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Karunakaran Coral, Jagaveerapandiyan Sarangapani, Vineet Ratra, Sulochana KN; In Vitro Proliferative Response Of Human Tenons And Scleral Cells From Patients Undergoing Glaucoma Filtration Surgery- A Clinical Correlation. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3870.
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Glaucoma is an optic neuropathy characterized by optic disc changes and corresponding visual field defects in which intra ocular pressure (IOP) is the only treatable factor. Failure of trabeculectomy surgery is due to excessive scarring in sclera and subconjunctiva. The purpose of this study is to look at the proliferative response of the sclera and tenons tissues removed from patients undergoing trabeculectomy surgery and also to screen the effect of compounds which inhibit excess wound healing.
The study was approved by the Ethics committee of the Institute and informed consent were obtained from patients. Detailed clinical proforma was filled. Scleral tissues (n=8) and tenons (n=7) removed surgically from patients undergoing trabeculectomy was processed for cell culture. The clinical outcome of the patients after surgery was assessed.The tissues were treated with 0.2 % collagenase and plated on 0.1 % gelatin coated dishes with EGM medium. The isolated cells were identified using specific markers viz Fibroblast specific protein (FSP) for human tenons fibroblast (HTF) and SOX9 for human scleral cells (HSC) by RT-PCR. Wound healing was done by scratch assay in cells using drugs like mitomycin C (MMC) and cyclosporine A (CsA) at concentrations of 10 ng to 100 μg/ml and endogenous extracellular matrix derived peptides at concentrations of 10 to 100 µM.
Sclera and tenons tissues from patients (n=2) who had post operatively corkscrew vessels on the bleb which is suggestive of increased scarring grew in vitro.These cells grew after 5-7 days and the confluent cells showed typical cell specific morphology. mRNA expression of HSC were positive for SOX 9 (213bp) and HTF were positive for FSP (153bp) by RT-PCR. Wound healing as evaluated by scratch assay showed inhibition of cell migration with a combination of MMC (100 μg/ml ie 300 μM) and CsA (50 μg/ml ie 40 μM) compared to MMC alone. Extracellular matrix derived peptides were effective in inhibiting cell migration at 10 μM concentration. No cytotoxicity was observed for these compounds at the tested concentrations by MTT assay.
In vitro cell growth of sclera and tenons could be a predictive marker for the poor outcome of glaucoma filtration surgery, such cases could be treated with endogenous extracellular matrix derived peptides which inhibit cell migration thereby preventing scar formation.
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