| Part Ⅰ:The expression of PSD-95 in brain following ICH in ratsObjectiveTo investigate the changes of expression of postsynaptic density protein-95(PSD-95)at different time points in the brain tissue surrounding the hematoma after intracerebral hemorrhage(ICH)in rats.MethodsForty-eight healthy adult male Sprague-Dawley(SD)rats were randomly divided into eight groups,including the Normal group,the Sham group,and the 6 different time-point groups(6h,12h,24h,48h,72h,1w),6 rats in each group.A model of cerebral hemorrhage of right basal ganglia was established by collagenase method in rats.The rats were sacrificed at corresponding time points,and brain tissue samples surrounding the hematoma were excised for examination.The protein level of PSD-95 was detected by dual immunofluorescence and Western blot analysis,and the mRNA level of PSD95 in the brain tissue around the hematoma was detected by RT-PCR.result:The level of PSD-95 protein in the neurons surrounding the hematoma decreased significantly at 6 hours,reached the lowest point at 12 hours,gradually recovered after 24 hours,and then decreased again at 72 hours after ICH.2.The mRNA level of PSD-95 in the brain tissue around the hematoma was significantly reduced from 6h after ICH modeling,and recovered to a level comparable to that of the Sham group after 48h.Conclusion:1.After ICH,the expression of PSD-95 in the brain tissue around the hematoma showed a trend of first decrease and then recovery.2.The decrease of PSD-95 protein level may be caused by the reduction of PSD-95 transcription induced by ICH.Part Ⅱ:Protective effect of blocking PSD-95 receptor interaction on secondary brain injury after intracerebral hemorrhage in ratsObjectiveTo explore the protective effect of rat tail vein injection of PSD-95 inhibitor Tat-NR2B9c on secondary brain injury after cerebral hemorrhage(ICH)in rats.Methods120 rats(135 rats were used,but only 120 rats survived after the surgery)were randomly assigned to into 4 groups of 30 rats each.include sham group,cerebral hemorrhage group(ICH),cerebral hemorrhage+solvent group(ICH+Vehicle)and cerebral hemorrhage+ PSD-95 inhibitor group(ICH+Tat-NR2B9c)A model of collagenase and autologous blood cerebral hemorrhage was established.0.5 hours after ICH modeling,rats in the ICH+Tat-NR2B9c group were injected with Tat-NR2B9c 2.6 mg/kg in the tail vein,and rats in the ICH+vehicle group were injected with the same dose of normal saline.At 24 hours after ICH,brain tissue was isolated from 6 rats in each group,and proteins were extracted for Western blot analysis,and paraffin sections(for TUNEL staining and FJB staining)were made.72 hours after modeling in each group,they were used for neurological deficit score and assessment of cerebral edema,and all serum was collected for enzyme-linked immunosorbent assay(ELISA)to assess the inflammatory response.The remaining 18 rats in each group were subjected to a Morris water maze experiment.Results:1.Compared with the Sham group,the number of TUNEL and FJB positive cells in the ICH group increased significantly(P<0.01),and the level of activated caspase-3 increased significantly(P<0.01).After Tat-NR2B9c intervention,compared with the ICH+Vehicle group,the number of TUNEL and FJB positive cells in the ICH+Tat-NR2B9c group was significantly reduced(P<0.01),and the level of Active caspase-3 was also significantly decreased(P<0.01).2.Compared with the Sham group,the albumin content in the brain tissue of the ICH group increased significantly(P<0.01).After the Tat-NR2B9c intervention,compared with the ICH+Vehicle group,the albumin content in the brain tissue of the ICH+Tat-NR2B9c group decreased significantly(P<0.01).3.Compared with the Sham group,the water content of brain tissue in the ICH group increased significantly(P<0.001).After Tat-NR2B9c intervention,compared with the ICH+Vehicle group,the brain water content in the ICH+Tat-NR2B9c group decreased significantly(P<0.01).4.Compared with Sham group,the inflammatory factors IL-1β and IL-17 in brain tissue of ICH group increased significantly(P<0.01).After Tat-NR2B9c intervention,compared with the ICH+Vehicle group,IL-1β and IL-17 decreased significantly(P<0.05).5.Compared with the Sham group,the neurological deficit score of the ICH group significantly increased(P<0.01).After Tat-NR2B9c intervention,compared with the ICH+Vehicle group,the neurological deficit score of rats significantly decreased(P<0.05).6.The Morris water maze experiment showed that compared with the Sham group,the escape latency and swimming distance of the ICH group were significantly increased(P<0.01).After Tat-NR2B9c intervention,compared with the ICH+Vehicle group,the Tat-NR2B9c group The escape latency and swimming distance were