrome or keratoconjuctivitis sicca (KCS) is a common condition affecting humans and animals. It has been estimated that 4.5 million people over the age of 50 suffer from dry eye.12 The disorder can be a consequence of poor tear quality or the lack of tear production. Causes include aging, medications, environment, and medical conditions.13,14 Sjögren’s syndrome (SS) is a systemic, autoimmune disease that often manifests with a form of dry eye, particularly in its early stages.12,15 Very little is known about the changes that may occur in the corneal innervation during the disease process, therefore, a better understanding of these changes is the first step towards the development of new therapeutic approaches to improve patient’s quality of life. Therefore, the goal of this study is to provide a detailed and comprehensive description of the corneal nerve architecture, using multifluorescence confocal microscopy, with emphasis on the corneal nerve distribution of the main sensory neuropeptides SP and CGRP, and to elucidate innervation changes in the corneal tissue of a rodent model of Sjögren’s syndrome.

CHAPTER 2 LITERATURE REVIEW

The subject of corneal innervation has taken on heightened importance in recent years due to the observation that corneal nerves are routinely injured following modern refractive surgical procedures, repeated exposure to air-conditioned environments or following certain corneal diseases. Descriptions of the anatomy and physiology of the corneal innervation in different species are numerous, yet many aspects of corneal nerve architecture and function remain incompletely understood.

Schlemm first discovered corneal nerves in 1831, prior to this discovery, the cornea was thought to be entirely without nerves.16 Many years after Schlemm’s original observation, Bochdalek found that the ciliary nerves divide into corneal and iridal branches and that the corneal braches enter from the limbus into the anterior stroma and epithelium.17 Despite these initial findings, the first detailed description of corneal epithelial nerves was made after studying fresh central corneal buttons from enucleated globes and keratectomy specimens stained with gold chloride and examined by light microscopy.9,17 More recently, the architecture of the human corneal innervation was systematically described using immunohistochemically stained anterior-cornea whole mounts by Marfut et al. In this study the corneal nerves were stained with a primary antibody against neuronal-class III-tubulin and subjected to quantitative and qualitative analyses.5 Several groups have used immunochemical methods to study corneal innervation. This process allows the localization of different neuropeptides within the cell soma and peripheral axonal fibers of corneal neurons, which suggests that they are functionally heterogeneous.

In addition, image acquisition techniques play a critical role in nerve fiber identification. Most information on corneal nerve ultrastructure has been gleaned from electron microscopic observations. However, recently, IVCM has been used to image nerves in diseased and healthy corneas and has provided considerable new information on the density and morphology for quantitative and qualitative research, including cross- sectional and longitudinal studies.18-20

The objectives of this literature review are: to provide a comprehensive understanding of the corneal anatomy and physiology, corneal innervation, neuropeptides and their role in neuro-ophthalmology in both normal and diseased state.

Additionally, I will discuss dry eye syndrome and its clinical significance, immunopathology, epidemiology, diagnosis, treatment and prognosis.

The Cornea

For optimal vision to occur, light will travel unimpeded through the cornea, the transparent anterior tunic of the eye. Thus, the cornea must maintain an intact, smooth surface to best achieve its primary physiologic functions of transmitting incident light, and providing protection of the intraocular structures from trauma and pathogens.1,21

The corneal thickness varies among species and across different regions of the cornea (530  20m in humans, 106  3.4m in mice and 356.11 14.3m in rabbits).22

By comparison, corneal curvature values have a lower range of variation (2.1 0.019 mm in the mice and 11.7 mm in one human population).23 It is elliptical in shape, with a horizontal diameter greater than the vertical. (Figure 3-1) The cornea makes up the anterior aspect of the outer fibrous tunic of the globe. The posterior part of the fibrous tunic is the sclera, which joins the cornea and bulbar conjunctiva at the corneoscleral

junction (limbus). This transition zone is rich in blood vessels and nerves and is the location of epithelial stem cells.24,25

Anatomy and Physiology

The cornea is composed by several layers. These include a stratified epithelium and its basement membrane, stroma composed of a complex organized collagen matrix disperse with keratocytes, nerves and glycosaminoglycans, Descemet’s membrane which forms the basement layer for the endothelium, which is the latter and innermost layer, composed a monolayer of hexagonal cells.21,26-28

The precorneal tear film lies anterior to of the cornea, and contains many important elements which contribute to corneal health and function. The human eye can each accommodate 7-30L of tear fluid on the ocular surface with a turnover rate of approximately 0.5-2.2L/min. Spontaneous blinking mechanically spreads the tear film evenly and removes debris in contact with the corneal surface. Humans exhibit an unconsciously blinking rate of 6-15 times/min.29 The tear film itself is a trilaminar film composed of an aqueous fracture from the lacrimal gland containing cytokines, growth factors, fibronectin, plasminogen and inflammatory cells providing moistening and protection of the corneal epithelium, and mucin and lipid fractions that promote adhesions of the aqueous tears to the ocular surface and prevent premature evaporation.29-31

The epithelium is the outermost portion of the cornea that functions to support and protect the deeper tissue from injury and assault by pathogens.32,33 The corneal stratified epithelium comprises 8-15 cells layers of non-keratinized, squamous cells with intercellular tight junctional complexes, important for performing a barrier function.34,35

Beneath the superficial cell layers is the basement membrane, separated by a group of polyhedral wing cells (transition zone) and firmly attached by the basal cells via hemidesmosomes, lamina collagens, fibrils and laminin. Basal cells are tall and columnar with a flattened base, their nuclei is located in the apical region and are often forced into two due to crowding. These cells are transient amplifying cells because of their limited mitotic ability, replacing cells after desquamation of the superficial epithelial layer.32,33,36

The corneal stroma constitutes 90% of the corneal thickness and confers rigidity to the globe. In most species, it is composed of keratocytes, orderly collagen type I and type V, water and glycosaminoglycans including keratin sulfate, dermatan sulfate and chondroitin sulfate. In addition to the precise organization of the stromal collagen, the keratocytes have crystallins, which are believed to facilitate corneal transparency.37,38

Disruption of this complex arrangement can transform keratocytes into myofibroblasts and they may form scar tissue that is disorganized and opaque. In humans and a few other species, the most anterior stroma has a thin, acellular layer known as Bowman’s layer. The collagens fibrils in this layer are randomly dispersed and are smaller in diameter allowing communication between the epithelium with the underlying stroma.37

Descemet’s membrane is an inner protective barrier deep to the corneal stroma, which is contains a number of collagens including Type VIII.39 It is a homogenous, elastic and impermeable membrane underlying endothelium.32,40 Finally, the last layer of the cornea is the endothelium, which consist in a single layer of hexagonal cells lining the anterior chamber. These cells interdigitate laterally without a specific cell-cell junction mechanism. Its major role is to actively remove ions from the corneal stroma

into the aqueous humor in order to maintain corneal clarity and function. Loss or damage of the corneal endothelium such as genetic predisposition, trauma, corneal surgeries or inflammation, may cause permanent corneal opacification caused by edema.

Innervation

The corneal pain system is essential for maintaining the integrity of intraocular structures and functional vision. This is accomplished through early detection by primary sensory neurons of noxious or potentially noxious stimuli and subsequent responses by the organism to minimize damage.41 The ocular primary sensory neurons compose the afferent branch of protective reflexes including blinking, retraction of the globe, pupil constriction and tear production.42,43 In addition, sensory neurons produce a continuous effect on their target cells, which may contribute to the modulation of healing processes in injured tissues.44-46

Several studies have described the corneal sensory fibers originating from ipsilateral trigeminal ganglion (TG) as a part of the ophthalmic nerve, however few descriptions have shown that a portion of these neurons in monkeys and maybe in humans, reaches the eye through the maxillary nerve.44,47 According to Rozsa et al, the cornea contains the highest density of sensory endings of the body: 300 - 600 times mores than the skin and 20 - 40 times more than the tooth pulp.48

The morphological appearance of the ocular TG neurons is quite simple and can be classified as thinly myelinated (A- units) or unmyelinated (C-afferent units) axons that terminate peripherally as naked nerve endings called nociceptors.3,8 Each of these may respond differently to mechanical forces, temperature changes and chemical

agents which leads to their classification by the modality of stimulus that they are preferentially activated by.49 These so-called polymodal neurons have large receptive fields and are the most abundant class of corneal sensory units found in most of species. 50,51

Some of the corneal polymodal nociceptors fibers belong to the thin myelinated group, however 70% of them are of the slower-conducting unmyelinated C type. They are activated by mechanical energy near or above noxious level, such as contact with external objects, presence of foreign bodies or even air pressure on the surface of the eye.8,49,52 When polymodal nociceptors are stimulated they depolarize, releasing neuroptides at all the peripheral nerve terminals for that unit.53 Therefore, this process extends to axons that are directly exposed to the stimulus in addition to activating other units.54

Drying of the ocular surface can produce a mechanical distortion of the epithelial layers deforming the intracellular spaces where nerve endings are located, leading to their stimulation.55 A notable feature of corneal polymodal nociceptors is that when a noxious stimulus happens repeatedly, the mean firing frequency in response to that noxious stimulus increases over time, in a process called sensitization. This process is developed in response to inflammatory mediators released by damaged cells and the detection of the released neuropeptides.56,57 Morphological changes in the molecular structure of the affected neurons are not fully elucidated, however it is well known that unpleasant sensations caused by this neuropathic activity can be manifested as desiccation and ocular pain.58

Neuropeptides

Neuropeptides are a group of proteins, which are widely distributed in the central and peripheral nervous system that act as neurotransmitters and/or neuromodulators.59,60 The first neuropeptide to be discovered was substance P (SP) by von Euler and Gaddum in 1931. It was originally described as hypotensive and spasmogenic factor found in extracts of equine brain and intestine, which later was determined to be SP.61 The human skin is supplied with nerves that contain several neuropeptides. These neuropeptides include calcitonin gene-related peptide (CGRP) and substance P (SP).62-64 Further studies in the mammalian skin described that CGRP and SP were both found in unmyelinated sensory nerves fibers and may mediate the proliferation of endothelial cells, arterial smooth muscle cells and skin fibroblasts playing a role in the angiogenesis of healing tissues. 63,64

Ocular neurons may also contain several neuropeptides expressing to a variable degree a number of membrane proteins that act as receptor molecules for neurotransmitters, neuromodulators, cytokines and growth factors.3 In 1987, Stone et al. summarized findings of descriptive investigations with regard to neuropeptides within the human eye. Again, SP and CGRP which were the main peptides noted, both in cornea and in other tissues of the eye.65 In recent years, great progress has been made in the identification and interpretation of their mechanism of action. They act as pro- inflammatory substances, stimulating the release of other mediators that control the development of inflammation immediately after noxious stimulation.

