| Retinal degeneration (RD) is a group of severe diseases leading to blindness, which lack effective therapeutic measures. Among the RD, Retinitis pigmentosa (RP) is the most common retinal inherited disease, which featured in a progressing apoptosis of photoreceptors and disfunction of retinal pigmented epitheliums (RPE). The pathological mechanism and functional changes of neurons in the progress of RP is still unknown.Müller cells are main glial cells in the mammalian retina. They connect almost all the retinal neurons by their specialized morphology, which is the foundation of normal retinal function. Müller cells are involved in many retinal physiological activities including glycometabolism, blood regulation, neurotransmitter cycling, homeostasis and control of neuronal excitability.Under pathological conditions, Müller cells can be activated to perform a series of changes in morphology, amount and protein expression. It was reported that Müller cells could proliferate dramatically, becoming hypertrophy and fibrosis in retina detachment, PVR and PDR. Besides, the mature Müller cells may re-enter the cell cycling, and differentiate into retinal stem cells expressing PAX6, Nestin in some acute retinal injuries.Meanwhile, Müller cells showed obvious changes of the electrophysiological properties when being activated. The characteristic electrophysiological properties of Müller cells distinguished to other retinal cells are large amounts of potassium channels in the membrane, which is related to Müller cells'function of buffering K+. In retinal detachment and acute retinal injuries, K+ channels currents of Müller cells decreased significantly.The mechanism of Müller cells activation is still not clear. Extracellular signal - regulated kinase (ERK) signal pathway is the core in regulation of cells'growth, development and mitosis. Many articles reported that purinergic receptor P2Y1-ERK signal pathway was closely related to the proliferation and differentiation of astrocytes. So the P2Y1-ERK pathway is probably involved in the activation of Müller cells . As a chronic inflammatory disease, if RP can also activate Müller cells , and how Müller cells change in RP is still unknown.Our previous studies showed that Müller cells glial seal formed in the subretinal space in the progress of RCS rats'retinal degenetation, which is considered to block the connection between neuroretinal epitheliums and RPE.。Quantitive test of RCS retinal proteins showed, CHX10, a marker of retinal stem cells , increased significantly during the progress of retinal degeneration. Our patch clamp recording on RCS rats retinal neurons showed that a dramatically changes of ionic currents occurred in the ganglion cells and the bipolar cells. So as a bridge among retinal neurons, Müller cells'ionic currents may also changed in progress of RCS rats retinal degeneration.Based on above, we hypothesis that Müller cells also can be activated in the progress of RP to proliferate and re-differentiate, and display changes of ionic currents cross the membrane. P2Y1-ERK signal pathway may mediate the activation of Müller cells .Our main research contents and results are showed as following:1,Activated state of Müller cells in RCS rats at different development stage.Methods:frozen section, immunohistochemistry . Results:1)Müller cells of RCS rats developed more rapidly than control, and became hypertrophy , disorder. And a local fibril layer former in the subretinal space. 2)At P60d,90d,120d,amount of Müller cells in RCS rats retina was significantly higher than control(P<0.05 or P<0.01) which implied that Müller cells of RCS rats proliferated dramatically. 3)At P30d,60d,120d, the expression of GFAP and ERK in RCS rats retina was significantly higher than control ,which implied Müller cells were activated , and ERK might be involved in it. 4)Müller cells of RCS retina could express CHX10, a maker of retinal stem cells, which proved that differentiation of Müller cells happened in the progress of retinal degeneration.2,Properties of K+channels currents in Müller cells from RCS retina.Methods: patch clamp recording of the acute isolated Müller cells. Results:1)With theprogress of RD, RMP of Müller cells from RCS rats retina depolarized dramatically, and IR, Cm increased significantly higher than control (P<0.05), implying that Müller cells became hypertrophy in responds to RD.2)inward and outward currents were recorded when giving hyper- and depolarized voltage steps, which could be blocked by Ba2+ and TEA, implying a predominant distribution of K+ channels on Müller cells . 3)at the early stage of RCS rats retinal degeneration, the inward and outward currents increased, and were significantly higher than control, which is considered to eliminate the extracelluar K+ produced in the RD, to be helpful to neuron survival. At late stage, the inward and outward currents decreased, and were significantly lower than control, which worsened the retinal impairments. 4)At P30d the K+ currents density of Müller cells in RCS rats was significantly higher (P<0.05) than control; At P90d, the K+ currents density of Müller cells in RCS rats was significantly lower (P<0.05). The decease of K+ current density implied that the number of K+ channels might deceased during retinal degeneration. 5)The I-V curves of RCS rats'Müller cells showed, at P30d, with the increase of voltage impulse, the current amplitude of RCS rats Müller cells increased more rapidly than control, and both inward and outward current amplitudes were higher. At P90d, it changed adversely.3,Effect of RCS rats retinal mixed cells on the normal cultured Müller cells, and P2Y1-ERK signal pathway's involvement in it.Methods: cocluture of normal cultured Müller cells and RCS rats retinal mixed cells, immunohistochemistry, western-blot,RT-PCR. Results: 1)At 3d,7d after coculture, the normal Müller cells proliferated dramatically, and the expression of GFAP and ERK was significantly higher than control. Part of normal Müller cells expressed CHX10, which implying that normal Müller cells were activated by the RCS rats retinal mixed cells. 2)At 3d,7d after coculture, the proliferation rate of normal Müller cells increased dramatically(P<0.05), which could be completely blocked by PD98059, the blocker of ERK pathway. It is further proved that ERK pathway might mediate the activation o Müller cells . 3)The expression of GFAP and ERK was significantly decreased after blocking the ERK pathway by PD98059. RT-PCR results showed , at 3d,7d after coculture, the quantity of normal Müller cells'P2Y1 mRNA increased , and significantly higher than control and PD98059 group.Based on the results, we concluded:1,Müller cells could be activated during the progress of RCS rats'retinal degeneration. After being activated, Müller cells begin to proliferate, becoming hypertropy, and formed local glial seal in the subretinal space. The expression of GFAP increased with progress of retinal degeneration. 2,After being activated, Müller cells might differentiate into retinal stem cells in responds to activation by expressing CHX10. 3,At middle and late stage of retinal degeneration, RMP of RCS rats'Müller cells became more depolarized, the IR and Cm of RCS rats'Müller cells increased dramatically, which provided electrophysiological evidence to Müller cells activation. 4,At the early stage of RCS rats retinal degeneration, Müller cells enhanced their ability of buffering K+ by increasing the trans-membrane K+ currents. At late stage, trans-membrane K+ currents of RCS rats'Müller cells decreased significantly, implying a disorder of Müller cells function, which might worsen the retinal impairments. 5,Coincident with the change of K+ currents, the decease of K+ current density implied that the number of K+ channels might deceased during retinal degeneration. 6,In RCS rats, the expression of ERK in Müller cells increased with the progress of retinal degeneration, and quantity of P2Y1 mRNA increased under the stimulation of RCS rats'retinal mixed cells. So the P2Y1-ERK signal pathway might mediate the activation of Müller cells. |
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