Age-Related Differences in the Mouse Corneal Epithelial Transcriptome and Their Impact on Corneal Wound Healing.

Purpose: Aging is a risk factor for dry eye. We sought to identify changes in the aged mouse corneal epithelial transcriptome and determine how age affects corneal sensitivity, re-epithelialization, and barrier reformation after corneal debridement. Methods: Corneal epithelium of female C57BL/6J (B6) mice of different ages (2, 12, 18, and 24 months) was collected, RNA extracted, and bulk RNA sequencing performed. Cornea sensitivity was measured with an esthesiometer in 2- to 3-month-old, 12- to 13-month-old, 18- to 19-month-old, and 22- to 25-month-old female and male mice. The 2-month-old and 18-month-old female and male mice underwent unilateral corneal debridement using a blunt blade. Wound size and fluorescein staining were visualized and photographed at different time points, and a re-epithelialization rate curve was calculated. Results: There were 157 differentially expressed genes in aged mice compared with young mice. Several pathways downregulated with age control cell migration, proteoglycan synthesis, and collagen trimerization, assembly, biosynthesis, and degradation. Male mice had decreased corneal sensitivity compared with female mice at 12 and 24 months of age. Aged mice, irrespective of sex, had delayed corneal re-epithelialization in the first 48 hours and worse corneal fluorescein staining intensity at day 14 than young mice. Conclusions: Aged corneal epithelium has an altered transcriptome. Aged mice regardless of sex heal more slowly and displayed more signs of corneal epithelial defects after wounding than young mice. These results indicate that aging significantly alters the corneal epithelium and its ability to coordinate healing.

Immune Cells Localize to Sites of Corneal Erosions in C57BL/6 Mice.

Recurrent epithelial erosions develop in the cornea due to prior injury or genetic predisposition. Studies of recurrent erosions in animal models allow us to gain insight into how erosions form and are resolved. While slowing corneal epithelial cell migration and reducing their proliferation following treatment with mitomycin C reduce erosion formation in mice after sterile debridement injury, additional factors have been identified related to cytokine expression and immune cell activation. The relationship between recruitment of immune cells to the region of the cornea where erosions form and their potential roles in erosion formation and/or erosion repair remains unexplored in the C57BL/6 mouse recurrent erosion model. Here, high resolution imaging of mouse corneas was performed at D1, D7, and D28 after dulled-blade debridement injury in C57BL/6 mice. Around 50% of these mice have frank corneal erosions at D28 after wounding. A detailed assessment of corneas revealed the involvement of M2 macrophages in both frank and developing erosions at early stages of their formation.

Conditional deletion of CD25 in the corneal epithelium reveals sex differences in barrier disruption.

PURPOSE: IL-2 promotes activation, clonal expansion, and deletion of T cells. IL-2 signals through its heterotrimeric receptor (IL-2R) consisting of the CD25, CD122 and CD132 chains. CD25 knockout (KO) mice develop Sjögren Syndrome-like disease. This study investigates whether corneal CD25/IL-2 signaling is critical for ocular health. METHODS: Eyes from C57BL/6 mice were collected and prepared for immunostaining or in-situ hybridization. Bulk RNA sequencing was performed on the corneal epithelium from wild-type and CD25KO mice. We generated a conditional corneal-specific deletion of CD25 in the corneal epithelium (CD25Δ/ΔCEpi). Corneal barrier function was evaluated based on the uptake of a fluorescent dye. Mice were subjected to unilateral corneal debridement, followed by epithelial closure over time. RESULTS: In C57BL/6 mice, CD25 mRNA was expressed in ocular tissues. Protein expression of CD25, CD122, and CD132 was confirmed in the corneal epithelium. Delayed corneal re-epithelization was seen in female but not male CD25KO mice. There were 771 differentially expressed genes in the corneal epithelium of CD25KO compared to wild-type mice. While barrier function is disrupted in CD25Δ/ΔCEpi mice, re-epithelialization rates are not delayed. CONCLUSIONS: All three chains of the IL-2R are expressed in the corneal epithelium. Our results indicate for the first time, deleting CD25 systemically in all tissues in the mouse and deleting CD25 locally in just the corneal epithelium compromises corneal epithelial barrier function, leading to dry eye disease in female mice. Future studies are needed to delineate the pathways used by IL-2 signaling to influence cornea homeostasis.

Molecular mechanisms regulating wound repair: Evidence for paracrine signaling from corneal epithelial cells to fibroblasts and immune cells following transient epithelial cell treatment with Mitomycin C.

