The Role Of Late Endosome Rab7 In The Formation Of Glial Scar Induced By Ischemic Stroke And Its Mechanisms

Aim:Rab7,a member of the small GTPase family,is mainly found in late endosomes and organelles such as lysosomes.It has been reported that Rab7 may contribute to ischemic-reperfusion(I/R)injury of the brain,but the relevant mechanism has not yet been established.Therefore,the purpose of this study is to investigate the effect of Rab7 on glial scar formation induced by cerebral ischemia and reperfusion in vivo and in vitro and its mechanisms.Methods:The model of transient middle cerebral artery occlusion(tMCAO)was established in vivo.The Sprague-Dawley(SD)male rats were reperfused for 2 h,4 h,6 h,1 d,7 d,14 d and 28 d following ischemia for 90 min.Western blotting and immunohistochemistry was used to examine the expression of Rab7 and glial scar-related proteins GFAP,phosphacan and neurocan.Rats were intraperitoneally injected with Rab7 receptor antagonist CID1067700(250?g/kg)once a week for 4 weeks(28 days)after ischemia-reperfusion.The rats were subjected to behavioral tests once a week.After reperfusion for 7 days,glial scar-related proteins GFAP and phosphacan were detected with the treatment of CID 1067700 by Western blotting.After reperfusion for 28 days,the volumes of brain atrophy were measured.Primary astrocyte cultures were performed in vitro and oxygen and glucose deprivation(OGD)-reoxygenation(OGD/Re)model was established.CID 1067700 was given 30 minutes prior to OGD.Up on reoxygenation,CID 1067700 was given again.The cells were treated with OGD6h-Re12h and OGD6h-Re24h,respectively.The effect of OGD/Re-induced astrocyte injury with the treatment of CID 1067700 was detected by the lactate dehydrogenase(LDH)assay,which checked the protection of CID 1067700 after OGD/Re of astrocytes.We also used Western blotting and immunofluorescence to detect the protein levels of glial scar marker GFAP,phosphacan and neurocan,and checked the colocalization of Rab7-Lamp1,Rab7-cathepsin B and Lampl-cathepsin B with the treatment of CID 1067700.At the same time,the effect of CID 1067700 on the permeability of lysosomal membrane in astrocytes after OGD/Re was detected by AO staining.Results:In vivo,the expression of Rab7 in GFAP+cells in the peri-infarct region of cerebral cortex reached the highest level at 7 days after I/R,and at the same time the expression levels of glial scar-related proteins GFAP,phosphacan and neurocan also reached the highest level and decreased at 14 days of reperfusion.And CID1067700 significantly reduced the expression of glial scar-related proteins GFAP,phosphacan and neurocan in the peri-infarct region after 7 days of reperfusion,and significantly reduced the volumes of brain atrophy after 28 days of reperfusion and improved neurobehavioral symptoms.Consistent with the data in vivo,the in vitro data showed that Rab7 had the same changes together with glial scar-related proteins GFAP and phosphacan and peaked at OGD6h-Re24h.Similarly,CID1067700 could reduce the expression of GFAP,phosphacan and neurocan and decrease the OGD/Re-induced LDH leakage rate of astrocytes.AO staining showed that CID1067700 could stable lysosomal membrane and further experimental studies showed that CID1067700 decreased the expression of Lampl,cathepsin B and inhibited the release of cathepsin B from lysosomes into the cytoplasm.Conclusion:CID1067700 can effectively reduce the volumes of brain atrophy after cerebral ischemia and reperfusion and improve neurobehavioral symptoms.CID1067700 inhibits the formation of astrogliosis and glial scar induced by ischemic stroke and protects astrocytes after ischemia-reperfusion brain injury,associating with inhibition of the release of cathepsin B from lysosomes into the cytoplasm and played a key role in stabilizing of lysosomes membrane.