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P.N. Martin, P. Roon, V. Ganapathy, S.B. Smith; Sigma Receptor Ligands as Neuroprotectants Against Apoptotic Retinal Ganglion Cell (RGC) Death in a Streptozotocin (STZ)-Induced Mouse Model of Diabetes . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3880.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: In diabetic retinopathy, RGCs die by apoptosis early after disease onset. This phenomenon was reported in humans and rats, but it is not known whether it occurs in mice. The RGC death is thought to occur via overstimulation of the NMDA receptor. Type 1 sigma receptor (σR1) is a nonopiate, nonphencyclidine binding site that demonstrates robust neuroprotective properties by modulating NMDA-receptors. In this study, we asked whether RGCs die by apoptosis in STZ-diabetic mice, and whether the σR1-specific ligand (+)-pentazocine (PTZ) might prevent this death. Methods: C57Bl/6 mice (age: 3 wks) were made diabetic via intraperitoneal injection of 75 mg/kg STZ dissolved in sodium citrate buffer (0.01M, pH 4.5) on 3 successive days. Mice were sacrificed 2, 4, 6, 8, 10, 12 and 14 weeks post-onset of diabetes. Eyes were harvested for cryosectioning. RGC apoptosis was analyzed in cryosectioned eyes by the TUNEL assay and caspase-3 immunohistochemistry. EM was used to confirm the classical ultrastructural features of apoptosis. The total number of RGCs was determined in each section. A second group of diabetic mice received PTZ (0.5, 1.0 mg/kg 3 X/wk beginning 1 wk post-onset of diabetes). Their eyes were analyzed also for RGC death. Results: TUNEL-positive RGCs were numerous in diabetic retinas during the first few weeks of diabetes (average: 2-3 cells/100 µm length of retina, compared to <0.15 in normal retinas.) Analysis of active caspase-3 showed many positive RGCs in retinas of diabetic, but not control, mice. Ultrastructural analysis of RGCs of diabetic mice revealed chromatin margination and crenated nuclei, classical morphological features of apoptosis. In diabetic mice, there were significantly fewer (~25-30%) RGCs by 10-14 weeks post-onset of diabetes compared to controls (p<0.05). Mice treated with PTZ retained significantly more RGCs than untreated diabetic mice (p<0.01) and the number of caspase-3-positive RGCs was similar to that of non-diabetic controls. Conclusions: RGCs die by apoptosis in the STZ-induced diabetic mouse model in a manner similar to that observed in retinas of rats and human patients with diabetic retinopathy. Treatment with PTZ may protect against this apoptotic RGC death.
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