In this paper, we use RNAseq to identify senescence and phagocytosis as key factors to understanding how mitomyin C (MMC) stimulates regenerative wound repair. We use conditioned media (CM) from untreated (CMC) and MMC treated (CMM) human and mouse corneal epithelial cells to show that corneal epithelial cells indirectly exposed to MMC secrete elevated levels of immunomodulatory proteins including IL-1α and TGFß1 compared to cells exposed to CMC. These factors increase epithelial and macrophage phagocytosis and promote ECM turnover. IL-1α supplementation can increase phagocytosis in control epithelial cells and attenuate TGFß1 induced αSMA expression by corneal fibroblasts. Yet, we show that epithelial cell CM contains factors besides IL-1α that regulate phagocytosis and αSMA expression by fibroblasts. Exposure to CMM also impacts the activation of bone marrow derived dendritic cells and their ability to present antigen. These in vitro studies show how a brief exposure to MMC induces corneal epithelial cells to release proteins and other factors that function in a paracrine way to enhance debris removal and enlist resident epithelial and immune cells as well as stromal fibroblasts to support regenerative and not fibrotic wound healing.

Uveitis-mediated immune cell invasion through the extracellular matrix of the lens capsule.

While the eye is considered an immune privileged site, its privilege is abrogated when immune cells are recruited from the surrounding vasculature in response to trauma, infection, aging, and autoimmune diseases like uveitis. Here, we investigate whether in uveitis immune cells become associated with the lens capsule and compromise its privilege in studies of C57BL/6J mice with experimental autoimmune uveitis. These studies show that at D14, the peak of uveitis in these mice, T cells, macrophages, and Ly6G/Ly6C+ immune cells associate with the lens basement membrane capsule, burrow into the capsule matrix, and remain integrated with the capsule as immune resolution is occurring at D26. 3D surface rendering image analytics of confocal z-stacks and scanning electron microscopy imaging of the lens surface show the degradation of the lens capsule as these lens-associated immune cells integrate with and invade the lens capsule, with a subset infiltrating both epithelial and fiber cell regions of lens tissue, abrogating its immune privilege. Those immune cells that remain on the surface often become entwined with a fibrillar net-like structure. Immune cell invasion of the lens capsule in uveitis has not been described previously and may play a role in induction of lens and other eye pathologies associated with autoimmunity.

Corneal nonmyelinating Schwann cells illuminated by single-cell transcriptomics and visualized by protein biomarkers.

The cornea is the most innervated tissue in the human body. Myelinated axons upon inserting into the peripheral corneal stroma lose their myelin sheaths and continue into the central cornea wrapped by only nonmyelinating corneal Schwann cells (nm-cSCs). This anatomical organization is believed to be important for central vision. Here we employed single-cell RNA sequencing (scRNA-seq), microscopy, and transgenics to characterize these nm-cSCs of the central cornea. Using principal component analysis, uniform manifold approximation and projection, and unsupervised hierarchal cell clustering of scRNA-seq data derived from central corneal cells of male rabbits, we successfully identified several clusters representing different corneal cell types, including a unique cell cluster representing nm-cSCs. To confirm protein expression of cSC genes, we performed cross-species validation, employing corneal whole-mount immunostaining with confocal microscopy in mouse corneas. The expression of several representative proteins of nm-cSCs were validated. As the proteolipid protein 1 (PLP1) gene was also expressed in nm-cSCs, we explored the Plp1-eGFP transgenic reporter mouse line to visualize cSCs. Specific and efficient eGFP expression was observed in cSCs in adult mice of different ages. Of several putative cornea-specific SC genes identified, Dickkopf-related protein 1 was shown to be present in nm-cSCs. Taken together, our findings, for the first time, identify important insights and tools toward the study nm-cSCs in isolated tissue and adult animals. We expect that our results will advance the future study of nm-cSCs in applications of nerve repair, and provide a resource for the study of corneal sensory function.

Combination therapy of cold atmospheric plasma (CAP) with temozolomide in the treatment of U87MG glioblastoma cells.

