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Rationale: The cornea is a unique structure that maintains its clarity by remaining avascular. Corneal injuries can lead to neovascularisation (CNV) and fibrosis and are the third most common cause of blindness worldwide., Objective: Corneal injuries induce an immune cell infiltration to initiate reparative processes. However, inflammation caused by sustained immune cell infiltration is known to be detrimental and can delay the healing process. This study was designed to understand the potential role of neutrophil and epithelial cell crosstalk in post-injury CNV., Methods and Results: Western blotting and immunostaining assays demonstrated that neutrophils infiltrated corneas and underwent pyroptosis following acute alkali injury. In vivo studies showed that genetic ablation of Gasdermin D (GsdmD), a key effector of pyroptosis, enhanced corneal re-epithelialisation and suppressed post-injury CNV. In vitro co-culture experiments revealed that interleukin-1β (IL-1β) was released from pyroptotic neutrophils which suppressed migration of murine corneal epithelial cells. Real-time RT-PCR and immunostaining assays identified two factors, Wnt5a and soluble fms-like tyrosine kinase-1 (sflt-1), highly expressed in newly healed epithelial cells. sflt-1 is known to promote corneal avascularity. Bone marrow transplantation, antibody mediated neutrophil depletion, and pharmacological inhibition of pyroptosis promoted corneal wound healing and inhibited CNV in an in vivo murine corneal injury model., Conclusion: Taken together, our study reveals the importance of neutrophil/epithelium crosstalk and neutrophil pyroptosis in response to corneal injuries. Inhibition of neutrophil pyroptosis may serve as a potential treatment to promote corneal healing without CNV., Key Points: Neutrophil pyroptosis delays re-epithelialization after corneal injury Compromised re-epithelialization promotes corneal neovascularization after injury Inhibition of post-injury pyroptosis could be an effective therapy to promote corneal wound healing., (© 2024 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

The cornea plays an important role in transmitting light and providing protection to the eye, but is susceptible to injury and infection. Standard treatments for corneal wounds include topical lubricants, antibiotics, bandage contact lens, and surgery. However, these measures are often ineffective. Here we show that MG53, a protein with an essential role in cell membrane repair, contributes to the corneal injury-repair process. Native MG53 is present in the corneal epithelia, tear film, and aqueous humor, suggesting its potential function in corneal homeostasis. Knockout of MG53 in mice causes impaired healing and regenerative capacity following injury. Exogenous recombinant human MG53 (rhMG53) protein protects the corneal epithelia against mechanical injury and enhances healing by promoting migration of corneal fibroblasts. Using in vivo alkaline-induced injury to the rat cornea, we show that rhMG53 promotes re-epithelialization and reduces post-injury fibrosis and vascularization. Finally, we show that rhMG53 modulates TGF-β-mediated fibrotic remodeling associated with corneal injury. Overall, our data support the bi-functional role of MG53 in facilitating corneal healing and maintaining corneal transparency by reducing fibrosis and vascularization associated with corneal injuries., Competing Interests: J.M. is a founder of TRIM-edicine, Inc. and T.T. has an equity interest in TRIM-edicine, Inc., a biotechnology company developing rhMG53 as a therapeutic protein. The remaining authors declare no competing interests.

Objective: Heat-shock proteins, particularly the 70-kDa member (Hsp70), have been implicated in facilitating wound healing in multiple tissues. Expression and localization of three HSPs were assessed in normal and wounded canine corneas to elucidate a role in epithelial healing., Methods: Paraffin-embedded normal corneas, acute and repeatedly abraded corneas, and keratectomies of spontaneous chronic corneal epithelial defects (SCCEDs) were subjected to routine immunohistochemistry for Hsp27, 47, and 70 expression. Ex vivo corneal defects were created and treated with anti-HSPs or IgG controls, and wound healing was monitored. Primary cultures of canine corneal stromal fibroblasts and corneal epithelial cells were treated with exogenous Hsp70, and an artificial wound was created in vitro to monitor restoration of the monolayer., Results: Normal canine corneas exhibited constitutive expression of all HSPs evaluated. Inducible expression was demonstrated in acutely wounded tissues, and expression in the chronically abraded corneas was relocalized. All HSP expression was below the limits of detection in the epithelium of SCCED samples. Inhibition of HSPs in culture resulted in delayed wound healing when compared to controls. Hsp70-treated fibroblasts demonstrated significantly (P < 0.001) increased migration and proliferation compared to the vehicle control; however, there was no significant effect of exogenous Hsp70 on corneal epithelial cells., Conclusions: These findings suggest that HSPs are induced in the normal canine cornea during re-epithelialization. Hsp70 expression is likely important for inducing the cytoarchitectural remodeling, migration, and proliferation necessary early in the canine corneal healing response, and suppressed expression may contribute to the pathophysiology of nonhealing defects., (© 2015 American College of Veterinary Ophthalmologists.)