CGRP

Calcitonin gene-related peptide (CGRP) is a neuro-peptide composed of 37 amino acids, whose presence is widespread in the nervous tissue of the body.66,67 It is

thought to be the result of alternative processing of the primary RNA transcript of the calcitonin gene in the rat.68 CGRP-like immunoreactivity was shown to be present in neurons of the rat brain and in peripheral areas known to be rich in nerves and terminals of primary sensory axons, such as tongue, blood vessels, and skin and central axons processes found in the spinal cord and medulla oblongata.66,69,70 The presence of

CGRP in the sensory neuronal cell bodies in the trigeminal and dorsal root ganglia, which are known to relay nociceptive information from the periphery to the central nervous system, suggests involvement of this peptide in pain perception or modulation of nociceptive information.71,72 Hence, in addition to its peripheral release, CGRP released in the central nervous system may be involved in migrane pain pathophysiology.73 Studies using cultured rat trigeminal neurons indicated that prostaglandins, which are inflammatory mediators implicated in migrane, cause CGRP release.74 Moreover, this peptide, which seems to be bound to other peptides, was found to coexist with SP in sensory ganglia of the rat and in guinea-pig corneas.72,75,76

CGRP is also a potent vasodilator. Henderson et al. quantified the regeneration patterns of blood vessels and nerves in excisional skin wounds in mice and found that CGRP is important for the formation of the new vessels in ischemia, inflammation, and in wound healing77.

Substance P

Of all the peptides, SP has the longest history and is probably the best characterized as far as distribution, release, and biological properties are concerned.

Detailed studies on the occurrence of SP in various organs have shown that it contributes to diverse physiological functions, including neurogenic inflammation, vasodilation, contraction of smooth muscles, and modulation of immune cell activity.78

Substance P neurons have also been found to be involved in the transmission of pain- related information, primarily via polymodal C-fibers nociceptors located on the cornea.79-83 Substance P has been identified in the corneal nerves with immunocytochemistry.84 Substance P neurons are known to participate in low-grade inflammatory responses for several months post corneal injury85. Therefore, the presence of high levels of SP-positive fibers in damaged tissues suggests an increased sensitivity to noxious stimuli and the ability to participate in the neurogenic inflammatory process.

The neurokinins are mammalian peptides found in the central and peripheral nervous system, and the receptors for neurokinins have been designated as NK-1, NK-

2 and NK-3.86,87 These receptors are small proteins composed of 350 to 500 amino acid residues that are expressed on nerves, immune cells, and epithelial cells. Maggio et al. identified two homologues of SP, NKA (neurokinin ) and NKB (neurokinin ).

Substance P exerts its biological effects through the binding to G protein-coupled receptors, and has the highest affinity for the neurokinin type 1 (NK-1) receptor. The binding of ligand to NK receptors results in the activation of phospholipase C (PLC) and adenylate cyclase and the consequent production of the second messengers inositol

1,4,5-triphosphate (IP3) and cAMP, respectively, mediating neurogenic inflammation.87,88

Protein Gene Product 9.5 – PGP 9.5

Protein gene product 9.5 (PGP 9.5), also known as ubiquitin carboxyl-terminal hydrolase-1 (UCH-L1), is a cytoplasmatic protein present in large amounts in the neurons of central and peripheral nervous systems. Jackson et al. described the initial

detection of PGP 9.5 in human brain in 1981, and shown to be a marker of certain neurons in the central nervous system.89 PGP 9.5 was found to have a molecular weight of 27.000, and to be present in human brain at concentrations at least 50-fold greater than in other organs. Subsequent studies demonstrated that traces of this protein were also seen in large intestine, kidney, ovary and testis, showing the same specific protein patterns, excluding the unlikely possibility of individual polymorphism.

Immunohistochemical analysis demonstrated the presence of PGP 9.5 not only in neurons in the central and peripheral nervous system but also in neuroendocrine cells.90 It is believed that PGP 9.5 plays an important role during proliferation of glial cells in the nervous system, during reactive gliosis, and/or in connection with tumorigenesis of the glial lineage. In fact, PGP 9.5 immunohistochemical phenotype is not only a diagnostic marker but also a prognostic marker when described in pancreatic endocrine tumors.91 Further studies using antibodies against PGP 9.5 have demonstrated its extensive presence in the skin of healthy and diabetic patients,92,93 as well as in the canine corneal nerve architecture.7

The distribution of PGP 9.5 immunoreactive nerve fibers has been compared to the distribution of nerve fibers immunoreactive to others neuronal markers such as neuron specific enolase, neurofilament proteins, calcitonin gene related peptide, vasoactive intestinal polypeptide and neuropeptide Y, and the number of positive fibers and the intensity of the fluorescence was greater with PGP 9.5 antibodies.92 Therefore, staining for PGP 9.5 is a valuable probe in the exploration of innervation architecture.

Dry Eye Syndrome

Dry eye, also known as keratoconjunctivitis sicca (KCS), is a common cause of chronic ocular pain and it is exceedingly common in human patients.12,94 Chronic

irritation and exposure of the ocular surface and subsequent irritation can result in profound discomfort and may lead to changes in corneal clarity. The condition may be the consequence of either lack of tear production (quantitative tear deficiency) or of poor tear quality (qualitative tear deficiency). Postulated mechanisms include inflammation and sensory neuronal dyregulation.95 Chronic ocular surface irritation and subsequent inflammation are associated with pain, corneal fibrosis, edema, vascularization and pigmentation, all of which impair vision. Etiologies may include aging, medications or toxicities, environmental conditions and some medical conditions, including immune- mediated diseases.96 Sjögren syndrome (SS) is a generalized autoimmune condition that manifests as a form of aqueous-deficient dry eye, particularly in the early stages, and xerostomia. Symptoms of dry eye and dry mouth are considered hallmark indicators of patients suffering from SS. Patients diagnosed with SS experience mild discomfort to severe chronic pain. Pain relief is momentary and fleeting with tear supplements and oral moistening agents, and long-term relief is elusive. The ocular surface has a rich sensory innervation, which is responsible for modulating healing processes and eliciting pain, such as that experienced during the dry eye condition. Little is known about the changes that occur in corneal innervation during disease, particularly in sicca.

Understanding the innervation of the cornea in the normal state and characterizing the differences that occur during KCS are critical for a better understanding of the disease state, which may lead to the development of new therapeutic approaches for this condition that affects so many individuals. Although studies have previously demonstrated that exposure to a desiccating stress in a mouse model of experimental dry eye significantly increases the cell proliferation rate and the central thickness of the

corneal epithelium, studies on the corneal innervation in SS are lacking and warrant further investigation.

Clinical Significance of Sjögren’s Syndrome

Sjögren's syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration of the exocrine glands reducing lacrimal and salivary secretion.

The disease can occur alone − primary Sjögren’s syndrome (pSS) − or concomitantly with another autoimmune disease, most often a connective tissue disease like rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) or systemic sclerosis – secondary SS. Even though salivary and lacrimal glands are the principal targets of the

T cell mediated chronic inflammation that gives rise to impaired function and to dry eyes

(keratoconjunctivis sicca) and mouth (xerostomia), systemic manifestations have been described. Abnormalities with the musculoskeletal, pulmonary, gastric, hematologic, dermatologic, renal and/or nervous system are observed in a majority of patients.97,98

Analysis of data concerning the involvement of particular organs indicates that approximately 20% - 30% of patients with primary SS have clinical pulmonary involvement, which is associated with significantly greater impairment in quality of life.99

Patients with SS have an estimated 16 – 37.5 times increased risk of lymphoma compared to the general population.100 Moreover, studies regarding kidney and liver diseases in SS patients suggest that that the majority of extraglandular manifestations of SS are due to the attraction of lymphocytes by different epithelial tissues. The main histopathologic manifestation of the affected exocrine glands is a focal round-cell infiltrate, which in the early lesions localizes around ductal epithelium. In advanced stages the round-cell infiltrate extends and occupies the acinar epithelium and leads,

though unknown mechanisms, to a glandular dysfunction which manifests in dry eyes, dry mouth, and enlargement of the major salivary glands.101

Immunopathology

In the 1970s, intense research evaluating the immunopathologic lesions of the affected exocrine glands, although indirect, indicated that the majority of the infiltrating lymphocytes were T cells and that the B cells in the lesion were activated. Since then, the development of molecular tools has allowed for a better understanding of the immunophatologic lesions in the exocrine gland of SS patients.

As early as 1972, it was found that lymphocytes from labial salivary gland are able to produce increased amounts of immunoglobulins in vitro with autoantibody activity that indicated the activation status of the B-lymphocytes infiltrating the salivary glands. 15 Salivary and lacrimal gland dysfunction in SS is believed to result initially from the production of pro-inflammatory cytokines capable of inducing cellular apoptosis and auto-antibodies. Aberrant expression of the cytokine (interleukin-18) was observed in tissues of SS patients. The Interferon (IFN) system was also found to be involved in the disease process.102,103 After investigation of the salivary glands from a large population of SS patients, IFN activity was associated with a more severe disease phenotype of salivary dysfunction and ocular dryness.102

Both genetic and non-genetic factors are involved in disease susceptibility and initiation, leading to a malfunction of the glandular epithelial cells and to an autoimmune response. Beyond genetics, epigenetic alterations, especially those related to DNA methylation, have been shown to play key roles in the pathogenesis of SS. Association between low platelet serotonin level in patients with SS and polymorphism in the

serotonin-transported gene (5-HTT), demonstrate that variants of the 5-HTT gene significantly contribute to decreased platelet serotonin levels in theses patients.104

Examinations of biomarkers such as SS-A/Ro, SS-B/La, ANA (antinuclear antibody), and RF (rheumatoid factor) are considered important in the diagnosis of SS.

Studies have showed that patients expressing SS-A/Ro antibodies have an increased risk of developing extraglandular manifestations. Primary SS was significantly more likely in patients positive for ANA or RF. SS-A/Ro and SS-B/La are positive in approximately half of patients with SS who exhibit dry eye symptoms in early stages.105

Epidemiology

Despite expanding efforts to define the genetic, environmental and immunological basis of SS, the underlying etiology of this disease remains undefined.

For many years after SS was characterized in 1933, it was considered relatively rare and primarily a disease of older women that was often associated with RA. Indeed, SS occurs in 30-50% of patients with RA, but also in 10-25% of patients with SLE, and in substantial numbers of patients with primary cirrhosis, myasthenia gravis, and pernicious anemia.106-108 The female: male ratio is generally estimated to be around 9:1, with peak onset occurring between the ages of 40 and 60.98,109 A number of studies have shown great variation in the frequency of SS. A cautious but realistic estimate from studies presented so far is that pSS likely has a prevalence rate of 0.6% of the general population.110 However, the geoepidemiological distribution of systemic autoimmune diseases is highly variable in global population, relative to the prevalence of specific diseases, M/F ratio, organ involvement and severity.