Cold atmospheric plasma (CAP) technology, a relatively novel technique mainly investigated as a stand-alone cancer treatment method in vivo and in vitro, is being proposed for application in conjunction with chemotherapy. In this study, we explore whether CAP, an ionized gas produced in laboratory settings and that operates at near room temperature, can enhance Temozolomide (TMZ) cytotoxicity on a glioblastoma cell line (U87MG). Temozolomide is the first line of treatment for glioblastoma, one of the most aggressive brain tumors that remains incurable despite advancements with treatment modalities. The cellular response to a single CAP treatment followed by three treatments with TMZ was monitored with a cell viability assay. According to the cell viability results, CAP treatment successfully augmented the effect of a cytotoxic TMZ dose (50 µM) and further restored the effect of a non-cytotoxic TMZ dose (10 µM). Application of CAP in conjunction TMZ increased DNA damage measured by the phosphorylation of H2AX and induced G2/M cell cycle arrest. These findings were supported by additional data indicating reduced cell migration and increased αvß3 and αvß5 cell surface integrin expression as a result of combined CAP-TMZ treatment. The data presented in this study serve as evidence that CAP technology can be a suitable candidate for combination therapy with existing chemotherapeutic drugs. CAP can also be investigated in future studies for sensitizing glioblastoma cells to TMZ and other drugs available in the market.

Diurnal Control of Sensory Axon Growth and Shedding in the Mouse Cornea.

Purpose: The circadian clock plays an important role in the expression and regulation of various genes and cellular processes in the body. Here, we study diurnal regulation of the growth and shedding of the sensory axons in the mouse cornea. Methods: Male and female BALB/cN mice were euthanized 90 minutes before and after the lights are turned on and off; at 5:30 AM, 8:30 AM, 5:30 PM, and 8:30 PM. Nerve terminal growth, shedding and overall axon density were assessed at these four time points using confocal imaging after staining axons in en face whole mount corneas with antibodies against ßIII tubulin, GAP43, and L1CAM. In addition, corneal epithelial cell proliferation, thickness, and desquamation were assessed using ki67, LAMP1, Involucrin, and ZO1. Results: Nerve terminal shedding took place between 5:30 AM and 8:30 AM and correlated positively with the timing of apical cell desquamation. After shedding the tips of the nerve terminals, axonal growth increased as indicated by increased axonal GAP43 expression. At 5:30 PM and 8:30 PM before and after the lights are turned off, cell proliferation was reduced, and epithelial thickness was maximal. Conclusions: Intraepithelial corneal nerve growth and shedding are under diurnal control regulated by the time of day and whether lights are on or off. Axons extend during the day and are shed within 90 minutes after lights are turned on. The data presented in this article shed light on the potential role that circadian clock plays in corneal pain and discomfort.

The impact of euthanasia and enucleation on mouse corneal epithelial axon density and nerve terminal morphology.

INTRODUCTION: Here we study the impact of using either CO2 gas or cervical dislocation (CD) for euthanasia and using different techniques to enucleate the eye on preserving axonal density and morphology of the intraepithelial corneal nerves (ICNs). OBJECTIVES: To determine whether using CO2 gas or CD for euthanasia and enucleating by cutting or pulling eyes out impacts axon density and nerve terminal morphology in the mouse cornea. METHODS: Mice were euthanized by CO2 gas or CD; the impact of delaying fixation for 5 min post-euthanasia was also assessed. We tested two different techniques to enucleate the eyes: cutting the optic nerve by curved scissors or pulling the eye out. A minimum of 10 corneas from 5 male and female BALB/c mice were used for each variable. Axons and intraepithelial corneal nerve terminals (ICNTs) were visualized utilizing ßIII tubulin and L1CAM and quantified using confocal microscopy. RESULTS: The variations seen in axon density between individual mice are not gender- or euthanasia-dependent. A significant reduction in axon density and loss of ICNT morphology are observed in eyes enucleated by pulling the optic nerve out. Similar results are obtained in male and female mice. CONCLUSION: While the variations tested in euthanasia do not affect axon density in male and female mouse corneas, enucleation by proptosing and gently cutting out the eyes yields increased axon density and improved ICNT morphology compared to pulling eyes out and leaving the optic nerve attached.

Parity Attenuates Intraepithelial Corneal Sensory Nerve Loss in Female Mice.

