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.

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.

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.

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.

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.

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 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.

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.

Corneal epithelial cells function as surrogate Schwann cells for their sensory nerves.

The eye is innervated by neurons derived from both the central nervous system and peripheral nervous system (PNS). While much is known about retinal neurobiology and phototransduction, less attention has been paid to the innervation of the eye by the PNS and the roles it plays in maintaining a functioning visual system. The ophthalmic branch of the trigeminal ganglion contains somas of neurons that innervate the cornea. These nerves provide sensory functions for the cornea and are referred to as intraepithelial corneal nerves (ICNs) consisting of subbasal nerves and their associated intraepithelial nerve terminals. ICNs project for several millimeters within the corneal epithelium without Schwann cell support. Here, we present evidence for the hypothesis that corneal epithelial cells function as glial cells to support the ICNs. Much of the data supporting this hypothesis is derived from studies of corneal development and the reinnervation of the ICNs in the rodent and rabbit cornea after superficial wounds. Corneal epithelial cells activate in response to injury via mechanisms similar to those induced in Schwann cells during Wallerian Degeneration. Corneal epithelial cells phagocytize distal axon fragments within hours of ICN crush wounds. During aging, the proteins, lipids, and mitochondria within the ICNs become damaged in a process exacerbated by UV light. We propose that ICNs shed their aged and damaged termini and continuously elongate to maintain their density. Available evidence points to new unexpected roles for corneal epithelial cells functioning as surrogate Schwann cells for the ICNs during homeostasis and in response to injury. GLIA 2017;65:851-863.

K14 + compound niches are present on the mouse cornea early after birth and expand after debridement wounds.

BACKGROUND: We previously identified compound niches (CNs) at the limbal:corneal border of the mouse cornea that contain corneal epithelial progenitor cells, express Keratin 8 (K8), and goblet cell mucin Muc5AC. During re-epithelialization after 2.5 mm epithelial debridement wounds, CNs migrate onto the cornea and expand in number mimicking conjunctivalization. When CNs form during development and whether they express corneal epithelial progenitor cell enriched K14 was not known. RESULTS: To provide insight into corneal epithelial homeostasis, we quantify changes in expression of simple (K8, K18, K19) and stratified squamous epithelial keratins (K5, K12, K14, and K15) during postnatal development and in response to 2.5 mm wounds using quantitative polymerase chain reaction (Q-PCR), confocal imaging and immunoblots. K14 + CNs are present 7 days after birth. By 21 days, when the eyelids are open, K8, K19, and Muc5AC are also expressed in CNs. By 28 days after wounding, the corneal epithelium shows enhanced mRNA and protein expression for K14 and retains mRNA and protein for corneal epithelial specific K12. CONCLUSIONS: The keratin phenotype observed in corneal epithelial cells before eyelid opening is similar to that seen during wound healing. Data show K14 + corneal epithelial progenitor cells expand in number after 2.5 mm wounds.

Topical Mitomycin-C enhances subbasal nerve regeneration and reduces erosion frequency in the debridement wounded mouse cornea.

Corneal epithelial basement membrane dystrophies and superficial injuries caused by scratches can lead to recurrent corneal erosion syndrome (RCES). Patients and animals with reduced corneal sensory nerve innervation can also develop recurrent erosions. Multiple wild-type mouse strains will spontaneously develop recurrent corneal erosions after single 1.5 mm debridement wounds. Here we show that this wound is accompanied by an increase in corneal epithelial cell proliferation after wound closure but without a commensurate increase in corneal epithelial thickness. We investigated whether excess corneal epithelial cell proliferation contributes to erosion formation. We found that topical application of Mitomycin C (MMC), a drug used clinically to improve healing after glaucoma and refractive surgery, reduces erosion frequency, enhances subbasal axon density to levels seen in unwounded corneas, and prevents excess epithelial cell proliferation after debridement wounding. These results suggest that topically applied MMC, which successfully reduces corneal haze and scarring after PRK, may also function to enhance subbasal nerve regeneration and epithelial adhesion when used to treat RCES.

Partial denervation of sub-basal axons persists following debridement wounds to the mouse cornea.

Although sensory reinnervation occurs after injury in the peripheral nervous system, poor reinnervation in the elderly and those with diabetes often leads to pathology. Here we quantify sub-basal axon density in the central and peripheral mouse cornea over time after three different types of injury. The mouse cornea is highly innervated with a dense array of sub-basal nerves that form a spiral called the vortex at the corneal center or apex; these nerves are readily detected within flat mounted corneas. After anesthesia, corneal epithelial cells were removed using either a dulled blade or a rotating burr within an area demarcated centrally with a 1.5 mm trephine. A third wound type, superficial trephination, involved demarcating the area with the 1.5 mm trephine but not removing cells. By 7 days after superficial trephination, sub-basal axon density returns to control levels; by 28 days the vortex reforms. Although axon density is similar to control 14 days after dulled blade and rotating burr wounding, defects in axon morphology at the corneal apex remain. After 14 days, axons retract from the center leaving the sub-basal axon density reduced by 37.2 and 36.8% at 28 days after dulled blade and rotating burr wounding, respectively, compared with control. Assessment of inflammation using flow cytometry shows that persistent inflammation is not a factor in the incomplete reinnervation. Expression of mRNAs encoding 22 regeneration-associated genes involved in axon targeting assessed by QPCR reveals that netrin-1 and ephrin signaling are altered after wounding. Subpopulations of corneal epithelial basal cells at the corneal apex stop expressing ki67 as early as 7 days after injury and by 14 and 28 days after wounding, many of these basal cells undergo apoptosis and die. Although sub-basal axons are restored to their normal density and morphology after superficial trephination, sub-basal axon recovery is partial after debridement wounds. The increase in corneal epithelial basal cell apoptosis at the apex observed at 14 days after corneal debridement may destabilize newly reinnervated sub-basal axons and lead to their retraction toward the periphery.

