Involvement And Mechanism Of M(u|")ller Cells In Retinal Ischemic Postconditioning

Objective: This study was aimed at the involvement of Müller cells, themajor glial cells in retina tissue, in the positive retinal ischemicpostconditioning (PostC) and its mechanism.Methods:1. Animals were divided into three groups: sham, ischemiaand ischemic PostC. The thickness of retina, apoptosis within retina anddistribution of Müller cells in retina was assayed by light microscopy,terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick endlabeling assay (TUNEL) and immunohistochemistry respectively.2. Toestablish the model of retinal ischemia and PostC in vitro, we set three groups:(1) taking normal Müller cells as control;(2) Müller cells of hypoxia;(3)Müller cells of hypoxic PostC. Supernatant of cultured Müller cells wascollected and proteins were extracted at each time point. The level of BDNF protein was determined by enzyme linked immunosorbent assay (ELISA) andthe expression of glutamine synthetase (GS) and glial fibrillary acidic protein(GFAP) was examined by western blotting analysis (WB).3. Up-regulatedand down-regulated the expression of GFAP by GFAP over-expression andGFAP-siRNA respectively.4. Müller cells with up-regulated anddown-regulated GFAP were treated by hypoxia, PostC, post-hypoxia andnormal condition. Then we analyzed the viability of Müller cells, level ofsecreted BDNF and GS function respectively.Results:1. Thickness of layers of retina was examined which showed adecrease after PostC than that of hypoxia, especially the ganglion cells layer(GCL) and inner nuclear layer (INL) with a remarkably decrease. It wasshowed by TUNEL assay that apoptosis within GCL and INL of PostC wasattenuated than that of ischemia. Müller cells of PostC and ischemia werefound stretched into GCL and INL more than that in control.2. Müller cellswere treated by hypoxia and PostC and were observed at0h and12h. Theboundary of Müller cells of hypoxia became blurred at12h and the cells werearranged loosely with an increase of granules in the cytoplasm. However,Müller cells of PostC were better-organized with good structure at12h aftertreated. In addition, compared with hypoxia group, the number of apoptoticMüller cells of PostC was significantly fewer with a higher viability.3. There was a sharply decrease of BDNF in hypoxia and PostC groups than that incontrol group, however the decreased level of BDNF in Müller cells ofhypoxia was up-regulated by PostC and the peak prolonged from0.5h to6hin PostC group. Meanwhile GS was up-regulated in PostC group than that inhypoxia group.4. Müller cells with up-regulated GFAP were kept goodcondition and the viability was even higher than that of normal Müller cells.The protective ability was elevated which was evidenced by increasedsecreted BDNF level and GS function while was opposite to the Müller cellswith down-regulated GFAP.Conclusions:1．Müller cells were activated positively in PostC and inwhich process their protective effects were augmented.2．PostC kept Müllercells good conditions which enable Müller cells exert their protective effectsby recovering the secretion of BDNF and enhancing the function of GS.3．GFAP takes a key role in PostC and the viability and protective function ofMüller cells could be elevated by increased expression of GFAP whileopposite when down-regulated GFAP in Müller cells which illustrated thatPostC induced GFAP up-regulation was a key role in protective PostC.