Expression Of TRPC Channels In Rat Retina And The Effects Of TRPC6 On Retinal Ganglion Cell Apoptosis In Rat Chronic Ocular Hypertension Model

Glaucoma is the second leading cause of blindness worldwide. Among many reasons that cause glaucoma, elevated intraocular pressure (IOP) is commonly regarded as the major risk factor. It is believed that apoptotic death of retinal ganglion cells (RGCs) is the primary reason for severe and irreversible visual damage in glaucoma. However, the pathogenesis of RGC death following intraocular hypertension is still poorly understood, similar to the other neurodegenerative diseases. TRPC (canonical transient receptor potential) channels, widely distributed in the CNS, are a family of non-selective cation channels, which play important roles in modulation of cellular functions and in a variety of diseases. However, express and distribution of these channels in retina and their potential roles in RGCs death in glaucoma are largely unknown.In the present work, by using immunohistochemistry, Western blot, intravitreal injection and TUNEL assay techniques, we first examined expression and distribution of TRPC channel subtypes (TRPC1-TRPC7) in rat retina. We further studied the changes of expression of TRPC6 subunit in rat chronic ocular hypertension (COH) model, reproduced by blocking the episcleral veins, and explored the potential roles of TRPC6 channels in RGCs apoptotic death in the COH retinas. The results were showed as follow. (1) TRPC1, TRPC3 TRPC5 and TRPC6 proteins were expressed extensively in rat retina by using immunohistochemistry. The faint TRPC2-positive signals were also detected, while TRPC4 and TRPC7 proteins were not detectable. Consistent with these results, Western blot analysis confirmed the expression of these proteins in rat retina, and identified the specificity of antibodies we used. (2) To identify the exact localization of the expressed subtypes in the retina, especially in RGCs and Muller cells, double immunofluorescent staining was performed with the antibodies against glutamine synthetase (GS), a specific Muller cell marker, or CTB in the CTB retrograded retinal slices (RGCs were labeled). The results showed that TRPC1, TRPC3 and TRPC5 were expressed in both RGCs and Muller cells, but the expression of TRPC6 proteins was likely restricted to cells in the ganglion cell layer (GCL) even though very weak signals were detected in cells of the inner nuclear layer (INL). (3) Since our results showed that TRPC6 subunits were mainly expressed in RGCs and TRPC6 was found to be involved in the neuronal pathological injury in the CNS, we examined the changes of TRPC6 protein levels in retina obtained from the COH rats by immunohistochemistry and Western blotting. Unfortunately, we did not detected significant changes of TRPC6 proteins in the COH rats within 6 weeks after the operation, as compared to that obtained from control, even though the protein expression kept at a higher level at different time after the operation. However, intravitreal injection of OAG or SAG, two TRPC6 channel agonists, in the COH rats significantly reduced the total numbers of TUNEL-positive signals of RGCs. In contrast, intravitreal injection of SKF96365, a non-selective TRPC channel antagonist, in the COH rats did not show significant influence on the total numbers of TUNEL-positive signals of RGCs. These results suggest that activation of TRPC6 channels may prevent RGCs from apoptotic death in the COH rats.In conclusion, our present work shows that TRPC1, TRPC3 and TRPC5 are widely expressed and distributed in the cells of rat retina, including Muller cells and RGCs. The expression of TRPC6 subunit is selectively enriched in the RGC layer of the retina, suggesting that this subunit may be involved in modulation RGCs functions. Total proteins of TRPC6 channels keep at a high level and has no significant changes in the COH model. Endogenous TRPC6 channels may not be involved in RGC injury in the COH model. However, activating TRPC6 channels may provide a potential neuroprotective role for RGCs in glaucoma through inhibiting RGC apoptotic death. Our findings may help us to understand the pathogenesis of glaucoma and improving the therapeutic strategy.