Aging impacts the ocular surface and reduces intraepithelial corneal nerve (ICN) density in male and female mice. Many researchers use retired breeders to study naturally aged female mice. Yet, the impact of parity and the length of time since breeders were retired on age-related changes in the intraepithelial corneal nerves is not known. Here we study 2 month (M) nulliparous (NP) females as well as 9M, 10M, and 11M NP and multiparous (MP) female mice to determine whether parity impacts the age-related decline seen in corneal axon density; 9M male mice are also included in these assessments. After showing that parity attenuates age-related loss in axon density, we also assess the impact of parity on corneal epithelial cell proliferation and find that it impacts cell proliferation and axon density normalized by cell proliferation. Stromal nerve arborization is also impacted by aging with parity enhancing stromal nerves in older mice. qPCR was performed on 20 genes implicated in ICN density using corneal epithelial RNA isolated from 10M NP and MP mice and showed that NGF expression was significantly elevated in MP corneal epithelium. Corneal sensitivity was significantly higher in 9M MP mice compared to NP mice and increased sensitivity in MP mice was accompanied by increased nerve terminals in the apical and middle cell layers. Together, these data show that parity in mice attenuates several aspects of the age-related decline seen on the ocular surface by retaining sensory axons and corneal sensitivity as mice age.

Integrin: Basement membrane adhesion by corneal epithelial and endothelial cells.

Integrins mediate adhesion of cells to substrates and maintain tissue integrity by facilitating mechanotransduction between cells, the extracellular matrix, and gene expression in the nucleus. Changes in integrin expression in corneal epithelial cells and corneal endothelial cells impacts their adhesion to the epithelial basement membrane (EpBM) and Descemet's membrane, respectively. Integrins also play roles in assembly of basement membranes by both activating TGFß1 and other growth factors. Over the past two decades, this knowledge has been translated into methods to grow corneal epithelial and endothelial cells in vitro for transplantation in the clinic thereby transforming clinical practice and quality of life for patients. Current knowledge on the expression and function of the integrins that mediate adhesion to the basement membrane expressed by corneal epithelial and endothelial cells in health and disease is summarized. This is the first review to discuss similarities and differences in the integrins expressed by both cell types.

An immune response to the avascular lens following wounding of the cornea involves ciliary zonule fibrils.

The lens and central cornea are avascular. It was assumed that the adult lens had no source of immune cells and that the basement membrane capsule surrounding the lens was a barrier to immune cell migration. Yet, microfibril-associated protein-1 (MAGP1)-rich ciliary zonules that originate from the vasculature-rich ciliary body and extend along the surface of the lens capsule, form a potential conduit for immune cells to the lens. In response to cornea debridement wounding, we find increased expression of MAGP1 throughout the central corneal stroma. The immune cells that populate this typically avascular region after wounding closely associate with this MAGP1-rich matrix. These results suggest that MAGP1-rich microfibrils support immune cell migration post-injury. Using this cornea wound model, we investigated whether there is an immune response to the lens following cornea injury involving the lens-associated MAGP1-rich ciliary zonules. Our results provide the first evidence that following corneal wounding immune cells are activated to travel along zonule fibers that extend anteriorly along the equatorial surface of the lens, from where they migrate across the anterior lens capsule. These results demonstrate that lens-associated ciliary zonules are directly involved in the lens immune response and suggest the ciliary body as a source of immune cells to the avascular lens.

Corneal Epithelial "Neuromas": A Case of Mistaken Identity?

Laser scanning in vivo confocal microscopy is a useful clinical tool to assess the corneal nerves in human and laboratory animals. With this new technology, the use of terms such as "neuromas" and "microneuromas" is becoming popular to describe nerve structures seen in humans. Here, we point out that the sites where stromal nerves enter the corneal epithelium are often hyperreflective and can appear dysmorphic when imaged using in vivo confocal microscopy. Furthermore, we clarify what is known anatomically about how the nerves enter the corneal epithelium from the stroma, and we urge colleagues to differentiate between hyperreflective foci at the corneal stromal-epithelial nerve penetration sites and alterations in nerve morphology secondary to injury or disease.

Axonal debris accumulates in corneal epithelial cells after intraepithelial corneal nerves are damaged: A focused Ion Beam Scanning Electron Microscopy (FIB-SEM) study.

