Figure 2.

NS deficiency aggravates glaucomatous degenerative changes in the retina and ON

Induction of chronic glaucoma causes inner retinal function and structure damage in WT and NS^−/− mice. (A) Non-injected mice (control) had a steady IOP level that was maintained at an average value of 11.6 ± 2.1 mm Hg (WT mice) and 11.4 ± 1.9 mm Hg (NS^−/− mice) throughout the period (n = 10 in each group). Weekly injections of microbeads induced an elevation of IOP and were maintained for 8 weeks (n = 10 in each group). Values are mean ± SEM. (B) pSTR traces in a normal (dark magenta) and chronic glaucoma (pink) model of WT retinas. (C) pSTR traces in a normal (dark green) and chronic glaucoma (cyan) model of NS^−/− retinas. (D) Significant differences in pSTR amplitudes were observed in WT mice exposed to chronically elevated IOP (n = 10 animals in each group, p < 0.001), and NS^−/− mice are more susceptible to glaucoma damage compared with WT counterparts (n = 10 animals in each group, p < 0.009 and p < 0.02). (E and F) Histological analysis of paraffin-embedded retinal sections from WT and NS^−/− mice stained by H&E under control and glaucoma conditions. Arrows indicate GCL. Scale bars, 50 μm). (G) There was a significant decrease in GCL cells in WT mice under chronic elevation of IOP (n = 4 animal, 3 sections/animal, p < 0.0001). The decrease in GCL density was significantly higher in NS^−/− mice under normal and glaucoma conditions (n = 4 animals, 3 sections/animal; p < 0.0001 and p < 0.004). (H and I) ON axonal appearance in WT and NS^−/− mice under control and experimental glaucoma conditions, stained with TB (10× and 63× resolution). (J) Quantification of axonal density revealed significant axonal loss in WT mice exposed to experimental glaucoma (n = 4 animal, 3 sections/animal, p < 0.0001). NS^−/− mice showed a significant lower axon density compared with WT mice, and experimental glaucoma further increases significant axon loss in these knockout mice (n = 4 animal, 3 sections/animal, p < 0.0001). (K) Increased TUNEL⁺ staining (red) was observed in retinal sections exposed to microbead injections in WT and NS^−/− mice in the inner retinal layers (white arrows). Shown are DAPI-stained cell nuclei (blue). Scale bars, 50 μm. (L) Quantification of TUNEL⁺ cells showing significantly increased numbers in WT glaucoma (n = 3 animals in each group, p < 0.0008). NS^−/− mice showed a significant increase in TUNEL⁺ cells in a control and experimental glaucoma model (n = 3 animals in each group, p < 0.002, p < 0.004). Graphs show means ± SEM, and p values were obtained using Student’s t test.