Diagnostic, Treatment and Prognosis

There is no simple diagnostic test for SS, although several classification criteria have been designed. Sjögren's syndrome is a heterogeneous disease, characterized by a broad spectrum of clinical and serological manifestations. Therefore, due to its complexity the diagnosis of SS remains challenging. To date, effective therapy is not available and treatment has been mainly symptomatic (tear and saliva substitutes, saliva stimulating agents such as pilocarpine or cevimeline and analgesics). Thus, further unraveling of the pathophysiology of pSS is essential for finding novel biomarkers and identifying new treatment targets. Ongoing studies in murine models are aimed at developing more effective and targeted therapies in SS. The heterogeneity of SS will most probably benefit from optimizing therapies, tailored to specific subgroups of the disease in an attempt to better clarify the innate and adaptive mechanisms involved in the disease initiation and perpetuation.

Understanding of Sjögren’s syndrome has increased since its first identification in

1933, resulting in improved recognition. Treatment primarily targets control of mucous membrane dryness, fatigue and pain.

C57BL/6.NOD-Aec1Aec2 Mouse Model

Different murine models for SS have been previously described.111 For years, the nonobese diabetic mice (NOD) strain was the most extensively characterized model of both SS and Type 1 Diabetes (T1D). Although some genetic loci related to diabetes contribute to the inflammatory changes in the exocrine glands, it seems that diabetes and SS develop independently of each other.112,113 The NOD mouse model has provided important insight into the genetics of human SS. The development of T1D in the NOD mouse is controlled by more than 18 chromosomal regions.114 Early studies

involving replacement of individual insulin dependent diabetes (idd) susceptibility intervals such as Idd3, Idd5, Idd13, Idd1, and Idd9 had minimal effect on the development of autoimmune exocrinopathy or SS-like disease. Both Idd3 and Idd5 are required for development of salivary and lacrimal dysfunction.115 When both NOD- derived genetic regions were introduced to the SS non-susceptible C57BL/6 strain by crossing C57BL/6.NODc3 mice carrying Idd3 (Autoimmune exocrinopathy 1 [Aec1]) locus and C57BL/6.NODc1t mice carrying Idd5 (Aec2) locus, the

C57BL/6.NODc3.NODc1t or C57BL/6.NOD-Aec1Aec2 mouse strain was produced which is homozygous for both Idd3 and Idd5 chromosomal intervals.112 This double congenic strain fully recapitulated the SS disease process, exhibiting pathophysiological changes at early age, followed by lymphocytic infiltrations of the salivary and lacrimal glands at 12-16 weeks of age, then accompanied by the production of autoantibodies to nuclear antigens (SSA/Ro, SSB/La) and acetylcholine muscarinic type 3 receptor in the absence of T1D. The lymphocytic foci consisted mainly of CD4+ and CD8+ T cells, as well as B lymphocytes and loss of saliva production by noted 20 weeks of age. Due to the presence of T cells and sporadic numbers of dendritic cells and macrophages within infiltrates, an increase in the levels of proinflammatory cytokines such as interleukin-17

(IL-17), IL-22, and IL-23 was also detected locally and systemically. Similar observations are observed in human SS patients.116

The differential gene expression in the lacrimal gland during development and onset of KCS in SS using the same mouse model was also described.117 However, the anatomical changes in the corneal innervation pattern have never been described.

Therefore, in the present study, we have used the C57BL/6.NOD-Aec1Aec2 model as an animal model for pSS to examine the innervation changes in corneal tissue.

CHAPTER 3 CHARACTERIZATION OF THE ARCHITECTURAL PATTERN OF CORNEAL INNERVATION IN C57BL/6.NOD-AEC1AEC2 MICE WITH SJÖGREN`S SYNDROME COMPARED TO WILD TYPE

Background

Dry eye is a common condition affecting the cornea of an estimated 4.5 million people over the age of 50.118 This condition can be a consequence of poor quality of the tearing or the lack of its production. Proposed causes include aging, medications, environmental conditions and some systemic medical conditions. One of these includes

Sjögren syndrome (SS), a systemic, autoimmune disease that often manifests as a form of aqueous-deficient dry eye, particularly in the early stages. Symptoms of dry eye and dry mouth are considered hallmark indicators of SS 12. Patients diagnosed with SS can experience mild to severe chronic ocular discomfort, with relief only momentarily accomplished by application of topical lubricants. Very few medical therapies are available to treat this condition beyond rescue lubricants. Therefore, finding new therapies to improve patient quality of life and the clinical signs symptoms of this condition is imperative. Little is known about the changes that may occur in the corneal innervation during the disease process; therefore, achieving a better understanding of this pathophysiology is an important first step towards new therapeutic approaches.

The mammal corneal surface, containing an estimated 7000 sensory nerve terminals per square millimeter of epithelium, is the most sensitive and densely innervated peripheral tissue in the human body.5 Corneal nerves are responsible for promoting and maintaining the health of the ocular surface by activating brain stem circuits that stimulate tear production, and by releasing trophic substances that protect and support physiologic renewal of the corneal epithelium (e.g. neuropeptides).

The corneal neuroanatomy has been studied for many years by a variety of methods. Light and electron microscopic investigations of human corneal nerve distribution, density, and ultrastructure have generated most of the data on which current models of corneal innervation are based.2-4 Descriptions of the neuroanatomy and neurochemistry of the normal mouse cornea have been previously performed with immunohistochemistry techniques using antibodies to neuronal-class III-tubulin, calcitonin gene-related peptide (CGRP), and substance P (SP).119 Which revealed that sub-basal nerve density and nerve terminals were greater in the center of cornea rather than its periphery, and that CGRP-positive nerve fibers and terminals were more abundant than SP-positive nerves. The functional significance of the peptidergic nerves in the cornea is still not fully elucidated, but SP and CGRP may be involved in the initiation of acute inflammation within the corneal epithelium after injury or infection.120,121

When the cornea is injured either accidentally or as a consequence of its surgical manipulation, the axons of neurons that form the corneal nerves are severed to a variable degree.9 The functional and morphological properties of corneal neurons change substantially following a peripheral axotomy. The area of the cornea that became denervated as a consequence of the lesion is invaded in the next days by outgrowths of neighboring non-injured nerve fibers.58 Also, the central stump of cut axons dilates and their surrounding glial and connective tissue cells proliferate, so that microneuromas are formed and the cut axon starts to regenerate, producing sprouts that grow and try to cross the scar tissue to penetrate the denervated area.122 Different studies demonstrate that exposure to a desiccating stress in a mouse model of

experimental dry eye significantly increases the cell proliferation rate and the central thickness of the corneal epithelium 123, and suggests that abnormally rapid tissue turnover and hyperplasia is a common response to the epithelial ‘‘stress response’’ throughout the entire ocular surface. Previous studies in humans have focused on total corneal, not just epithelial, thickness, and have reported a reduction in central corneal thickness in Sjögren’s and non-Sjögren’s dry eye. However, to the authors’ knowledge, there are no studies reporting the changes on corneal innervation during SS or the consequences of dry eye on corneal healing.

Several imaging techniques have been described for qualification and quantification of corneal nerve fibers such as fluorescence microscopy, light and electron microscopy and In Vivo Confocal Microscopy.7,9,119,124 Corneal confocal microscopy is a relatively novel technique that has been originally developed to quantify the severity of neuropathy in several diseases.125,126 However, to the author’s knowledge, a comprehensive study of mouse corneal nerves using Ex Vivo Confocal

Microscopy for imaging and analyses is yet to be performed.

Therefore, using a modified method of immunofluorescence and imaging based on the previously published successful methodologies, the objective of this study is to provide comprehensive description of the corneal nerve architecture in the C57B6 mouse, using multifluorescence confocal microscopy, with emphasis on the corneal nerve distribution of the main sensory neuropeptides SP and CGRP, in order to elucidate the innervation changes in the corneal tissue of a mouse model of Sjögren’s syndrome.

Materials and Methods

Animals

Fourteen C57BL/6 mice aged 3 to 24 weeks (both sexes) were purchased from

Jackson Laboratory (Bar Harbor, ME, USA) and housed at Biomedical Sciences

Building, University of Florida (UF; Gainesville, FL, USA). Additional six C57BL/6.NOD-

Aec1Aec2 mice aged 16 to 31 weeks were acquired from a C57BL/6.NOD-Aec1Aec2 mouse model colony, and bred and maintained within the Department of Pathology’s

Mouse Facility at University of Florida (UF; Gainesville, FL, USA). All animals used in the experiment reported herein were handled in a manner consistent with ARVO

Statement for the Use of Animals in Ophthalmic and Vision Research, and in protocol that was reviewed and approved by the University of Florida Institutional Animal Care and Use Committee.

Antibodies

Rabbit polyclonal anti-PGP 9.5 antibody (AB1761) (Chemicon International, Inc.,

Temecula CA, USA); guinea pig polyclonal anti-SP (GP14103) (Neuromics Inc,

Minneapolis MN, USA); and rabbit polyclonal anti-CGRP (RA24112) (Neuromics Inc.,

Minneapolis MN, USA) were employed as primary antibodies. Secondary antibodies were AlexaFluor 488 (green fluorphore) goat anti-rabbit IgG (H+L) and AlexaFluor 594

(red fluorophore) goat anti-guinea pig IgG (H+L) (Molecular Probes, Inc., Eugene, OR,

USA).

Tissue Preparation and Immunofluorescence Staining

Mice were euthanized via CO2 inhalation in a closed chamber. Enucleations were performed promptly after euthanasia and globes were immersion-fixed whole in freshly prepared 4% paraformaldehyde (PFA) at room temperature for 20 minutes. The

corneas were carefully excised along the corneoscleral junction. After removal of iris and ciliary body from the corneoscleral rim, incisions using a razor blade were made to produce a cloverleaf-shaped flat mount of the cornea. Tissues were immersion-fixed for additional 2 hours at 4C followed by three washes with 0.1 M phosphate buffer (pH

7.2). In order to block nonspecific binding, corneas were placed in a 96-well plate (one cornea per well) and then incubated with 30% normal goat serum for 1 hour at room temperature. Immunohistochemistry process was performed using a double labeling indirect immunofluorescent method on free-floating, whole mount sections. Rabbit polyclonal anti-PGP 9.5 antibody was used diluted to 1:1500 as a pan-neuronal marker.

Guinea pig polyclonal anti-SP antibody was used diluted to 1:200, and both antibodies were applied into the well simultaneously in order to demonstrate co-localization of PGP

9.5 and SP. Similar procedure was performed using rabbit polyclonal anti-CGRP diluted to 1:1000 and SP at the same concentration described above to demonstrate co- localization of SP and CGRP. Following incubation of 48 hours at 4C in primary antibody solution, tissues were extensively rinsed in with 10% normal goat serum.

Corneas were incubated with corresponding secondary antibodies for 3.5 hours at room temperature followed by rinsing three times with 10% normal goat serum. Whole corneas were stretched and mounted using anti-fade mounting medium (Beyotime

Institute of Biotechnology, Jiangsu, China) on a glass slide, and coversliped with the endothelium positioned down.

