Thesis

Purpose: To evaluate the topical and intravitreal use of alpha B-Crystalline minipeptides for ameliorating inflammation, rendering cytoprotection and neuroprotection to the cornea and retina post injury. Methods: Internalization of the FICT tagged alpha B-crystalline minipeptides was assessed. Cytoprotection by minipeptides to corneal cells was evaluated by MTT assay and neuroprotection to trigeminal neurons was evaluated by beading quantification. Neurite elongation in trigeminal ganglion with the minipeptides was quantified. Rabbit corneal nerve ablation model was performed; topical therapy with alpha B-crystalline peptide was instilled for thrice daily for 14days. Corneal healing was evaluated by slit lamp biomicroscopy and tissues were harvested for gene expression, histopathology and immunostaining. Retinal injury was induced in eye of rabbits which received alpha B-crystalline peptide, by intravitreal injection immediately after inducing injury. The inflammatory response was monitored clinically, by gene expression, histopathology and immunohistochemistry. Results: There was evidence of internalization of fluroscence tagged minipeptides in the cytosol and nucleus of cultured corneal cells. The MTT assay recorded significant number of live cells in stressed corneal cells cotreated with the minipeptides versus the stressed cells without treatment (p<0.05). Although there was no significant evidence of elongation of trigeminal neurites with the minipeptides, beading was significantly (p<0.05) reduced in the stressed neurons. The cornea receiving treatment showed marked improvement with reduced hyperreflective zones. There was suppression in the gene expressions of proinflammatory cytokines and oxidative stress gene. Histopathology was depictive of rescue from detrimental changes and immunostaining for expression of alpha B-crystalline was high in normal and lowest in untreated injured cornea. In the retinal injury study, there was significant (p<0.05) reduction in gene expression of inflammatory cytokines and oxidative stress markers. Histopathology revealed reduced inflammatory response with the therapy. GFAP positive cells in the retina and vitreous were enhanced in the untreated versus the treated eye. Conclusion: Our study shows the cytoprotective and neuroprotective role of alpha B-crystalline in corneal cells and trigeminal ganglion neurons, its profound anti-inflammatory, anti-oxidant activity as exogenous therapy following corneal and retinal injury. This natural protein emerges as an interesting and promising agent with therapeutic potency which entails further investigation.

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