The intraepithelial corneal nerves (ICNs) that innervate the corneal epithelium are maintained through interactions with corneal epithelial cells and the extracellular matrix they produce. One to several axons bundle together within the basal cell layer and extend parallel to the ocular surface or branch and extend apically. Here we use 3-dimentional (3D) ultrastructural reconstructions of control and trephine injured mouse corneal epithelium and stroma produced using Focused Ion Beam Scanning Electron Microscope (FIB-SEM) to determine whether corneal epithelial or immune cells resident in the epithelium remove axonal debris and degrade it in their lysosomes after trephine injury to the cornea. We demonstrate that axonal fragments are internalized in the corneal epithelium and accumulate within electron dense structures consistent with lysosomes 3 h after trephine injury in both epithelial and immune cells located among the basal cells of the trephine injured cornea. Confocal imaging showed fewer CD45+ immune cells within the corneal epithelium after trephine injury compared to controls. The resolution obtained using FIB-SEM also allowed us to show that the presence of sensory axons at the basal aspect of the epithelial basal cells close to the anterior aspect of the epithelial basement membrane (EBM) is associated with a focal reduction in EBM thickness. In addition, we show using FIB-SEM and confocal imaging that superficial trephine injuries that do not penetrate the stroma, damage the integrity of anterior stromal nerves. These studies are the first to look at the mouse cornea following nerve injury using FIB-SEM.

Transient Mitomycin C-treatment of human corneal epithelial cells and fibroblasts alters cell migration, cytokine secretion, and matrix accumulation.

A single application of Mitomycin C (MMC) is used clinically in ophthalmology to reduce scarring and enhance wound resolution after surgery. Here we show in vitro that a 3-hour MMC treatment of primary and telomerase immortalized human corneal limbal epithelial (HCLE) cells impacts their migration and adhesion. Transient MMC treatment induces HCLE expression of senescence associated secretory factors, cytokine secretion, and deposition of laminin 332 for several days. Transient MMC treatment also reduces migration and deposition of transforming growth factor-ß1 (TGFß1)-stimulated collagen by corneal fibroblasts. Using conditioned media from control and MMC treated cells, we demonstrate that factors secreted by MMC-treated corneal epithelial cells attenuate collagen deposition by HCFs whereas those secreted by MMC-treated HCFs do not. These studies are the first to probe the roles played by corneal epithelial cells in reducing collagen deposition by corneal fibroblasts in response to MMC.

Desmin deficiency is not sufficient to prevent corneal fibrosis.

The type III intermediate filament (IF) proteins vimentin and desmin are sequentially overexpressed in stromal myofibroblasts over the period when fibrosis sets in after corneal injury. Prior findings have revealed vimentin-deficient mice are significantly protected from corneal fibrosis after alkali injury, which has implicated this IF protein as an important regulator of corneal fibrosis. It has remained as yet unproven whether desmin contributes in any significant manner to corneal fibrosis. Here we have employed desmin-deficient (Des KO) mice in the corneal alkali injury model and show that injured Des KO mice develop fibrosis and show similar levels of corneal opacity at 14 days post-injury as wild type (WT) mice and retain this phenotype even at 30d post injury. Des KO corneas from injured mice show upregulation of vimentin and alpha-smooth muscle actin expression to equivalent levels as WT corneas, illuminating that desmin deficiency does not interfere with myofibrobast differentiation. Employing the small molecule withaferin A (WFA), an inhibitor of vimentin, we show that WFA treatment causes the decrease in steady state levels of vimentin and serine 38 phosphorylated vimentin, the latter a biomarker associated with corneal fibrosis, and improved corneal clarity through blockade of myofibroblast differentiation. To investigate further the mechanism of fibrosis in desmin deficiency, we examined keratin 8 expression in the epithelium, and found reduced levels of this cytokeratin in injured Des KO corneas compared to WT corneas. This finding also corroborates the decrease of cell proliferation in injured Des KO corneas compared to that in WT corneas. The fibrotic phenotype of Des KO corneas also features abundant vascularization, further exemplifying the magnitude of corneal pathology. Together, these findings illuminate that desmin does not contribute significantly to corneal fibrosis in this injury model.

Reduced Corneal Innervation in the CD25 Null Model of Sjögren Syndrome.

Decreased corneal innervation is frequent in patients with Sjögren Syndrome (SS). To investigate the density and morphology of the intraepithelial corneal nerves (ICNs), corneal sensitivity, epithelial cell proliferation, and changes in mRNA expression of genes that are involved in autophagy and axon targeting and extension were assessed using the IL-2 receptor alpha chain (CD25 null) model of SS. ICN density and thickness in male and female wt and CD25 null corneas were assessed at 4, 6, 8, and 10/11 wk of age. Cell proliferation was assessed using ki67. Mechanical corneal sensitivity was measured. Quantitative PCR was performed to quantify expression of beclin 1, LC3, Lamp-1, Lamp-2, CXCL-1, BDNF, NTN1, DCC, Unc5b1, Efna4, Efna5, Rgma, and p21 in corneal epithelial mRNA. A significant reduction in corneal axon density and mechanical sensitivity were observed, which negatively correlate with epithelial cell proliferation. CD25 null mice have increased expression of genes regulating autophagy (beclin-1, LC3, LAMP-1, LAMP-2, CXCL1, and BDNF) and no change was observed in genes that were related to axonal targeting and extension. Decreased anatomic corneal innervation in the CD25 null SS model is accompanied by reduced corneal sensitivity, increased corneal epithelial cell proliferation, and increased expression of genes regulating phagocytosis and autophagy.