Imaging

Images were acquired using an Olympus Disk Scanning Unit (DSU) –IX81

Spinning Disc Confocal equipped with a camera (Hamamatsu C4742-80-12AG

Monochrome CCD) equipped with imaging software (Slide Book Reader, Intelligent

Imaging Innovations Inc, Denver CO, USA). The camera used allowed full frame, high resolution imaging at up to 15 f.p.s and 1344 x 1024 resolution (1.37 million pixels).

To obtain orthogonal section displays of whole corneal thickness, series of Z- sections of the central and peripheral corneal zones (one image per quadrant) were generated using a 20x/0.75 objective lens. Automated mode was applied to obtain full thickness scans from each area of interest (AOI). During scanning, the microscope recorded a mean of 5 frames/s as the focal plane advanced anteriorly, with 1m between z axis frames, until a mean of 25 frames was recorded. A projection image of all frames for each AOI was generated for quantitative analysis. The images comprised

384 x 384 pixels covering an area of 400 x 400m.

Data Analysis

Quantitative and qualitative analyses of corneal nerve architecture were performed by a single observer, using Image Intensity Processing (pixel intensity),

ImageJ software (Rasband, W.S., ImageJ, U.S. National institutes of Health, Bethesda,

Maryland, USA,../, 1997-2016). ImageJ was developed at the National Institute of

Health by Wayne Rasband and can be extended by adding features in the form of plugins, macros or scripts. The images were analyzed using a Mean Gray Value, which consists of the sum of the gray values of all the pixels in the selection area divided by the number of pixels. To obtain better contrast, the red channel was subtracted from the green channel using Image Calculator tool. The fluorescent images were then changed to grayscale mode and placed against a black background using the Auto Threshold

tool. The percentage for each nerve area was quantified using the image analysis program.

In order to compare nerve densities in the central and peripheral zones, 5 images from each cornea encompassing the zones of interest were randomly chosen for analysis (1 image/quadrant). A total of 50 images for each zone from 10 corneas were averaged.

Basal epithelial and stromal nerves were identified by their previously described characteristics in rodents.7,124,127,128 The examined parameters for evaluation of corneal nerves were as follows: (i) density of corneal nerves, defined as total of the pixel intensity observed within a frame; and (ii) relative content of sensory neuropeptides - co-localization of PGP 9.5 and SP as well as SP and CGRP - in the central cornea and periphery.

Results

No differences in nerve densities were found either in the central or peripheral cornea between sexes. Therefore, all subsequent data presented represents a combination of both sexes. Relative to the description which follows, the anatomical features of the mice corneal innervation will be presented first, followed by a description of the density changes that occurred in diseased corneas.

Stromal Nerve Architecture

Our findings describing the major anatomical features of mouse corneal innervation are similar to those described previously.119 A mounted complete view of the nerve distribution of a normal mouse cornea revealed that the nerves penetrate the stroma in a radial pattern from the dense innervation of the corneoscleral limbus (Figure

3-1).

Projection images of green-fluorescent nerves (PGP 9.5 positive) in the normal mouse cornea showed that the stromal network is formed by thick nerve trunks that travel through the cornea, branching often and anastomosing with other nerve trunks.

The majority of axons in this location were long, straight and filamentous. The anterior portion of the stroma was much more densely innervated and morphologically complex.

In contrast, the posterior half, adjacent to the corneal endothelium, seemed to entirely lack innervation.

Epithelial Nerve Architecture

Thinner nerves that arose from the limbal and stromal nerve trunks characterized the sub-basal network as they project centripetally and run parallel to one another. A swirl pattern was noted in 2 of the 8 corneas stained for PGP 9.5 (Figure 3-2). Nerve fibers immerging from this network innervated most of the peripheral area, dividing into numerous smaller branches that connected with other, and composed a fine nerve plexus within the epithelial layer. The corneal epithelium contained a dense accumulation of PGP 9.5, CGRP and SP- positive nerve fibers.

PGP 9.5 and SP

Eight whole corneas from wild type mice were double labeled for PGP 9.5 and

SP. Figures 3-3A, 3-3B, and 3-3C are representative images of sub-basal and stromal networks in the central cornea. The images suggest a greater innervation density of

PGP 9.5-positive nerves than of SP-positive nerves. A similar pattern was visualized at the periphery of the cornea (Figures 3-3D, 3-3E, and 3-3F). Although no statically difference was found, fluorescence intensity processing confirmed that PGP 9.5-positive nerves were indeed more numerous than SP-positive neurons in both locations, central and peripheral. In the central cornea, the intensity of fluorescence for PGP 9.5 tended

(P = 0.08) to be greater than SP, with mean gray value (MGV) of 3.48 ± 0.50 and 2.07 ±

0.54 respectively. It is suggested that SP stained only 60% of the central corneal axons

(Figure 3-4). Moreover, at the periphery, PGP 9.5-positive nerve fibers had a greater (P

< 0.05) mean gray value (4.38 ± 0.40) when compared with SP (2.36 ± 0.54). Fifty five percent (55%) of the peripheral corneal axons were SP-positive (Figure 3-4).

Co-Localization of SP and CGRP

Five whole corneas of wild type mice were double labeled with antibody against

CGRP and SP. Immunoreactive nerve fibers were observed for both peptides though out the corneas stained simultaneously for CGRP and SP. Most axons showed co- localization within the same nerve fiber. Although nerve fibers CGRP-positive were present in greater number in the peripheral area (2.70 ± 0.44) when compared with the central cornea (2.75 ± 0.65), and similar pattern occurred with SP-positive fibers, where a greater intensity value was observed in the peripheral cornea (3.37 ± 0.54) than in the central cornea (1.84 ± 0.64; Figure 3-5), there were no statistically significant differences between those regions. Figure 3-6 represents the distribution of the two neuropeptides, CGRP and SP, at the central cornea and periphery.

Sjögren’s Syndrome-Related Changes in Nerve Density

We found that dry eye-affected mice had lower (P < 0.01) nerve density in the periphery of the cornea when compared with wild type control (Figure 3-7), showing a reduction of 68% of nerve fibers in this area of the cornea. In addition, a decrease in innervation density of the central cornea in dry eye-affected mice compared with wild type was noted; however, no statistical difference was established (P = 0.17). Figure 3-8 represents cornea innervation labeled for PGP 9.5 in both groups of mice. It suggests that the disease condition can be associated with a different innervation pattern, as the

dry eye mice exhibited fewer epithelial nerve fibers when compared with wild type mice, within both the central and peripheral cornea.

Discussion

Corneal Nerve Anatomy

The results of this study provide a detailed and comprehensive description of mouse corneal innervation that adds to existing knowledge in the field, and describe for the first time the quantitative changes in corneal nerve fibers in a genetically modified mouse model for Sjögren’s Syndrome. Novel methodology approaches used in this study in the mouse cornea included immunofluorescence using PGP 9.5 antibody as an indicator of overall innervation density, spinning disk confocal microscopy (SDCM), which provided more detailed images, and multilabel neuropeptide immunohistochemistry.

The major anatomic features of mouse corneal innervation demonstrated in the present study are similar to those described previously,119 and suggest that comparable patterns of corneal innervation may apply, with minor differences, to all mammals. The mouse cornea presents with dense innervation around the limbus, with nerves running horizontally and penetrating the stoma in a radial pattern. A single stromal nerve trunk gives rise to numerous branched sub-basal nerves and project toward the apical surface. Most sub-basal nerves appear thinner than the stromal nerves, and were predominantly observed in the anterior stroma at the corneal periphery. They run parallel to the ocular surface forming a distinct swirl pattern.

When comparing to the density of nerve fibers stained for PGP 9.5 in the center and periphery of the cornea, we did not find a statistically significant difference between the two areas. In contrast, He et al. using an antibody specific for neuronal-class III-

tubulin found that mice corneas have higher nerve density in the central area versus periphery. Similar immunohistochemistry findings have been also described in human corneas 119,129 Moreover, Puhakka et al. used antibodies for Beta-tubulin and PGP9.5 aiming to evaluate intraepidermal nerve fibers in tongue mucosa. Results in this study showed that both antibodies stained nerves in a similar manner, however, beta-tubulin was a slightly more sensitive marker than PGP 9.5 for epithelial nerve fibers in the tongue (0.45 for fiber mm-2 and 0.47 for fibers mm-2).130 The failure of our results to identify a significant difference between central and peripheral corneal nerve density may be attributed to the possible lack of sensitivity of PGP 9.5 in identifying sub-basal nerve fibers, which is the predominant nerve fiber in the central cornea. More recently,

Dennis et al. used a double staining technique using both ‘pan-neuronal’ markers, Beta- tubulin and PGP 9.5, to characterize the expression of proteins in the canine vomeronasal organ. Results in that study showed although double labeling with these antibodies stained axons similarly, the intensity of signal varied between preparations from different individuals. Thus, it was not asserted that most or all neurons express both proteins. Findings from rat vomeronasal sections run in parallel with the canine results, double labeled sections showed a mosaic of nerves positive for one or the other antibody but, in many cases not both. In that regard, some of the samples labeled with

Beta-tubulin did not present a uniform pattern suggesting that not all sensory axons express Beta-tubulin or that this protein is not expressed in the axons of all sensory neurons. Findings followed by age-related differences investigation in the same study suggest that this apparent mosaic expression pattern may mean that Beta-tubulin is expressed by younger sensory cells but not mature neurons.131 Moreover, PGP 9.5

antibody has already been described by immunohistochemical studies of cutaneous innervation and distribution of neuropeptides in commonly employed laboratory animals suggesting that PGP 9.5 is the most appropriate and practical marker for the demonstration of peripheral sensory nerves.132 To further support this presumption, an earlier study on the morphology and distribution of canine corneal nerves suggests that

PGP 9.5 is an accurate baseline indicator of overall innervation density in this species.7

Despite these findings, whether or not antibodies for Beta-tubulin and PGP 9.5 are reliable markers of all mouse nerve fibers is yet to be determined.

Methodological Considerations

Several imaging techniques have been described for quantification of corneal nerve fibers.5,133,134 Confocal imaging has been demonstrated both theoretically and practically to give improved resolution when compared to conventional imaging by

Fluorescence Light Microscopy used by previous immunostained corneal innervation descriptions. 119,135,136 The improvements are such that is possible to serially produce thin optical sections through thick fluorescent specimens with little degradation in quality of the interior sections resulting in three-dimensional images.136,137 As a novel approach, spinning disk confocal microscopy (SDCM) used in the present study additionally promotes more detailed images by using a large number of pinholes punched into a disk, so that fluorophores can be excited and the light detected for many different non- adjacent pixels simultaneously. By recording adjacent pixels at different time points, pinholes can be used to block most of the out-of-focus light for each pixel, as it originates close to the region of interest for that pixel, but sufficiently spaced pixels can still be recorded at the same time with little influence on one another.

