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Nishant Sinha, Ratnakar Tripathi, Suneel Gupta, Praveen Balne, Nathan P. Hesemann, Jason Rodier, Elizabeth A Giuliano, Prashant R. Sinha, Lynn M Martin, Shyam S Chaurasia, Rajiv R Mohan; Topical ophthalmic formulation to counteract sulfur mustardinduced corneal damage. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4672.
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© ARVO (1962-2015); The Authors (2016-present)
Sulfur mustard gas (SM) was used in two World Wars and recently inSyria. It causes blindness called mustard gas keratopathy (MGK) by inducing severe ocular inflammation, recurrent corneal epithelial erosions/ulcerations, corneal fibrosis, and neovascularization. No effective antidote for MGK currently exists. We tested therapeutic potential and safety of a topical ophthalmic solution to treat acute MGK using human corneal cells and ex vivo organ culture.
Human corneal stromal fibroblasts (CSFs), SV40-immortalized human corneal epithelial (HCE) and endothelial (HCN) cells,and ex vivohuman cornea organ culture model were used. Nitrogen mustard (NM; a primary SM analog) for SM toxicity and a topical ophthalmic Turbo Eye Drop (TED) developed in our lab (patent pending) were used for all studies. Cultures were exposed to NM (200μM) for 2h, washed, and then grown in +/- of TED (60μl) for specific times. Immunofluorescence, western blotting, H&E, qRT-PCR, confocal microscopy, and commercial kits were used to study cellular, molecular and toxicological parameters.
Time-dependent cytotoxicity assays found NM treatment caused significant cytotoxicity to the HCE, CSF and HCN; and TED 60μl (2 drops) treatment successfully mitigated adverse cellular changes to the levels seen in vehicle-control. Also, TED alone showed no significant cytotoxicity to these cells and human cornea. Asevere corneal damage and opacification to human cornea in organ cultures was noted after NM treatment, and TED application (8h/day for 3 days 2h after NM) effectively prevented NM-toxicity. H&E staining of NM-exposed serial corneal sections demonstrated that NM caused epithelial-stromal separation and damage to basement membrane, and TED treatment markedly decreased these adverse effects. qRT-PCR demonstrated that TED treatment significantly reduced NM-increased mRNA levels of inflammatory, fibrotic and angiogenic factors vital in corneal wound healing: αSMA (2.5 fold, p<0.001), iNOS (3.8 fold, p<0.001), COX2 (3.5 fold, p<0.001), TGFβ (3.5 fold, p<0.001), VEGF (3.5 fold, p<0.001) and MMP9 (3.7 fold, p<0.001) compared to vehicle treated control. Western blot analyses validated reduction in these factors at the protein level.
Novel topical eye drops developed in this study demonstrated excellent promise for the treatment of corneal cell SMG-toxicity. In vivorabbit studies are underway.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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