Molecular basis of Mitomycin C enhanced corneal sensory nerve repair after debridement wounding.

The ocular surface is covered by stratified squamous corneal epithelial cells that are in cell:cell contact with the axonal membranes of a dense collection of sensory nerve fibers that act as sentinels to detect chemical and mechanical injuries which could lead to blindness. The sheerness of the cornea makes it susceptible to superficial abrasions and recurrent erosions which demand continuous regrowth of the axons throughout life. We showed previously that topical application of the antibiotic and anticancer drug Mitomycin C (MMC) enhances reinnervation of the corneal nerves and reduces recurrent erosions in mice via an unknown mechanism. Here we show using RNA-seq and confocal imaging that wounding the corneal epithelium by debridement upregulates proteases and protease inhibitors within the epithelium and leads to stromal nerve disruption. MMC attenuates these effects after debridement injury by increasing serpine1 gene and protein expression preserving L1CAM on axon surfaces of reinnervating sensory nerves. These data demonstrate at the molecular level that gene expression changes in the corneal epithelium and stroma modulate sensory axon integrity. By preserving the ability of axons to adhere to corneal epithelial cells, MMC enhances sensory nerve recovery after mechanical debridement injury.

Adaptation of Operational Parameters of Cold Atmospheric Plasma for in Vitro Treatment of Cancer Cells.

Cold atmospheric plasma (CAP), an ionized gas operated at near-ambient temperatures, has been introduced as a new therapeutic opportunity for treating cancers. The effectiveness of the therapy has been linked to CAP-generated reactive oxygen and nitrogen species such as hydrogen peroxide and nitrite. In this study, we monitor in real-time cancer cell response to CAP over the course of 48 h. The results demonstrate a correlation between cell viability, exposure time (30, 60, 90, and 180 s), and discharge voltage (3.16 and 3.71 kV), while stressing the likely therapeutic role of plasma-generated reactive species. A 30-60 s increase in CAP exposure time and/or a discharge voltage adjustment from 3.16 to 3.71 kV is consistently accompanied by a significant reduction in cell viability. Comparably, levels of hydrogen peroxide and nitrite vary as a function of voltage with elevated levels detected at the highest tested voltage condition of 3.71 kV. CAP ultimately initiates a reduction in cell viability and triggers apoptosis via damage to the mitochondrial membrane, while also deregulating protein synthesis. The findings presented in this study are discussed in the context of facilitating the development of an adaptive CAP platform which could improve treatment outcomes.

Reduced intraepithelial corneal nerve density and sensitivity accompany desiccating stress and aging in C57BL/6 mice.

Dry Eye disease causes discomfort and pain in millions of patients. Using a mouse acute desiccating stress (DS) model we show that DS induces a reduction in intraepithelial corneal nerve (ICN) density, corneal sensitivity, and apical extension of the intraepithelial nerve terminals (INTs) that branch from the subbasal nerves (SBNs). Topical application of 0.02% Mitomycin C (MMC) or vehicle alone has no impact on the overall loss of axon density due to acute DS. Chronic dry eye, which develops progressively as C57BL/6 mice age, is accompanied by significant loss of the ICNs and corneal sensitivity between 2 and 24 months of age. QPCR studies show that mRNAs for several proteins that regulate axon growth and extension are reduced in corneal epithelial cells by 24 months of age but those that regulate phagocytosis and autophagy are not altered. Taken together, these data demonstrate that dry eye disease is accompanied by alterations in intraepithelial sensory nerve morphology and function and by reduced expression in corneal epithelial cells of mRNAs encoding genes mediating axon extension. Précis: Acute and chronic mouse models of dry eye disease are used to evaluate the pathologic effects of dry eye on the intraepithelial corneal nerves (ICNs) and corneal epithelial cells. Data show reduced numbers of sensory nerves and alterations in nerve morphology, sensitivity, corneal epithelial cell proliferation, and expression of mRNAs for proteins mediating axon extension accompany the pathology induced by dry eye.