The immunohistochemical staining procedure in this study used a member of the ubiquitin hydrolase family of proteins, a “pan-neuronal” marker that stains neural tissues. PGP 9.5 is an ubiquitin C-terminal hydrolase that potentially plays a role in proofreading by removing ubiquitin from proteins that are not designated for degradation.138 Ubiquitin, also known as “stress protein” is a highly conserved eukaryotic protein that has been suggested to be involved in intracellular protein degradation, cell cycle regulation, DNA repair, recombination, stress response and programed cell death. For all these circumstances, ubiquitin appears to exert its function by being attached to C-terminal glycine and an amino group (- or - amino) and has been observed in immunostained neural tissues involving deposition of abnormal proteins, particularly cytoskeletal and neurofilament proteins characteristic of degenerative processes.139 Moreover, a few studies have demonstrated postmortem changes of human corneal nerves, reporting that the majority of the sub-basal nerve fibers had degenerated or disappeared rapidly after death.140,141 In addition, a study using ex vivo confocal microscopy in different areas of the cornea confirmed this fact and demonstrated changes in stromal nerves and branching patterns.142 Therefore, we conclude that the PGP 9.5 may be an efficient tool to study the corneal nerves during degeneration of sensory axons followed by a corneal disease (e.g. Sjögren’s Syndrome and dry eye).

Several attempts have been made to measure the number and size of the individual fibers, but due to the variation in size and branching, it is difficult to draw accurate conclusions. The images were analyzed using a Mean Gray Value, which is calculated from the sum of the gray values of all the pixels in the selection area divided

by the number of pixels. The mean was calculated by converting each pixel to grayscale using the formula gray=0.299*red+0.587*green+0.114*blue. To obtain better contrast and minimize nonspecific background, the red channel was subtracted from the green channel and threshold adjusted highlighting the area to be analyzed. Although density is a relative magnitude and is not able to assess objectively the number of nerve fibers, the Regions of Interest (ROIs) can be used to define specific parts of an image and so pixels within a ROI will be included in the calculations and measurements. Thus, we conclude that we successfully extracted useful information from the fluorescence images, overcoming limitations in image (e.g. non specific background, noise) in order to make image content more clearly visible, and computing meaningful measurements through analysis to obtain our quantitative values.

CGRP and SP

The expression of the sensory neuropeptides CGRP and SP in corneal tissue has been studied previously by immunofluorescence in several species.143-145 These two neuropeptides have been described to be the best markers for the neuropeptidergic subpopulation, compromising mostly neurons with unmyelinated axons (C fibers) and innervating mainly polymodal nociceptors.146 However, the ocular sensory neurons are functionally heterogeneous, and the expression of other neuropeptides in the cornea, such as galanin (GAL), neuropeptide Y (NPY), methionine-enkephalin (M-ENK) and vasoactive intestinal polypeptide (VIP) was already described.147 In the present investigation, corneas double labeled for PGP 9.5 and SP suggest colocalization within most neurons. Therefore, PGP 9.5 positive fibers that were not labeled for SP most likely express different neuropeptides such the ones previously described. To further support this supposition, our results confirm and extend previous immunohistochemical

demonstration of SP and CGRP within same nerve fibers in the mammalian cornea.5,148

The similarities between the CGRP- and SP- immunoreactive innervation patterns shown in the current investigation suggest that these two neuropeptides colocalize in the same sensory axon. In agreement with this hypothesis, at a ultrastrutural level,

CGRP and SP coexist in the same synaptic vesicles, and likely are release together following appropriate stimulation.149 When comparing the densities between CGRP- and

SP- positive fibers in different regions of the cornea, we found that the density of CGRP is higher in the central cornea, while the density of SP-positive fibers is higher in the periphery. A trophic role for corneal sensory nerves involving the release of axonally transported peptides has been recognized for many years.150-152 Reid et al. has shown that SP stimulates DNA synthesis in rabbit corneal epithelial cells, and that CGRP alone has no proliferative effect; however the latter neuropeptide acts synergistically with SP to increase epithelial proliferation.153 In addition to these findings, also in rabbit corneal tissue, Garcia-Hirsschfeld describes that CGRP induces epithelial differentiation. Based on these results, the authors have suggested that corneal sensory nerves modulate both epithelial proliferation and differentiation and that neuronally-released CGRP and

SP mediate these effects.

Disease-Related Changes in Corneal Nerve Density

To examine the effect of SS on corneal nerves, we compared the innervation density in corneas of wild type mice and corneas of C57BL/6.NOD-Aec1Aec2 mice. We found that corneas from the C57BL/6.NOD-Aec1Aec2 mouse model have significantly decreased nerve fiber density and structural alterations of sub-basal and stromal axons labeled for PGP 9.5 in the peripheral cornea compared to wild type. Similar findings were observed in the central cornea, however with no statistical difference. These

results may have implications for understanding changes in corneal sensitivity in human subjects. It has been reported that the absence of corneal nerves results in neurotrophic keratitis, a clinical condition characterized by corneal anesthesia and desiccation, and by abnormal epithelial activity.5 Moreover, Bourcier et al. explored changes in corneal sensitivity that develop in SS patients and in not SS-related-dry eye patients, and the relationship between sensitivity and severity of the disease. Findings of that study demonstrated no differences in corneal sensitivity between patients, suggesting that the degree of functional impairment of transducing properties of the corneal nerve endings is similar in both conditions. Although the higher level of dryness in patients with SS increased the exposure of injured nerve fibers, thus evoking more discomfort. This is in agreement with an important component of the pathogenesis of SS, which consists of extensive lymphoplasmocytic infiltration of tear glands that is believed to disrupt the reflex circuits that drive blinking and lacrimation, and also diminish essential corneal nerve-supplied trophic factors, thus leading to dry eye.154-156 This theory could be confirmed by measuring both levels of pro-inflammatory cytokines in the tears of patients with SS and their tear fluid osmolarities. Elevated levels of pro-inflammatory cytokines in non-hyperosmolar tear would lead to another type of inflammation such as neuroinflammation caused by the hyperactivity of dysfunctional corneal nociceptors.157

This would also be consistent with the increased incidence of peripheral neuropathies in this disease. 154 However, idiopathic keratoconjunctivis sicca, originally not thought to be based on generalized autoimmune disease but, a local immune driven inflammatory reaction of the lacrimal gland is also believed to interfere with the functional unit of the ocular surface.155

It has been confirmed that other diseases, such as diabetes, keratitis, and glaucoma also demonstrated corneal nerve dysfunction. 158-160 Bitirgen et al. showed statistically significant reductions in nerve fiber density, nerve branch density and nerve fiber length in patients with diabetes mellitus compared with control subjects.161 Corneal sensation in dry eye syndrome and its correlation to nerve fiber alteration is still a subject of debate. In the present study, SS mouse corneas exhibited fewer sub-basal nerve fibers when compared to the wild type, and these results may be relevant to the pathophysiology of the disease since others have shown that mechanical damage to the corneal nerves by surgical trauma or disease can deprive the corneal epithelium of essential neurotrophic influences, which in turn may result in the development of severe degenerative conditions.162,163 Therefore, SS-associated corneal nerve fiber loss observed in this study may be clinically related to initial ocular discomfort such as itchiness and stinging cause by a desiccating effect, followed by a decrease in sensitivity in more chronic conditions. However, to our knowledge, this hypothesis has not been investigated in this mouse model. Further studies involving clinical examination and ocular surface tests such as phenol red thread test, fluorescein tear break-up time, fluorescein corneal staining and esthesiometry are required in order to correlate clinical signs of the severity of keratoconjunctivitis sicca (KCS) with loss of corneal innervation.

We acknowledge that the small sample size as well as the nerve density quantification method used in the present study may account for the fact that no statistical difference in innervation density stained for PGP 9.5 was appreciated between central and peripheral cornea. Additional corneas are currently being

investigated to clarify our findings. In summary, the results of the present study have demonstrated in a SS-mouse model a significant decrease in corneal nerve density compared with wild type. We may infer from these data and previous descriptions that similar changes occur in human corneas in patients with SS and potentially in animals with dry eye syndrome.164 The role of neurons in the health, healing, scarring, and immunology of the cornea, as well as the responsible molecular and cellular mechanisms, has not been fully determined. Therefore, the molecular and cellular changes that occur in primary sensory neurons as a result of disease, including their regenerative mechanisms, constitute high priority needs and opportunities in the field and require further study.

Figure 3-1. Immunofluorescence imaging of wild type mouse cornea stained with PGP 9.5. Arrow shows stromal nerve bundles entering the cornea at corneoscleral limbus. The cornea was artificially flattened by making four radial slits.

Figure 3-2. Subbasal nerves in a wild type mouse cornea with whorl-like structure or vortex, labeled with PGP 9.5 antibody.

Figure 3-3. Representative images showing the expression of PGP 9.5-and-SubP positive nerves in the central (A-C) and peripheral cornea (D-F).

Figure 3-4. Difference of corneal nerve density between the central and the peripheral zones. Wild type mouse corneas (n = 8) were double stained with protein gene product 9.5 (PGP 9.5) and Substance P (SP). Images acquisition was performed using confocal microscopy and analyzed using image intensity processing with ImageJ. Results are expressed as mean gray value ± SEM. t P < 0.10 and * P < 0.05 by Student t-test.

Figure 3-5. Difference of corneal nerve density between the central and the peripheral zones. Wild type mouse corneas (n = 5) were double stained with calcitonin gene-related peptide (CGRP) and Substance P (SP). Images acquisition was performed using confocal microscopy and analyzed using image intensity processing with ImageJ. Results are expressed as mean gray value ± SEM.

Figure 3-6. Representative images showing the expression of CGRP-and-SubP positive nerves in the central (A-C) and peripheral cornea (D-F).

Figure 3-7. Difference of corneal nerve density between the C57BL/6.NOD-Aec1Aec2 (SS) and wild type mouse. SS mouse corneas (n = 6) and wild type corneas (n = 8) were stained with protein gene product 9.5 (PGP 9.5). Images acquisition was performed using confocal microscopy and analyzed using image intensity processing with ImageJ. Results are expressed as mean gray value ± SEM. * P < 0.05 by Student t-test.

Figure 3-8. Representative images showing decrease in the expression of PGP 9.5 positive nerves in normal mice and C57BL/6.NOD-Aec1Aec2 mice at the central (A-C) and peripheral cornea (D-F).

CHAPTER 4 CONCLUSIONS

In recent years, ocular discomfort in humans has been reported with increasing frequency, likely related to exposure to air-conditioned environments, contact lens use, and the increasing popularity of refractive surgery. This has stimulated a renewed interest in the neural mechanisms involved in ocular sensation. The normal anatomy of corneal innervation has been described in several mammalian species, including humans, rabbits, cats, mice and rats. However, to our knowledge, the innervation of the mouse cornea under desiccant conditions, such those associated with SS disease has not been described.