Wounding the cornea to learn how it heals.

Corneal wound healing studies have a long history and rich literature that describes the data obtained over the past 70 years using many different species of animals and methods of injury. These studies have lead to reduced suffering and provided clues to treatments that are now helping patients live more productive lives. In spite of the progress made, further research is required since blindness and reduced quality of life due to corneal scarring still happens. The purpose of this review is to summarize what is known about different types of wound and animal models used to study corneal wound healing. The subject of corneal wound healing is broad and includes chemical and mechanical wound models. This review focuses on mechanical injury models involving debridement and keratectomy wounds to reflect the authors' expertise.

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.

MMP9 cleavage of the ß4 integrin ectodomain leads to recurrent epithelial erosions in mice.

Integrin α6ß4 is an integral membrane protein within hemidesmosomes and it mediates adhesion of epithelial cells to their underlying basement membrane. During wound healing, disassembly of hemidesmosomes must occur for sheet movement-mediated cell migration. The mechanisms of disassembly and reassembly of hemidesmosomes are not fully understood. The current study was initiated to understand the underlying cause of recurrent corneal erosions in the mouse. Here, we show that in vivo: (1) MMP9 levels are elevated and ß4 integrin is partially cleaved in epithelial cell extracts derived from debridement wounded corneas; (2) the ß4 ectodomain is missing from sites where erosions develop; and (3) ß4 cleavage can be reduced by inhibiting MMP activity. Although ß4, α3 and ß1 integrins were all cleaved by several MMPs, only MMP9 was elevated in cell extracts derived from corneas with erosions. Coimmunoprecipitation studies showed that ß4 integrin associates with MMP9, and protein clustering during immunoprecipitation induced proteolytic cleavage of the ß4 integrin extracellular domain, generating a 100 kDa ß4 integrin cytoplasmic domain fragment. Confocal imaging with three-dimensional reconstruction showed that MMP9 localizes at erosion sites in vivo where the ectodomain of ß4 integrin is reduced or absent. MMP activation experiments using cultured corneal and epidermal keratinocytes showed reduced levels of α6ß4 and ß1 integrins within 20 minutes of phorbol ester treatment. This report is the first to show that ß4 integrin associates with MMP9 and that its ectodomain is a target for cleavage by MMP9 in vivo under pathological conditions.

Spontaneous skin erosions and reduced skin and corneal wound healing characterize CLIC4(NULL) mice.

Cutaneous wound healing is a complex process involving blood clotting, inflammation, migration of keratinocytes, angiogenesis, and, ultimately, tissue remodeling and wound closure. Many of these processes involve transforming growth factor-ß (TGF-ß) signaling, and mice lacking components of the TGF-ß signaling pathway are defective in wound healing. We show herein that CLIC4, an integral component of the TGF-ß pathway, is highly up-regulated in skin wounds. We genetically deleted murine CLIC4 and generated a colony on a C57Bl/6 background. CLIC4(NULL) mice were viable and fertile but had smaller litters than did wild-type mice. After 6 months of age, up to 40% of null mice developed spontaneous skin erosions. Reepithelialization of induced full-thickness skin wounds and superficial corneal wounds was delayed in CLIC4(NULL) mice, resolution of inflammation was delayed, and expression of ß4 integrin and p21 was reduced in lysates of constitutive and wounded CLIC4(NULL) skin. The induced level of phosphorylated Smad2 in response to TGF-ß was reduced in cultured CLIC4(NULL) keratinocytes relative to in wild-type cells, and CLIC4(NULL) keratinocytes migrated slower than did wild-type keratinocytes and did not increase migration in response to TGF-ß. CLIC4(NULL) keratinocytes were also less adherent on plates coated with matrix secreted by wild-type keratinocytes. These results indicate that CLIC4 participates in skin healing and corneal wound reepithelialization through enhancement of epithelial migration by a mechanism that may involve a compromised TGF-ß pathway.

Corneal goblet cells and their niche: implications for corneal stem cell deficiency.

Goblet cells are terminally differentiated cells secreting mucins and antibacterial peptides that play an important role in maintaining the health of the cornea. In corneal stem cell deficiency, the progenitor cells giving rise to goblet cells on the cornea are presumed to arise from differentiation of cells that migrate onto the cornea from the neighboring conjunctiva. This occurs in response to the inability of corneal epithelial progenitor cells at the limbus to maintain an intact corneal epithelium. This study characterizes clusters of cells we refer to as compound niches at the limbal:corneal border in the unwounded mouse. Compound niches are identified by high expression of simple epithelial keratin 8 (K8) and 19 (K19). They contain variable numbers of cells in one of several differentiation states: slow-cycling corneal progenitor cells, proliferating cells, nonproliferating cells, and postmitotic differentiated K12+Muc5ac+ goblet cells. Expression of K12 differentiates these goblet cells from those in the conjunctival epithelium and suggests that corneal epithelial progenitor cells give rise to both corneal epithelial and goblet cells. After wounds that remove corneal epithelial cells near the limbus, compound niches migrate from the limbal:corneal border onto the cornea where K8+ cells proliferate and goblet cells increase in number. By contrast, no migration of goblet cells from the bulbar conjunctiva onto the cornea is observed. This study is the first description of compound niches and corneal goblet cells and demonstration of a role for these cells in the pathology typically associated with corneal stem cell deficiency.

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.