Inducible Nitric Oxide Synthase (INOS) in a Rat Glaucoma Model with Chronic Elevated Intraocular Pressure Resulting from Aqueous Humor Outflow Obstruction

Abstract

Abstract: : Purpose: It has been proposed that INOS induction may play a destructive role in the pathogenesis of glaucoma. The purpose of this study was to determine the presence and responsiveness of INOS in retina and optic nerve head tissues from rats with experimental glaucoma due to aqueous outflow obstruction. Methods: Brown Norway rats received unilateral injections of hypertonic saline into episcleral veins to produce chronic intraocular pressure (IOP) elevation. Following up to five weeks of pressure elevation (mean tonopen IOP, 2 to 12 mm Hg above normal) tissues were collected. Optic nerve injury was graded in a masked fashion. For gene array analysis, tissues were from animals with 1 and 5 weeks of IOP elevation (N=10 rats /group). Within groups, tissues were analyzed according to the amount of optic nerve injury. RNA was extracted from individual retinas with associated optic nerve heads and INOS expression levels compared using rat Affymetrix gene arrays. Retinal INOS mRNA levels were also quantitated by both Sybr green and multiplex real-time RT-PCR protocols in RNA preparations from paired retinas from 14 additional animals with 5 weeks IOP elevation. INOS protein presence and distribution in the nerve head and retina were evaluated immunohistochemically (N=9 paired globes) using INOS antibodies (and control IgG) from both Santa Cruz and Transduction Laboratories at 1 and 4 ug/ml. Results: For eyes with elevated IOP, optic nerve damage was non-linearly correlated with mean IOP, r2=0.82. Rat Affymetrix arrays (which contain 6 different rat INOS sequences) revealed no statistically significant level of INOS gene expression for any of these sequences in any of the elevated IOP or control samples. Quantitative RT-PCR studies indicated that INOS is expressed at very low levels in the normal retina. With IOP elevation, INOS levels were found to be 54% and 50% (by the two methods, respectively) of paired fellow eye values (p=0.08,both methods). Immunohistochemical analysis using both antibodies showed no difference in nerve head or retinal INOS labeling distribution or intensity between elevated IOP and control tissues. Conclusions: Gene microarray analysis, quantitative real time RT-PCR and immunohistochemical labeling all failed to demonstrate significant upregulation of INOS in retinas or optic nerve heads from rat eyes with chronic IOP elevation and optic nerve damage due to aqueous humor outflow obstruction.

Keywords: animal model • nitric oxide • intraocular pressure