Background: Keratinocytes at wound margins undergo partial epithelial to mesenchymal transition (EMT). Based on previous in vitro and ex vivo findings, Slug (Snai2), a transcriptional regulator of EMT in development, may play an important role in this process., Objectives: This study was designed to validate an in vivo role for Slug in wound healing., Methods: Excisional wounds in Slug null and wild type mice were examined histologically at 6, 24, 48, and 72h after wounding; reepithelialization was measured and immunohistochemistry for keratins 8, 10, 14, and 6 and E-cadherin was performed. In 20 Slug null and 20 wild type mice exposed three times weekly to two minimal erythemal doses of UVR, the development of non-healing cutaneous ulcers was documented. Ulcers were examined histologically and by immunohistochemistry., Results: The reepithelialization component of excisional wound healing was reduced 1.7-fold and expression of the Slug target genes keratin 8 and E-cadherin was increased at wound margins in Slug null compared to wild type mice. In contrast, no differences in expression of keratins 10 or 14 or in markers of proliferation K6 and Ki-67 were observed. Forty per cent of Slug null mice but no wild type mice developed non-healing cutaneous ulcers in response to chronic UVR. Keratinocytes at ulcer margins expressed high levels of keratin 8 and retained E-cadherin expression, thus resembling excisional wounds., Conclusion: Slug is an important modulator of successful wound repair in adult tissue and may be critical for maintaining epidermal integrity in response to chronic injury.

Epithelial cell migration during corneal wound re-epithelialization shares features with the developmental process of epithelial-mesenchymal transition (EMT) modulated by Snail family transcription factors, including Slug. Our studies demonstrated that Slug expression was enhanced at sites of epithelial cell migration at the margins of normally healing corneal wounds in dogs, but significantly decreased at the margins of non-healing canine corneal erosions. Increased Slug expression was associated with internalization of E-cadherin and beta-catenin from the cell membrane and with enhanced expression of smooth-muscle-specific alpha-actin, tropomyosin, and matrix metalloproteinases at wound margins. Enhanced Slug expression in corneal explants due to an adenoviral expression construct or to oxytetracycline treatment resulted in significantly higher rates of corneal epithelial cell migration. Oxytetracycline appeared to act by stimulating transforming growth factor-beta activity, thus increasing Slug expression and enhancing corneal epithelial migration. These findings highlight the similarities between epithelial migration during EMT and during successful corneal wound healing, support an important role for the Snail family in the process, and indicate that modulating Slug expression may be clinically useful in treating non-healing corneal wounds.

Purpose To evaluate the in vitro effects of an aloe vera solution on (i) the viability and wound healing response of corneal cells and (ii) the ability to alter collagenase and gelatinase activities. Methods Primary cultures of corneal epithelial cells and fibroblasts were prepared from grossly normal enucleated canine globes and treated with an aloe solution (doses ranging from 0.0-2 mg/mL). Cellular viability was evaluated using a colorimetric assay. A corneal wound healing model was used to quantify cellular ingrowth across a defect made on the confluent surface. Anticollagenase and antigelatinase activities were evaluated by incubating a bacterial collagenase/gelatinase with aloe solution (doses ranging from 0.0-500 μg/mL) and comparing outcome measures to a general metalloproteinase inhibitor, 1, 10-phenanthroline, and canine serum (doses ranging from 0.0-100%). Results None of the concentrations of aloe solution tested significantly affected the viability of corneal epithelial cells or fibroblasts. Concentrations ≤175 μg/mL slightly accelerated corneal epithelial cell wound closure; this change was not significant. Concentrations ≥175 μg/mL significantly ( P ≤ 0.001) slowed the rate of corneal fibroblast wound closure, while aloe concentrations <175 μg/mL did not significantly alter fibroblast wound closure. Aloe solution did not alter the ability for collagenase to degrade gelatin or collagen Type I but increased the ability for collagenase to degrade Type IV collagen. Conclusions Although additional experiments are required, lower concentrations of aloe solution may be beneficial in healing of superficial corneal wounds to help decrease fibrosis and speed epithelialization. An increase in collagenase activity with aloe vera warrants further testing before considering in vivo studies. [ABSTRACT FROM AUTHOR]

Objective--To assess the in vitro effects of various nalbuphine concentrations on viability and wound healing ability of corneal cells and potential drug transport through the corneal epithelium. Sample--Cultured canine and human corneal epithelial cells (CECs) and cultured canine corneal stromal fibroblasts. Procedures--CECs and stromal fibroblasts were exposed to nalbuphine (concentration of solutions ranged from 0% to 1.2%) for up to 30 minutes, and viability was assessed with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. A standard scratch test technique was used. Wound healing of CECs and stromal fibroblasts was evaluated following treatment with nalbuphine solutions < 0.1%. Liquid chromatography--mass spectrometry--mass spectrometry analysis was used to evaluate drug transport across a monolayer and a multilayer of human CECs. Results--A progressive decrease in viability was detected in canine CECs for all nalbuphine treatment groups, whereas treatment with only 0.5% or 1.2% nalbuphine significantly reduced corneal stromal fibroblast viability, compared with results for control cells. Within 24 hours, treatment with 0.1% nalbuphine solution significantly altered the healing rate of both canine CECs and stromal fibroblasts. Continuous increases in transport rates of nalbuphine were detected with time for both the monolayer and multilayer of human CECs. Conclusions and Clinical Relevance--In vitro, nalbuphine potentially could penetrate through corneal tissue, but it may cause damage to the corneal epithelium and stromal fibroblasts. Therefore, nalbuphine potentially may impair corneal wound healing. [ABSTRACT FROM AUTHOR]