The Mechanism Of Müller Cell Gliosis In A Rat Chronic Ocular Hypertension Model

Glaucoma, the second leading cause of blindness, is a neurodegenerative disease that is characterized by optic nerve degeneration resulting from apoptotic death of retinal ganglion cells (RGCs). Although elevated intraocular pressure (IOP) is commonly regarded as a hallmark risk factor, the pathogenesis of RGC death following intraocular hypertension is still poorly understood. Reactivation (gliosis) of glial cells occurs in almost all kinds of central nervous system injury and disease. Reactive Muller cells may be involved in two contradictory functions:protecting retinal tissue from further damage and contributing to neurodegeneration. As an early response to cell injury, Muller cells release antioxidants and neurotrophic factors, and remove extracellular excess glutamate, thus promoting axonal regeneration and synaptic remodeling. In contrast, reactivated Muller cells produce and release various cytotoxic factors such as nitric oxide, tumor necrosis factor-a, reactive oxygen species, and prostaglandin E2, which induce RGC apoptosis. Reactivated glial cells may also hamper axonal regeneration and neurite outgrowth by forming scar tissues.In the vertebrate retina, Muller cells are principal glial cells that stretch across the whole thickness of the retina. Muller cell gliosis is a common response in a variety of pathological alternations of the retina. Muller cell gliosis is characterized by the upregulated expression of glial cytoskeletal proteins, glial fibrillary acidic protein (GFAP), nestin and vimentin, which are cellular markers of an early gliotic response. Another hallmark of Muller cell gliosis is downregulation of membrane K+ conductance. However, it has not been clearly established whether K+ channels change following the onset of glaucoma. Furthermore, the mechanisms that underlie Muller cell gliosis and downregulation of K+ currents in glaucoma and other retinal injuries and diseases are largely unknown.In the present work, we studied the changes of Kir currents in Muller cells in a rat chronic intraocular hypertension (COH) model, explored the underlying mechanisms and analyzed the relationship between downregulation of Kir currents and gliosis of Muller cells using whole-cell patch-clamp, immunochemistry and Western blot techniques. The results are showed below. (1) Robust COH model was reproduced by blocking the episcleral veins. (2) A Kir K+ current with weak rectifying identified in Muller cells acutely isolated from rat retinas. (3) Within 6 weeks after the operation, Kir currents of Muller cells isolated from the operated eyes and the sham-operated eyes were recorded respectively at different time points. We observed a pronounced downregulation of Kir currents following the elevation of IOP. Although slightly recovered from 4th week after the operation, the current amplitudes were still remarkable lower than that of the control. (4) The selective mGluR I receptor agonist DHPG significantly inhibited Kir currents in isolated Muller cells. The selective mGluR5 antagonist MPEP, but not the selective mGluR1 antagonist MPEQ completely eliminated the DHPG-induced inhibition of Kir currents. (5) We investigated what intracellular signaling pathway(s) may mediate the mGluR I mediated downregulation of Kir currents. cAMP-PKA pathway seems not to be involved in DHPG-induced inhibition of Kir currents in rat Muller cells since forskolin did not induce any change in Kir currents and a cAMP inhibitor Rp-cAMP hardly change the DHPG effect. CaMK II is also not involved since CaMKⅡinhibitor KN62 and KN93 failed to influence the DHPG effect. We then examined if PLC may be involved in the DHPG-induced inhibition of Kir currents. We found that phosphatidylinositol (PI)-PLC is involved because a PI-PLC inhibitor U73122, but not a phosphatidylcholine (PC)-PLC inhibitor D609 blocked the DHPG effect. Moreover, intracellular dialysis of Bis IV or chelerythrine chloride, specific PKC inhibitors, eliminated the inhibitory effect of DHPG on Kir currents. (6) Intracellular IP3-sensitive and ryanodine-sensitive Ca2+ stores participated in the DHPG mediated inhibition of Kir currents. Intracellular dialysis of heparin, a competitive IP3 receptor antagonist, inhibition of Kir currents by DHPG was less than 20%. Caffeine (ryanodine receptor agonist) could induce a minor reduction of Kir currents. After ryanodine(100μM) was dialyzed intracellularly DHPG failed to induce a considerable reduction of Kir currents. We added BAPTA to confirm intracellular Ca2+ mediated the DHPG effect, and it blocked DHPG mediated Kir current inhibition. These data suggest that Ca2+-dependent PLC/IP3-ryanodine/PKC signaling pathway is involved in the DHPG effect. (7) Immunohistochemic and Western blot experiments showed that Kir4.1 mainly expressed in the endfeet of Muller cells was reduced in the COH model although a slight recovery was observed from 4th week to 6th week after operation. In contrast, GFAP expression in Muller cells was continuously enhanced accompanying with the elevated IOP. The total Kir4.1 proteins extracted from the COH retinas detected by Western blot were increased compared with the control group. (8) MPEP was Intravitreally injected three days before the operation. MPEP significantly reduced GFAP and inhibited the decrease of Kir4.1 expression in Muller cells as compared with those injected normal physiological solution (NS). (9) We further injected Intravitreally DHPG to test whether it may induce Muller cell gliosis in normal retinas. Two weeks after DHPG injection, strong GFAP expression was observed in Muller cells, while Kir4.1 expression was decreased as compared to NS-injected retinas. Western blot revealed that total Kir4.1 protein in DHPG-injected retinas was decreased, while total GFAP protein was remarkably increased compared to the NS-injected retinas. In conclusion, Kir currents of retinal Muller cells were reduced significantly in the rat COH model. Accompany with the downregulation of Kir current, the expression of GFAP increased, suggesting the gliosis of Muller cells. Activation of mGluR I downregulated the Kir currents, which was mediated by mGluR5, but not mGluR1 receptors. And Ca2+-dependent PI-PLC/IP3-ryanodine/PKC signaling pathway mediated the DHPG effect. An intravitreal injection of DHPG induced Muller cell gliosis, whereas an injection of MPEP eliminated COH-induced changes in Muller cells. These results suggest that mGluR5-mediated inhibition of Kir currents contributes to retinal Muller cell gliosis in the rat COH model.