| Objectives 1.Isolation of microglia after spinal cord injury(SCI)in rats;2.To investigate the effect of spinal cord injury on the expression of programmed cell death-1(PD-1)in microglia;3.To investigate the effects of dexmedetomidine(DEX)on microglial inflammatory response and polarization after SCI;4.To investigate the effect of DEX on PD-1 expression and 5' adenosine monophosphate-activated protein kinase(AMPK)signaling pathway transduction after SCI.Methods 1.Adoptive transfer of microglia and macrophages :GFP-transgenic 10-week old Sprague-Dawley rats,normal rat splenocytes and spinal cord cells were stained with 2 ?g/ml PE anti-granulocytes(Gr)antibody and APC anti-CD11 b antibody.Gr-CD11+bcells(macrophages or microglia)were sorted using flow cytometry.GFP+Gr-CD11 b + cells were resuspended in artificial cerebrospinal fluid.SD rats subjected to adoptive transfer experiments were divided into laminar decompression(Sham)group and SCI group;Before induction of SCI,injections were conducted into T10.Each injection center was in the intermediate region of the dorsal horn and close to the lateral funiculus.After injection,clean the incision with penicillin-streptomycin solution and suture two layers.the T10 spinal cord was collected and pressed through a 45-?m cell strainer to make a single cell suspension;2.According to different treatment factors,the rats were randomly divided into Sham group,SCI group,Sham+DEX group,and SCI+DEX group.The modified Allen's method was used to construct traumatic spinal cord injury model.3.SCI was then induced and 6 h,24 h and 72 h after SCI,the T10 spinal cord was collected in Sham group,SCI group,Sham+DEX group and SCI+DEX group.Pressing through a 45-?m cell strainer to make a single cell suspension.Microglia and macrophages expressing Gr-CD11 b + were isolated by flow cytometry(FCM).Microglial expressing GFP+ Gr-CD45+ and macrophages expressing GFP+ GrCD45-were screened from SCI group with adoptive transfer of microglia / macrophages at 6 h and 72 h after operation.4.The obtained microglia by western blot to detect PD-1,programmed cell death ligand,inducible nitric synthase and arginase-1 expression in the Sham group,SCI group and SCI+DEX group at 24 h and 72 h after spinal cord injury;simultaneously the expression of AMPK signal channel proteins(p ACC ?,ACC,p AMPK ?,AMPK ?)in Sham group,SCI group and SCI+DEX group were detected by Western blot at 72 h after operation.5.The expression of PD-1,PD-L1 and PD-L2 in microglia of Sham group and SCI group at 6 h,24 h and 72 h after spinal cord injury were detected by Quantitative Reverse Transcription-PCR.Pro-inflammatory cytokines,chemokines and antiinflammatory cytokines(IL-1?,IL-18,TNF-?,IL-6,CCL5,CCL20,CXCL1,CXCL10,TGF-?,IL-10)m RNA in microglia of Sham group,SCI group and SCI+DEX group were detected at 24 h and 72 h after spinal cord injury.Results 1.CD45 is a reliable marker for differentiating microglia from macrophages in the acute phase of spinal cord injury;Spinal microglia and macrophages were successfully obtained;2.The expression of PD-1,PD-L1 and PD-L2 was up-regulated by microglia in acute stage of traumatic spinal cord injury in a time-dependent manner(P<0.05);DEX enhanced the expression of PD-1 in microglia in acute stage of traumatic spinal cord injury,but did not change the expression of PD-L1 and PD-L2(P>0.05);And DEX could not induce PD-1 expression in microglia in Sham-operated rats(P>0.05).3.After SCI,DEX inhibited the expression of pro-inflammatory factors IL-1?,IL-18,TNF-?,CCL5 and CCL20 in microglia(P<0.05),but promoted the expression of anti-inflammatory factors TGF-? and IL-10(P<0.05);and driving microglia to M2 type polarization(P<0.05);4.SCI inhibited AMPK signaling in microglia(P<0.05),and DEX activated AMPK signaling pathway(P<0.05);Conclusions DEX promotes the transmission of AMPK signaling pathway in activated microglia by up-regulating PD-1 expression,which drives microglia to M2 polarization and attenuates SCI microglia-mediated neuroinflammatory. |
PDF Full Text Request