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M. Doat, J.-L. Bourges, G. Renard, C. Martin, F. F. Behar-Cohen; Effect of Peroxynitrite on the Endothelial Cell Density in the Rat Eye Assessed by in vivo Confocal Microscope. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2704.
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© ARVO (1962-2015); The Authors (2016-present)
We have previously reported the potential induced toxicity of nitrosative stress on corneal endothelial cells (EC) during corneal graft rejection. However, the direct role of peroxynitrite (ONOO-) on EC death has not been assessed. The aim of this study was to evaluate the effect ONOO- on EC after its direct injection in the anterior chamber (AC) of rat eyes.
Before testing, ONOO- concentration and decomposition curve were measured in the vehicles selected for in vivo injection. Lewis rats (n=18) were injected in the AC with one of the following solutions of ONOO- (1 eye/rat; 3 rats per solution): 500, 250, or 50 µM ONOO- in 0.05 M NaOH (vehicle), decomposed ONOO- (dONOO), vehicle alone and saline. The fellow eyes served as controls. The endothelial cell density (ECD) and pachymetry were measured after 6 and 24 hours by in vivo confocal tomography (HRT II; Heidelberg Engineering, Germany).
Six hours after injection, the mean corneal thicknesses (MCT) were 158 ± 16 µm in non-injected eyes, 136 ±8 µm for the saline; 198±9 µm for the vehicle, 192±14µm for the dONOO; 234 ± 13µm for 50µM; 306 ±33 µm for 250µM; and 272 ±20 µm for the 500 µM ONOO- injected groups. The MCT in the vehicle group increased vs saline or non-injected eyes (p=0.014). But, the MCT increased significantly in the 250 and 500µM ONOO- vs vehicle injected eyes (p<0.0001 and p=0.005 respectively) demonstrating the toxicity per se. The mean ECD did not differ in the saline and non-injected groups (2028 ± 27 and 2257±107 cells/mm²). A reduction in ECD was observed in ONOO- groups compared to the vehicle injected eyes for all concentrations (1290±178 cells/mm² for 50µM, 809±323 cells/mm² for 250µM, and 1028±61 cells/mm² for 500µM; P<0.0001) except for dONOO- (2390 ± 69 cells/mm², p=0.99) demonstrating direct toxicity of ONOO- on EC.After 24 H, the pachymetry was increased in the 50µM vs vehicle injected eyes (p<0.0001) and could not be assessed accurately in the other ONOO- groups due to the strong inflammatory and fibrinous reaction. 50µM ONOO- induced a significant loss of ECD vs vehicle injections (1350 ± 209 vs 609 ±85 cells/mm²;P<0.0001).
ONOO- injected in the AC of rats induce a severe corneal EC loss and subsequent corneal thickening suggesting that nitrosative stress could induce direct damages to EC in pathological conditions where nitric oxide is produced.
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