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.

The eye is innervated by neurons derived from both the central nervous system and peripheral nervous system. 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 Wallarian 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.

We have reported previously that syndecan-1 (Sdc1)-null mice show delayed re-epithelialization after skin and corneal wounding. Here, we show that primary keratinocytes obtained from Sdc1-null mice and grown for 3-5 days in culture are more proliferative, more adherent and migrate more slowly than wt keratinocytes. However, the migration rates of Sdc1-null keratinocytes can be restored to wild-type levels by replating Sdc1-null keratinocytes onto tissue culture plates coated with fibronectin and collagen I, laminin (LN)-332 or onto the matrices produced by wild-type cells. Migration rates can also be restored by treating Sdc1-null keratinocytes with antibodies that block α6 or αv integrin function, or with TGFβ1. Antagonizing either β1 integrin function using a function-blocking antibody or TGFβ1 using a neutralizing antibody reduced wild-type keratinocyte migration more than Sdc1-null keratinocyte migration. Cultures of Sdc1-null keratinocytes accumulated less collagen than wild-type cultures but their matrices contained the same amount of LN-332. The Sdc1-null keratinocytes expressed similar total amounts of eight different integrin subunits but showed increased surface expression of αvβ6, αvβ8, and α6β4 integrins compared with wild-type keratinocytes. Whereas wild-type keratinocytes increased their surface expression of α2β1, αvβ6, αvβ8, and α6β4 after treatment with TGFβ1, Sdc1-null keratinocytes did not. Additional data from a dual-reporter assay and quantification of phosphorylated Smad2 show that TGFβ1 signaling is constitutively elevated in Sdc1-null keratinocytes. Thus, our results identify TGFβ1 signaling and Sdc1 expression as important factors regulating integrin surface expression, activity and migration in keratinocyte and provide new insight into the functions regulated by Sdc1.

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.

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.

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Integrin α6β4 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 β4 integrin is partially cleaved in epithelial cell extracts derived from debridement wounded corneas; (2) the β4 ectodomain is missing from sites where erosions develop; and (3) β4 cleavage can be reduced by inhibiting MMP activity. Although β4, α3 and β1 integrins were all cleaved by several MMPs, only MMP9 was elevated in cell extracts derived from corneas with erosions. Coimmunoprecipitation studies showed that β4 integrin associates with MMP9, and protein clustering during immunoprecipitation induced proteolytic cleavage of the β4 integrin extracellular domain, generating a 100 kDa β4 integrin cytoplasmic domain fragment. Confocal imaging with three-dimensional reconstruction showed that MMP9 localizes at erosion sites in vivo where the ectodomain of β4 integrin is reduced or absent. MMP activation experiments using cultured corneal and epidermal keratinocytes showed reduced levels of α6β4 and β1 integrins within 20 minutes of phorbol ester treatment. This report is the first to show that β4 integrin associates with MMP9 and that its ectodomain is a target for cleavage by MMP9 in vivo under pathological conditions.

Goblet cells are terminally differentiated cells secreting mucins and anti-bacterial 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, non-proliferating cells, and post-mitotic 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.