Using a modified method of immunofluorescence staining and imaging, we constructed a three-dimensional map of the architecture of the corneal nerves in both wild type control and SS-affected mice. We measured the nerve density in different parts of the cornea and determined the effects of dry eye on changes in peripheral corneal nerve densities and central corneal sub basal fibers. The pan neuronal marker

PGP 9.5 successfully worked as an accurate baseline for the overall innervation demonstrating that the nerves penetrate the stroma in a radial pattern from dense innervation of the limbus, branching often and giving rise to numerous branched sub- basal nerves.

The findings in this study not only provide comprehensive descriptions of the architecture of mouse corneal sensory innervation including the distribution of the two major neuropeptides, but also provide evidence for their role in clinical symptoms of SS- related dry eye and other ocular disorders related to neurotropic keratitis.

While the understanding of SS-related dry eye has increased since its identification in 1933, resulting improved assessment and diagnosis, treatment strategies still primarily target the control of surface dryness. The mouse corneal innervation has many similarities to human cornea making the mouse an appropriate model to study pathology involving corneal nerves. Therefore, achieving a better understanding of the changes that occur in the corneal innervation during SS-related dry eye is an important step towards new therapeutic approaches aiming patient quality of life. Thus the potential clinical impact is great, as visual discomfort and impairment resulting from corneal nerve dysfunction adversely affects the quality of life in animals and people.

LIST OF REFERENCES

1. Qazi Y, Wong G, Monson B, et al. Corneal transparency: genesis, maintenance and dysfunction. Brain Res Bull 2010;81:198-210.

2. MacKay GGGaEO. Physiology of the Eye In: Gelatt KN, Gilger BC, Kern TJ, et al., eds. Veterinary ophthalmology volume I and volume II. 5th ed. Ames, Iowa: Wiley- Blackwell,, 2013;1 online resource.

3. Belmonte C, Aracil A, Acosta MC, et al. Nerves and sensations from the eye surface. Ocul Surf 2004;2:248-253.

4. Beuerman RW, Schimmelpfennig B. Sensory denervation of the rabbit cornea affects epithelial properties. Exp Neurol 1980;69:196-201.

5. Muller LJ, Marfurt CF, Kruse F, et al. Corneal nerves: structure, contents and function. Exp Eye Res 2003;76:521-542.

6. ten Tusscher MP, Klooster J, Vrensen GF. The innervation of the rabbit's anterior eye segment: a retrograde tracing study. Exp Eye Res 1988;46:717-730.

7. Marfurt CF, Murphy CJ, Florczak JL. Morphology and neurochemistry of canine corneal innervation. Invest Ophthalmol Vis Sci 2001;42:2242-2251.

8. Belmonte C, Giraldez F. Responses of cat corneal sensory receptors to mechanical and thermal stimulation. J Physiol 1981;321:355-368.

9. Shaheen BS, Bakir M, Jain S. Corneal nerves in health and disease. Surv Ophthalmol 2014;59:263-285.

10. Tuominen IS, Konttinen YT, Vesaluoma MH, et al. Corneal innervation and morphology in primary Sjogren's syndrome. Invest Ophthalmol Vis Sci 2003;44:2545- 2549.

11. Stern ME, Gao J, Siemasko KF, et al. The role of the lacrimal functional unit in the pathophysiology of dry eye. Exp Eye Res 2004;78:409-416.

12. Beckman KA. Detection of early markers for Sjogren syndrome in dry eye patients. Cornea 2014;33:1262-1264.

13. Williams DL. Immunopathogenesis of keratoconjunctivitis sicca in the dog. Vet Clin North Am Small Anim Pract 2008;38:251-268, vi.

14. Barabino S, Chen W, Dana MR. Tear film and ocular surface tests in animal models of dry eye: uses and limitations. Exp Eye Res 2004;79:613-621.

15. Fox RI, Howell FV, Bone RC, et al. Primary Sjogren syndrome: clinical and immunopathologic features. Semin Arthritis Rheum 1984;14:77-105.

16. Schimmelpfennig B. Nerve structures in human central corneal epithelium. Graefes Arch Clin Exp Ophthalmol 1982;218:14-20.

17. Bochdalek. Über die Nerven der durchsichtigen Hornhaut, und über einiges anderweitiges Verhalten der letztern: I. Medicinische Jahrbücher des kaiserlich- königlichen österreichischen Staates 1839;20:185.

18. Patel DV, McGhee CN. Mapping of the normal human corneal sub-Basal nerve plexus by in vivo laser scanning confocal microscopy. Invest Ophthalmol Vis Sci 2005;46:4485-4488.

19. Patel DV, McGhee CN. In vivo confocal microscopy of corneal stromal nerves in patients with peripheral neuropathy. Arch Neurol 2009;66:1179-1180; author reply 1180.

20. Erie JC, McLaren JW, Patel SV. Confocal microscopy in ophthalmology. Am J Ophthalmol 2009;148:639-646.

21. Xu KP, Li XF, Yu FS. Corneal organ culture model for assessing epithelial responses to surfactants. Toxicol Sci 2000;58:306-314.

22. Schulz D, Iliev ME, Frueh BE, et al. In vivo pachymetry in normal eyes of rats, mice and rabbits with the optical low coherence reflectometer. Vision Res 2003;43:723- 728.

23. Schmucker C, Schaeffel F. A paraxial schematic eye model for the growing C57BL/6 mouse. Vision Res 2004;44:1857-1867.

24. Moriyama H, Kasashima Y, Kuwano A, et al. Anatomical location and culture of equine corneal epithelial stem cells. Vet Ophthalmol 2014;17:106-112.

25. O'Callaghan AR, Daniels JT. Concise review: limbal epithelial stem cell therapy: controversies and challenges. Stem Cells 2011;29:1923-1932.

26. Bohnsack RN, Warejcka DJ, Wang L, et al. Expression of insulin-like growth factor 2 receptor in corneal keratocytes during differentiation and in response to wound healing. Invest Ophthalmol Vis Sci 2014;55:7697-7708.

27. Doughty MJ. Re-assessment of the potential impact of physiologically relevant pH changes on the hydration properties of the isolated mammalian corneal stroma. Biochim Biophys Acta 1999;1472:99-106.

28. Liu Q, Smith CW, Zhang W, et al. NK cells modulate the inflammatory response to corneal epithelial abrasion and thereby support wound healing. Am J Pathol 2012;181:452-462.

29. Mohammadi S, Postnikoff C, Wright AM, et al. Design and development of an in vitro tear replenishment system. Ann Biomed Eng 2014;42:1923-1931.

30. Foreman DM, Pancholi S, Jarvis-Evans J, et al. A simple organ culture model for assessing the effects of growth factors on corneal re-epithelialization. Exp Eye Res 1996;62:555-564.

31. Hutcheon AE, Sippel KC, Zieske JD. Examination of the restoration of epithelial barrier function following superficial keratectomy. Exp Eye Res 2007;84:32-38.

32. David JM. Cornea and Sclera In: Maggs DJ, Miller PE, Ofri R, et al., eds. Slatter's fundamentals of veterinary ophthalmology. 4th ed. Philadelphia, PA: Saunders Elsevier, 2008;xi, 478 p.

33. Samuelson DA. Ophthalmic Anatomy In: Gelatt KN, Gilger BC, Kern TJ, et al., eds. Veterinary ophthalmology volume I and volume II. 5th ed. Ames, Iowa: Wiley- Blackwell,, 2013;1 online resource.

34. Yamada M, Mashima Y, Tsubota K. Scanning electron microscopic observation of basal cells following corneal epithelial abrasion. Eye (Lond) 1996;10 ( Pt 5):569-574.

35. Nishida K. Tissue engineering of the cornea. Cornea 2003;22:S28-34.

36. Zhang YQ, Zhang WJ, Liu W, et al. Tissue engineering of corneal stromal layer with dermal fibroblasts: phenotypic and functional switch of differentiated cells in cornea. Tissue Eng Part A 2008;14:295-303.

37. Gordon MK, Foley JW, Birk DE, et al. Type V collagen and Bowman's membrane. Quantitation of mRNA in corneal epithelium and stroma. J Biol Chem 1994;269:24959-24966.

38. Jester JV. Corneal crystallins and the development of cellular transparency. Semin Cell Dev Biol 2008;19:82-93.

39. Tamura Y, Konomi H, Sawada H, et al. Tissue distribution of type VIII collagen in human adult and fetal eyes. Invest Ophthalmol Vis Sci 1991;32:2636-2644.

40. Jakus MA. Studies on the cornea. II. The fine structure of Descement's membrane. J Biophys Biochem Cytol 1956;2:243-252.

41. Hayek SM, Sweet JA, Miller JP, et al. Successful Management of Corneal Neuropathic Pain with Intrathecal Targeted Drug Delivery. Pain Med 2015.

42. Chen X, Gallar J, Belmonte C. Reduction by antiinflammatory drugs of the response of corneal sensory nerve fibers to chemical irritation. Invest Ophthalmol Vis Sci 1997;38:1944-1953.

43. Marfurt CF, Kingsley RE, Echtenkamp SE. Sensory and sympathetic innervation of the mammalian cornea. A retrograde tracing study. Invest Ophthalmol Vis Sci 1989;30:461-472.

44. Luhtala J, Uusitalo H. The distribution and origin of substance P immunoreactive nerve fibres in the rat conjunctiva. Exp Eye Res 1991;53:641-646.

45. Luhtala J, Palkama A, Uusitalo H. Calcitonin gene-related peptide immunoreactive nerve fibers in the rat conjunctiva. Invest Ophthalmol Vis Sci 1991;32:640-645.

46. Felipe CD, Gonzalez GG, Gallar J, et al. Quantification and immunocytochemical characteristics of trigeminal ganglion neurons projecting to the cornea: effect of corneal wounding. Eur J Pain 1999;3:31-39.

47. Marfurt CF, Echtenkamp SF. Central projections and trigeminal ganglion location of corneal afferent neurons in the monkey, Macaca fascicularis. J Comp Neurol 1988;272:370-382.

48. Rozsa AJ, Beuerman RW. Density and organization of free nerve endings in the corneal epithelium of the rabbit. Pain 1982;14:105-120.

49. Nichols JJ, Willcox MD, Bron AJ, et al. The TFOS International Workshop on Contact Lens Discomfort: executive summary. Invest Ophthalmol Vis Sci 2013;54:TFOS7-TFOS13.

50. Lele PP, Weddell G. Sensory nerves of the cornea and cutaneous sensibility. Exp Neurol 1959;1:334-359.

51. Gallar J, Pozo MA, Tuckett RP, et al. Response of sensory units with unmyelinated fibres to mechanical, thermal and chemical stimulation of the cat's cornea. J Physiol 1993;468:609-622.

