• Energy Research

Ethanol is widely used in ocular surface surgeries and for the treatment of corneal diseases. However, ethanol is a toxic agent that is related to the development of a number of alcohol-related diseases. Despite the common use of ethanol for therapeutic purposes in ophthalmology, effects of ethanol on the ocular surface have been poorly defined. Hence, we performed this study to investigate effects of ethanol on corneal epithelium from various aspects.We exposed corneal epithelial cells in culture to different concentrations of ethanol for 30 seconds and evaluated the cells for toxicity, survival, and expression of cell-specific markers and inflammatory cytokines at 24, 48, and 72 hours after ethanol exposure.We found that ethanol markedly decreased the viability of cells in a concentration-dependent manner by causing cell lysis, suppressing proliferation, and inducing apoptosis. Also, expression of corneal epithelial cell-specific markers, both stem cell and differentiation markers, was significantly reduced by ethanol exposure. Expression of proinflammatory cytokines and chemokines was highly increased in corneal epithelial and stromal cells that were exposed to ethanol.Together, data suggest that brief exposure of the corneal surface to ethanol may have long-term effects by disrupting the integrity of corneal epithelium and generating inflammation, both of which are precursors to a number of ocular surface diseases.

Purpose: To evaluate the therapeutic effects of topical 8-oxo-2ʹ-deoxyguanosine (8-oxo-dG) on experimental ocular chemical injury models. Methods: We created ocular chemical injury models with 8-week-old BALB/c mice (n = 70) by applying 100% ethanol; the mice were then treated with 8-oxo-dG eye drops 10 and 5 mg/mL and phosphate-buffered saline (PBS) twice daily. After 7 days, clinical findings such as corneal integrity, clarity, and neovascularization were assessed. Histology, immunohistochemistry findings, and inflammatory cytokine levels using real-time polymerase chain reactions in the corneas of the mice were also analyzed. Results: Topical application of 8-oxo-dG eye drops resulted in a significant improvement of epithelial defects and clarity, dose dependently (each P < 0.001). Inflammatory cell infiltration and corneal stromal edema were also decreased in the 8-oxo-dG-treated mice compared with PBS-treated controls, based on hematoxylin and eosin staining. The expressions of F4/80 and neutrophil elastase–positive inflammatory cells and IL-1 and TNF-α cytokine levels were significantly reduced in the 8-oxo-dG group compared with the PBS group (each P < 0.01). Conclusions: Topical 8-oxo-dG application showed an excellent therapeutic effect in ocular chemical injury models by suppressing inflammation.

Mesenchymal stem/stromal cells (MSCs) facilitate the regeneration of injured tissue. Our group has previously shown that human MSCs (hMSCs) or hMSC-derived factors suppress excessive inflammatory response in the cornea following chemical injury in vivo. We here investigated direct effects of hMSC-derived factors on cultures of chemically injured human corneal epithelial progenitor cells (hCEP), independent of systemic anti-inflammatory effects that hMSCs have been shown to have in vivo.We injured hCEP by incubation in 20% ethanol for 30 seconds, and cultured the cells in fresh medium or in medium derived from cultures of human dermal fibroblasts (hFbs), hMSCs, or TNF-α-activated hMSCs. After 24 hours, we evaluated the survival, proliferation, and apoptosis of the cells.The hMSC-conditioned medium enhanced survival and proliferation and inhibited apoptosis of chemically injured hCEP. In addition, the conditioned medium accelerated the wound healing of corneal epithelium in tissue cultures of rabbit corneas following injury. The effects of the hMSC-conditioned medium were increased by preincubating hMSCs with TNF-α. The increased effectiveness of the medium from the preactivated hMSCs was in part explained by increased concentration of the multifunctional protein stanniocalcin-1 that inhibits apoptosis and promotes survival of cells.Together, the data account for beneficial effects of hMSCs on tissue-endogenous stem cells involving hCEP, and provide a basis for using MSCs or MSC-derived factors to treat diseases of the cornea and other tissues.

To investigate the effect of bevacizumab in a model of corneal inflammation.Epithelium from the cornea and limbus was completely removed using 100% alcohol in rats. Bevacizumab (1.25 mg/0.05 ml) or normal saline was subconjunctivally injected. One week later, corneas were examined and subjected to hematoxylin-eosin and immunofluorescent staining for VEGF. Expression of IL-2, IFN-gamma, IL-6, and TGF-beta 1 were analyzed by ELISA.Bevacizumab showed a borderline reduction in corneal neovascularization (11.0 +/- 4.8% and 18.0 +/- 10.0% in the bevacizumab and control groups, respectively; p = 0.054), while the extent of epithelialization was not affected (5.0 +/- 3.4% and 6.1 +/- 5.2% in the bevacizumab and control groups, respectively; p = 0.715). The infiltration of inflammatory cells was reduced (99.3 +/- 22.3 cells/x 400 in the bevacizumab-injected corneas and 182.3 +/- 58.0 cells/x 400 in the controls; p = 0.013). The levels of IL-2, IFN-gamma, and IL-6 were decreased in the rats with the bevacizumab injections (44 +/- 6 and 79 +/- 9 pg/ml for IL-2 in the bevacizumab-injected group and control, respectively, p = 0.025; 45 +/- 5 and 67 +/- 6 pg/ml for IFN-gamma in the bevacizumab-injected group and controls, respectively, p = 0.043; 45 +/- 6 and 75 +/- 8 pg/ml for IL-6 in the bevacizumab-injected group and controls, respectively, p = 0.030).Bevacizumab showed a reduction in inflammatory cell infiltration and cytokines in chemically burned rat corneas.