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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.

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., (© 2014 American College of Veterinary Ophthalmologists.)

Purpose: To determine whether ultraviolet (UV) radiation can modulate expression and regulation of matrix metalloproteinases (MMP) in the canine cornea and to examine the expression of MMPs in canine chronic superficial keratitis (CSK)., Methods: Immunohistochemistry for MMP-2 and MMP-9 was performed on samples of CSK. In vitro, canine corneal epithelial cell (CEC) and stromal cell cultures were exposed to UV-irradiation. Following 2, 8 or 24 h, cells were harvested. MMP expression was examined by zymography, and RT-PCR was used to examine expression of Slug and Snail. CEC cultures treated with an EGFR inhibitor or a p38 inhibitor were UV-exposed and harvested 24 h later to examine expression of MMPs, Slug and Snail., Results: Canine CSK had increased immunopositivity for both MMP-2 and MMP-9 compared to normal canine corneas. In vitro, CEC and stromal cell cultures exposed to UV showed generally increased expression of MMP-2, -9, Slug, and Snail; this response was dose and time dependent. Inhibition of the EGFR pathway did not prevent increased expression of MMP-2, -9, Slug or Snail in UV-exposed CEC; however, p38 inhibition did attenuate UV induction., Conclusions: We have found increased expression of MMPs in clinical samples of CSK compared to normal corneas. In addition, we have shown that there is a temporal association and dose dependency between UV exposure and production of MMPs, Slug, and Snail. These findings suggest that overexpression of MMPs due to UV-exposure may be linked to changes in the cornea that allow an influx of inflammatory cells and vascularization.

Ultraviolet radiation (UVR) is a risk factor for the development of ocular disease in humans, including acute photokeratitis, chronic corneal spheroidal degeneration, and cataract formation. This report describes the ocular lesions seen in 21 mice chronically exposed to UVR as part of a skin carcinogenicity study. All globes were affected to varying degrees. The primary lesion, not previously reported in UVR-exposed mice, was marked loss of keratocytes relative to age-matched controls. Secondary lesions included corneal stromal thinning, keratoconus, corneal vascularization and fibrosis, keratitis, globe rupture, and phthisis bulbi. In addition, more than 90% of UVR-exposed and unexposed lenses had evidence of cataract formation; this is the first report of the occurrence of spontaneous cataracts in 129 mice. In a subsequent study, apoptotic cells were identified histologically and by cleaved caspase 3 immunoreactivity in the corneal epithelium and, less commonly, in the corneal stroma after acute UVR exposure. Based on this finding, we propose that the loss of keratocytes observed in the chronic study was due to UVR-induced apoptosis.

Purpose Ultraviolet irradiation (UVR) increases telomerase activity in various cell types including skin, a sun-exposed organ. The lens is also continually exposed to UVR and we hypothesized that lenses exposed to UVR would have increased telomerase activity, with up-regulated TERT and TR, the two main components of the telomerase holoenzyme. To evaluate whether the cornea would protect lenses from such changes, whole globes, as well as isolated lenses, were exposed to UVR, and lenses were evaluated for changes in telomerase activity. Methods There were three parts to this project. The first part of this experiment evaluated freshly harvested normal adult canine lenses exposed to 0, 300, 600, or 1200 J/m2 UVR, and then allowed to recover for 1, 2, 3 and 4 h. Since only 600 J/m2 UVR increased telomerase activity, four more postexposure recovery time-points for this UVR dose were evaluated: 10 min, 30 min, 8 h and 24 h. The second part of this experiment used freshly enucleated whole canine globes exposed to 0, 50, 100, 150, 300, 600 or 1200 J/m2 and incubated overnight; lens epithelial cells (LEC) were evaluated for telomerase activity. The third part evaluated lenses that were exposed to 0 or 600 J/m2 UVR, and then allowed to recover for 8 and 24 h, before TERT and TR mRNA levels were measured. Results Isolated lenses exposed to 600 J/m2 UVR had significantly higher telomerase activity than unexposed controls and other UVR doses, at all time-points except 24 h postexposure. Lenses from whole globes exposed to UVR showed a dose-dependent increase in telomerase activity except at 50 J/m2 and 1200 J/m2. Isolated lenses exposed to 600 J/m2 UVR and then allowed to recover for 8 and 24 h significantly up-regulated TERT and TR mRNAs compared to unexposed control lenses. Conclusions Telomerase activity is regulated at both the transcriptional and post-translational levels in canine LEC. Previous work in our laboratory showed dose, time, and age-dependent changes in telomerase activity in the lens. The present study showed that TERT and TR mRNA transcription was increased for up to 24 h following an acute dose of UVR. Both telomerase activity and TERT and TR mRNA levels were elevated until 24 h post-UVR exposure, TERT in combination with TR functions in proliferation-related telomerase activity, but TERT alone has an anti-apoptotic function and its up-regulation may protect LEC from the acute effects of UVR. We are continuing to evaluate the mechanisms by which telomerase is regulated in normal and cataractous LEC. [ABSTRACT FROM AUTHOR]

Introduction: The current study was designed to test the potential role of recombinant human MG53 (rhMG53) protein on protecting against alkaline-induced corneal injury in mice.Materials and Methods: A round filter paper with 2-mm diameter was soaked in 1 mol/L of NaOH solution. The mouse alkaline injury was generated by placing the filter paper directly on the cornea for 30 seconds and washed with 30-mL saline; 10 µL of rhMG53 solution (20 µg/mL) or saline control was topically administrated on the mouse corneas (twice per day for 10 days). Re-epithelialization was measured by fluorescein staining and imaged by a slit lamp equipped with a digital camera. Clinical neovascularization and opacity scores were measured every day after injury. Ten days after injury, mice were sacrificed and corneas were dissected out for flat mount staining of CD31 for neovascularization.Results: MG53 was present in both dog aqueous humor and human tears. mg53-/- corneas were more susceptible to alkaline-induced corneal injury. Topical treatment of rhMG53 improved re-epithelialization, suppressed neovascularization, and fibrosis induced by alkaline injury.Conclusions: rhMG53 may be an effective means to treat corneal wounding. [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]