52. Stapleton F, Marfurt C, Golebiowski B, et al. The TFOS International Workshop on Contact Lens Discomfort: report of the subcommittee on neurobiology. Invest Ophthalmol Vis Sci 2013;54:TFOS71-97.

53. Kessler F, Habelt C, Averbeck B, et al. Heat-induced release of CGRP from isolated rat skin and effects of bradykinin and the protein kinase C activator PMA. Pain 1999;83:289-295.

54. Launay PS, Reboussin E, Liang H, et al. Ocular inflammation induces trigeminal pain, peripheral and central neuroinflammatory mechanisms. Neurobiol Dis 2016;88:16- 28.

55. Rosenthal P, Borsook D. The corneal pain system. Part I: the missing piece of the dry eye puzzle. Ocul Surf 2012;10:2-14.

56. Ji RR, Woolf CJ. Neuronal plasticity and signal transduction in nociceptive neurons: implications for the initiation and maintenance of pathological pain. Neurobiol Dis 2001;8:1-10.

57. Acosta MC, Tan ME, Belmonte C, et al. Sensations evoked by selective mechanical, chemical, and thermal stimulation of the conjunctiva and cornea. Invest Ophthalmol Vis Sci 2001;42:2063-2067.

58. Belmonte C, Acosta MC, Gallar J. Neural basis of sensation in intact and injured corneas. Exp Eye Res 2004;78:513-525.

59. Troger J, Kieselbach G, Teuchner B, et al. Peptidergic nerves in the eye, their source and potential pathophysiological relevance. Brain Res Rev 2007;53:39-62.

60. Isik FF, Gibran NS, Jang YC, et al. Vitronectin decreases microvascular endothelial cell apoptosis. J Cell Physiol 1998;175:149-155.

61. US VE, Gaddum JH. An unidentified depressor substance in certain tissue extracts. J Physiol 1931;72:74-87.

62. Onuoha GN, Alpar EK. Calcitonin gene-related peptide and other neuropeptides in the plasma of patients with soft tissue injury. Life Sci 1999;65:1351-1358.

63. Trantor IR, Messer HH, Birner R. The effects of neuropeptides (calcitonin gene- related peptide and substance P) on cultured human pulp cells. J Dent Res 1995;74:1066-1071.

64. Aoki M, Tamai K, Saotome K. Substance P- and calcitonin gene-related peptide- immunofluorescent nerves in the repair of experimental bone defects. Int Orthop 1994;18:317-324.

65. Stone RA, Kuwayama Y, Laties AM. Regulatory peptides in the eye. Experientia 1987;43:791-800.

66. Wiesenfeld-Hallin Z, Hokfelt T, Lundberg JM, et al. Immunoreactive calcitonin gene-related peptide and substance P coexist in sensory neurons to the spinal cord and interact in spinal behavioral responses of the rat. Neurosci Lett 1984;52:199-204.

67. Sternini C, Brecha N. Immunocytochemical identification of islet cells and nerve fibers containing calcitonin gene-related peptide-like immunoreactivity in the rat pancreas. Gastroenterology 1986;90:1155-1163.

68. Amara SG, Jonas V, Rosenfeld MG, et al. Alternative RNA processing in calcitonin gene expression generates mRNAs encoding different polypeptide products. Nature 1982;298:240-244.

69. Gibson SJ, Polak JM, Bloom SR, et al. Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and of eight other species. J Neurosci 1984;4:3101-3111.

70. Lee Y, Kawai Y, Shiosaka S, et al. Coexistence of calcitonin gene-related peptide and substance P-like peptide in single cells of the trigeminal ganglion of the rat: immunohistochemical analysis. Brain Res 1985;330:194-196.

71. Jancso G, Kiraly E, Jancso-Gabor A. Pharmacologically induced selective degeneration of chemosensitive primary sensory neurones. Nature 1977;270:741-743.

72. Jancso G, Kiraly E. Distribution of chemosensitive primary sensory afferents in the central nervous system of the rat. J Comp Neurol 1980;190:781-792.

73. Sammes PG, Taylor JB, Hansch C. Comprehensive medicinal chemistry : the rational design, mechanistic study & therapeutic applications of chemical compounds. 1st ed. Oxford ; New York: Pergamon Press, 1990.

74. Jenkins DW, Feniuk W, Humphrey PP. Characterization of the prostanoid receptor types involved in mediating calcitonin gene-related peptide release from cultured rat trigeminal neurones. Br J Pharmacol 2001;134:1296-1302.

75. Stone RA, Kuwayama Y, Terenghi G, et al. Calcitonin gene-related peptide: occurrence in corneal sensory nerves. Exp Eye Res 1986;43:279-283.

76. Skofitsch G, Jacobowitz DM. Calcitonin gene-related peptide coexists with substance P in capsaicin sensitive neurons and sensory ganglia of the rat. Peptides 1985;6:747-754.

77. Haegerstrand A, Dalsgaard CJ, Jonzon B, et al. Calcitonin gene-related peptide stimulates proliferation of human endothelial cells. Proc Natl Acad Sci U S A 1990;87:3299-3303.

78. Holzer P. Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides. Neuroscience 1988;24:739-768.

79. Lynn B, Ye W, Cotsell B. The actions of capsaicin applied topically to the skin of the rat on C-fibre afferents, antidromic vasodilatation and substance P levels. Br J Pharmacol 1992;107:400-406.

80. Pini A, Lynn B. C-fibre Function During the 6 Weeks Following Brief Application of Capsaicin to a Cutaneous Nerve in the Rat. Eur J Neurosci 1991;3:274-284.

81. Hokfelt T, Kellerth JO, Nilsson G, et al. Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons. Brain Res 1975;100:235-252.

82. Nagy JI, Hunt SP, Iversen LL, et al. Biochemical and anatomical observations on the degeneration of peptide-containing primary afferent neurons after neonatal capsaicin. Neuroscience 1981;6:1923-1934.

83. Lawson SN, Crepps BA, Perl ER. Relationship of substance P to afferent characteristics of dorsal root ganglion neurones in guinea-pig. J Physiol 1997;505 ( Pt 1):177-191.

84. Miller A, Costa M, Furness JB, et al. Substance P immunoreactive sensory nerves supply the rat iris and cornea. Neurosci Lett 1981;23:243-249.

85. Ketchum LD, Cohen IK, Masters FW. Hypertrophic scars and keloids. A collective review. Plast Reconstr Surg 1974;53:140-154.

86. Cridland RA, Yashpal K, Romita VV, et al. Distribution of label after intrathecal administration of 125I-substance P in the rat. Peptides 1987;8:213-221.

87. Regoli D, Boudon A, Fauchere JL. Receptors and antagonists for substance P and related peptides. Pharmacol Rev 1994;46:551-599.

88. Yamada N, Yanai R, Inui M, et al. Sensitizing effect of substance P on corneal epithelial migration induced by IGF-1, fibronectin, or interleukin-6. Invest Ophthalmol Vis Sci 2005;46:833-839.

89. Thompson RJ, Doran JF, Jackson P, et al. PGP 9.5--a new marker for vertebrate neurons and neuroendocrine cells. Brain Res 1983;278:224-228.

90. Rode J, Dhillon AP, Doran JF, et al. PGP 9.5, a new marker for human neuroendocrine tumours. Histopathology 1985;9:147-158.

91. Tomita T. PGP 9.5 immunocytochemical staining for pancreatic endocrine tumors. Islets 2013;5:122-128.

92. Dalsgaard CJ, Rydh M, Haegerstrand A. Cutaneous innervation in man visualized with protein gene product 9.5 (PGP 9.5) antibodies. Histochemistry 1989;92:385-390.

93. Ebenezer G, Polydefkis M. Epidermal innervation in diabetes. Handb Clin Neurol 2014;126:261-274.

94. The epidemiology of dry eye disease: report of the Epidemiology Subcommittee of the International Dry Eye WorkShop (2007). Ocul Surf 2007;5:93-107.

95. The definition and classification of dry eye disease: report of the Definition and Classification Subcommittee of the International Dry Eye WorkShop (2007). Ocul Surf 2007;5:75-92.

96. Moss SE, Klein R, Klein BE. Incidence of dry eye in an older population. Arch Ophthalmol 2004;122:369-373.

97. Borchers AT, Naguwa SM, Keen CL, et al. Immunopathogenesis of Sjogren's syndrome. Clin Rev Allergy Immunol 2003;25:89-104.

98. Patel R, Shahane A. The epidemiology of Sjogren's syndrome. Clin Epidemiol 2014;6:247-255.

99. Palm O, Garen T, Berge Enger T, et al. Clinical pulmonary involvement in primary Sjogren's syndrome: prevalence, quality of life and mortality--a retrospective study based on registry data. Rheumatology (Oxford) 2013;52:173-179.

100. Solans-Laque R, Lopez-Hernandez A, Bosch-Gil JA, et al. Risk, predictors, and clinical characteristics of lymphoma development in primary Sjogren's syndrome. Semin Arthritis Rheum 2011;41:415-423.

101. Moutsopoulos HM. Sjogren's syndrome: autoimmune epithelitis. Clin Immunol Immunopathol 1994;72:162-165.

102. Hall JC, Baer AN, Shah AA, et al. Molecular Subsetting of Interferon Pathways in Sjogren's Syndrome. Arthritis Rheumatol 2015;67:2437-2446.

103. Ferro F, Vagelli R, Bruni C, et al. One year in review 2016: Sjogren's syndrome. Clin Exp Rheumatol 2016;34:161-171.

104. Sudzius G, Mieliauskaite D, Siaurys A, et al. Distribution of Peripheral Lymphocyte Populations in Primary Sjogren's Syndrome Patients. J Immunol Res 2015;2015:854706.

105. Akpek EK, Klimava A, Thorne JE, et al. Evaluation of patients with dry eye for presence of underlying Sjogren syndrome. Cornea 2009;28:493-497.

106. Nossent JC, Swaak AJ. Systemic lupus erythematosus VII: frequency and impact of secondary Sjogren's syndrome. Lupus 1998;7:231-234.

107. Massardo L, Aguirre V, Garcia ME, et al. Clinical expression of rheumatoid arthritis in Chilean patients. Semin Arthritis Rheum 1995;25:203-213.

108. Vissink A, Kallenberg CG, Bootsma H. Treatment approaches in primary Sjogren syndrome. JAMA 2010;304:2015-2016; author reply 2016.

109. Pillemer SR, Matteson EL, Jacobsson LT, et al. Incidence of physician- diagnosed primary Sjogren syndrome in residents of Olmsted County, Minnesota. Mayo Clin Proc 2001;76:593-599.

110. Helmick CG, Felson DT, Lawrence RC, et al. Estimates of the prevalence of arthritis and other rheumatic conditions in the United States. Part I. Arthritis Rheum 2008;58:15-25.

111. Delaleu N, Nguyen CQ, Peck AB, et al. Sjogren's syndrome: studying the disease in mice. Arthritis Res Ther 2011;13:217.

112. Cha S, Nagashima H, Brown VB, et al. Two NOD Idd-associated intervals contribute synergistically to the development of autoimmune exocrinopathy (Sjogren's syndrome) on a healthy murine background. Arthritis Rheum 2002;46:1390-1398.

113. Robinson CP, Yamachika S, Bounous DI, et al. A novel NOD-derived murine model of primary Sjogren's syndrome. Arthritis Rheum 1998;41:150-156.

114. Brayer J, Lowry J, Cha S, et al. Alleles from chromosomes 1 and 3 of NOD mice combine to influence Sjogren's syndrome-like autoimmune exocrinopathy. J Rheumatol 2000;27:1896-1904.

115. Ridgway WM, Peterson LB, Todd JA, et al. Gene-gene interactions in the NOD mouse model of type 1 diabetes. Adv Immunol 2008;100:151-175.

116. Nguyen CQ, Hu MH, Li Y, et al. Salivary gland tissue expression of interleukin-23 and interleukin-17 in Sjogren's syndrome: Findings in humans and mice. Arthritis Rheum 2008;58:734-743.

117. Nguyen CQ, Sharma A, She JX, et al. Differential gene expressions in the lacrimal gland during development and onset of keratoconjunctivitis sicca in Sjogren's syndrome (SJS)-like disease of the C57BL/6.NOD-Aec1Aec2 mouse. Exp Eye Res 2009;88:398-409.

118. Catanzaro J, Dinkel S. Sjogren's syndrome: the hidden disease. Medsurg Nurs 2014;23:219-223.

119. He J, Bazan HE. Neuroanatomy and Neurochemistry of Mouse Cornea. Invest Ophthalmol Vis Sci 2016;57:664-674.

120. Tran MT, Lausch RN, Oakes JE. Substance P differentially stimulates IL-8 synthesis in human corneal epithelial cells. Invest Ophthalmol Vis Sci 2000;41:3871- 3877.

121. Tran MT, Ritchie MH, Lausch RN, et al. Calcitonin gene-related peptide induces IL-8 synthesis in human corneal epithelial cells. J Immunol 2000;164:4307-4312.

122. Beuerman RW, Rozsa AJ. Collateral sprouts are replaced by regenerating neurites in the wounded corneal epithelium. Neurosci Lett 1984;44:99-104.

123. Fabiani C, Barabino S, Rashid S, et al. Corneal epithelial proliferation and thickness in a mouse model of dry eye. Exp Eye Res 2009;89:166-171.

124. McKenna CC, Lwigale PY. Innervation of the mouse cornea during development. Invest Ophthalmol Vis Sci 2011;52:30-35.

125. Oudejans L, He X, Niesters M, et al. Cornea nerve fiber quantification and construction of phenotypes in patients with fibromyalgia. Sci Rep 2016;6:23573.

126. Mainka T, Maier C, Enax-Krumova EK. Neuropathic pain assessment: update on laboratory diagnostic tools. Curr Opin Anaesthesiol 2015;28:537-545.

127. Hiura A, Nakagawa H. Innervation of TRPV1-, PGP-, and CGRP-immunoreactive nerve fibers in the subepithelial layer of a whole mount preparation of the rat cornea. Okajimas Folia Anat Jpn 2012;89:47-50.

128. Marfurt CF, Ellis LC, Jones MA. Sensory and sympathetic nerve sprouting in the rat cornea following neonatal administration of capsaicin. Somatosens Mot Res 1993;10:377-398.

129. He J, Bazan NG, Bazan HE. Mapping the entire human corneal nerve architecture. Exp Eye Res 2010;91:513-523.

130. Puhakka A, Forssell H, Soinila S, et al. Peripheral nervous system involvement in primary burning mouth syndrome-results of a pilot study. Oral Dis 2016;22:338-344.

131. Dennis JC, Allgier JG, Desouza LS, et al. Immunohistochemistry of the canine vomeronasal organ. J Anat 2003;203:329-338.

132. Karanth SS, Springall DR, Kuhn DM, et al. An immunocytochemical study of cutaneous innervation and the distribution of neuropeptides and protein gene product 9.5 in man and commonly employed laboratory animals. Am J Anat 1991;191:369-383.

133. Patel DV, McGhee CN. In vivo confocal microscopy of human corneal nerves in health, in ocular and systemic disease, and following corneal surgery: a review. Br J Ophthalmol 2009;93:853-860.

134. Muller LJ, Pels L, Vrensen GF. Ultrastructural organization of human corneal nerves. Invest Ophthalmol Vis Sci 1996;37:476-488.

135. White JG, Amos WB, Fordham M. An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy. J Cell Biol 1987;105:41-48.

136. Brakenhoff GJ, van der Voort HT, van Spronsen EA, et al. Three-dimensional imaging in fluorescence by confocal scanning microscopy. J Microsc 1989;153:151-159.

137. Brakenhoff GJ, van Spronsen EA, van der Voort HT, et al. Three-dimensional confocal fluorescence microscopy. Methods Cell Biol 1989;30:379-398.

138. Wilkinson KD, Deshpande S, Larsen CN. Comparisons of neuronal (PGP 9.5) and non-neuronal ubiquitin C-terminal hydrolases. Biochem Soc Trans 1992;20:631- 637.

139. Bond U, Schlesinger MJ. The chicken ubiquitin gene contains a heat shock promoter and expresses an unstable mRNA in heat-shocked cells. Mol Cell Biol 1986;6:4602-4610.

140. Muller LJ, Vrensen GF, Pels L, et al. Architecture of human corneal nerves. Invest Ophthalmol Vis Sci 1997;38:985-994.

141. Ueda S, del Cerro M, LoCascio JA, et al. Peptidergic and catecholaminergic fibers in the human corneal epithelium. An immunohistochemical and electron microscopic study. Acta Ophthalmol Suppl 1989;192:80-90.

142. Al-Aqaba MA, Alomar T, Miri A, et al. Ex vivo confocal microscopy of human corneal nerves. Br J Ophthalmol 2010;94:1251-1257.

143. Jones MA, Marfurt CF. Calcitonin gene-related peptide and corneal innervation: a developmental study in the rat. J Comp Neurol 1991;313:132-150.

144. Uusitalo H, Krootila K, Palkama A. Calcitonin gene-related peptide (CGRP) immunoreactive sensory nerves in the human and guinea pig uvea and cornea. Exp Eye Res 1989;48:467-475.

145. Tervo K, Tervo T, Eranko L, et al. Substance P immunoreactive nerves in the rodent cornea. Neurosci Lett 1981;25:95-97.

146. Priestley JV, Michael GJ, Averill S, et al. Regulation of nociceptive neurons by nerve growth factor and glial cell line derived neurotrophic factor. Can J Physiol Pharmacol 2002;80:495-505.

147. Jones MA, Marfurt CF. Peptidergic innervation of the rat cornea. Exp Eye Res 1998;66:421-435.

148. Kuwayama Y, Stone RA. Distinct substance P and calcitonin gene-related peptide immunoreactive nerves in the guinea pig eye. Invest Ophthalmol Vis Sci 1987;28:1947-1954.

149. Gulbenkian S, Merighi A, Wharton J, et al. Ultrastructural evidence for the coexistence of calcitonin gene-related peptide and substance P in secretory vesicles of peripheral nerves in the guinea pig. J Neurocytol 1986;15:535-542.

150. Sigelman S, Friedenwald JS. Mitotic and wound-healing activities of the corneal epithelium; effect of sensory denervation. AMA Arch Ophthalmol 1954;52:46-57.

151. Sugiura S, Matsuda H. [Electron microscopic observations on the corneal epithelium after sensory denervation]. Nippon Ganka Gakkai Zasshi 1967;71:1123- 1137.

152. Stone RA, McGlinn AM. Calcitonin gene-related peptide immunoreactive nerves in human and rhesus monkey eyes. Invest Ophthalmol Vis Sci 1988;29:305-310.

153. Reid TW, Murphy CJ, Iwahashi CK, et al. Stimulation of epithelial cell growth by the neuropeptide substance P. J Cell Biochem 1993;52:476-485.

154. Hebbar M, Lassalle P, Janin A, et al. E-selectin expression in salivary endothelial cells and sera from patients with systemic sclerosis. Role of resident mast cell-derived tumor necrosis factor alpha. Arthritis Rheum 1995;38:406-412.

155. Augustin AJ, Spitznas M, Kaviani N, et al. Oxidative reactions in the tear fluid of patients suffering from dry eyes. Graefes Arch Clin Exp Ophthalmol 1995;233:694-698.

156. Alexander E. Central nervous system disease in Sjogren's syndrome. New insights into immunopathogenesis. Rheum Dis Clin North Am 1992;18:637-672.

157. Watkins LR, Wiertelak EP, Goehler LE, et al. Characterization of cytokine- induced hyperalgesia. Brain Res 1994;654:15-26.

158. De Cilla S, Ranno S, Carini E, et al. Corneal subbasal nerves changes in patients with diabetic retinopathy: an in vivo confocal study. Invest Ophthalmol Vis Sci 2009;50:5155-5158.

159. Malik RA, Kallinikos P, Abbott CA, et al. Corneal confocal microscopy: a noninvasive surrogate of nerve fibre damage and repair in diabetic patients. Diabetologia 2003;46:683-688.

160. Rosenberg ME, Tervo TM, Immonen IJ, et al. Corneal structure and sensitivity in type 1 diabetes mellitus. Invest Ophthalmol Vis Sci 2000;41:2915-2921.

161. Bitirgen G, Ozkagnici A, Malik RA, et al. Corneal nerve fibre damage precedes diabetic retinopathy in patients with type 2 diabetes mellitus. Diabet Med 2014;31:431- 438.

162. Paton L. The Trigeminal and Its Ocular Lesions. Br J Ophthalmol 1926;10:305- 342.

163. Araki K, Ohashi Y, Kinoshita S, et al. Epithelial wound healing in the denervated cornea. Curr Eye Res 1994;13:203-211.

164. Bianciardi G, Latronico ME, Traversi C. Entropy of corneal nerve fibers distribution observed by laser scanning confocal microscopy: A noninvasive quantitative method to characterize the corneal innervation in Sjogren's syndrome patients. Microsc Res Tech 2015;78:1069-1074.

BIOGRAPHICAL SKETCH

Renata Velloso Ramos was born in Brasília, Brazil, and completed her doctorate of veterinary medicine at União Pioneira de Integração Social, UPIS in 2010. She completed a small animal clinical skills internship followed by a large animal rotating internship in 2012 and 2013, at The Ontario Veterinary College, University of Guelph,

Canada. At which time, she moved to Gainesville to begin a combined master’s degree program in veterinary medical sciences and a comparative ophthalmology residency.

Renata will continue to peruse research in areas of interest including neuroanatomy and neurochemistry of the cornea in health and disease as well as simultaneously completing her residency program in the spring of 2019.