significantly reduced(P<0.01).Conclusion:After ICH,the use of Tat-NR2B9c via tail vein injection to inhibit the function of PSD-95 can effectively improve secondary brain injury such as ICH neuron degradation,apoptosis,cerebral edema,blood-brain barrier disruption and inflammatory response.Part Ⅲ:The mechanism of blocking PSD-95 receptor interaction on neuroprotection after cerebral hemorrhage in ratsObjective:To explore the mechanism of PSD-95 inhibitor Tat-NR2B9C blocking PSD-95 receptor interaction on neuroprotection after cerebral hemorrhage in ratsMethods:144 rats were randomly divided into 6 groups:sham operation group(sham),cerebral hemorrhage group(ICH),cerebral hemorrhage+solvent group(ICH+Vehicle),cerebral hemorrhage+PSD-95 inhibitor group(ICH+Tat-NR2B9C)),intracerebral hemorrhage+overexpression PSD-95 group(ICH+PSD-95 plasmid),intracerebral hemorrhage+PSD-95 inhibitor+overexpression PSD-95 group(ICH+Tat-NR2B9C+PSD-95 plasmid),n = 24.Establish a model of cerebral hemorrhage in vivo with collagenase.And primary rat neurons to establish an in vitro model of ICH.0.5 hours after the ICH model was established,rats in the ICH+Tat-NR2B9C group were injected with Tat-NR2B9C2.6 mg/kg through the tail vein,and rats in the ICH+vehicle group were injected with the same dose of normal saline.In the ICH+PSD-95 overexpression group,the PSD-95 plasmid was injected into the ventricle 48h before modeling.Rats in the ICH+Tat-NR2B9C+PSD-95plasmid group were injected with PSD-95 plasmid into the ventricle 48h before modeling,and Tat-NR2B9C2.6 mg/kg was injected into the tail vein 0.5 hours after modeling.24h after ICH,the brain tissues of 6 rats in each group were separated and protein was extracted(co-immunoprecipitation analysis).Six rats prepared quick frozen sections of brain tissue for in situ gelatin zymography to detect the activity of MMP2/9.The remaining 12 rats were subjected to a water maze test.Results:1.Both in vivo and in vitro experiments indicated that compared with the Sham group,the PSD95-NR2B-nNOS complex was significantly increased in the ICH group(P<0.001),and the PSD95-neurexin-l-nueroligin-1 complex was significantly reduced(P<0.01).).After intervention with Tat-NR2B9C,compared with the ICH+Vehicle group,the PSD95-NR2B-nNOS complex was significantly reduced in the ICH+Tat-NR2B9C group,and the PSD95-neurexin-l-nueroligin-1 complex was significantly increased.Double immunofluorescence staining of neurons in vitro also showed a consistent trend.2.Compared with the Sham group,the MMP2/9 activity in the brain tissue of the ICH group increased significantly after 24 hours(P<0.01).After Tat-NR2B9C intervention,compared with ICH+Vehicle group,Tat-NR2B9C group can significantly inhibit the activity of MMP2/9(P<0.05).3.Compared with the ICH group,the PSD-95 level in the Tat-NR2B9C intervention group did not change significantly,and the PSD-95 level in the PSD-95 overexpression intervention group and the PSD-95 overexpression+Tat-NR2B9C intervention group was significantly increased(p<0.001).Compared with the ICH group,the PSD95-NR2B-nNOS complex production in the Tat-NR2B9c intervention group was significantly reduced(p<0.001,p<0.01),and the PSD95-neurexin-l-nueuroligin-1 complex production was significantly increased(p<0.001,p<0.01).The generation of PSD95-NR2B-nNOS complex in the PSD-95 overexpression intervention group was significantly increased(p<0.001),and the generation of PSD95-neurexin-l-nueuroligin-1 complex was higher than that in the ICH group(p<0.05),but compared with Tat-NR2B9C The intervention group decreased.PSD-95 overexpression+Tat-NR2B9C intervention group SD95-NR2B-nNOS complex production decreased(p<0.01,p<0.05),PSD95-neurexin-1-nueuroligin-1 complex production was significantly increased(p<0.001).Compared with the ICH group,apoptotic cells and FJB positive cells were significantly reduced in the Tat-NR2B9C intervention group(P<0.01).The PSD-95 overexpression intervention group decreased apoptotic cells and FJB positive cells around the hematoma(P<0.05),but there was no significant difference in FJB positive cells in the cortex.PSD-95 overexpression+Tat-NR2B9C intervention group apoptotic cells and FJB positive cells were significantly reduced(P<0.001,P<0.01).4.Compared with the Sham group,the escape latency of the ICH group was significantly shorter(P<0.0001).Compared with the ICH+Vehicle group,the escape latency was significantly shorter in the Tat-NR2B9c intervention group(P<0.01),there was no significant difference in the PSD-95 overexpression intervention group,and the PSD-95 overexpression+Tat-NR2B9C intervention group was significantly shorter in the escape latency(P<0.0001).n=12.Conclusion:The PSD-95 inhibitor Tat-NR2B9c inhibits the formation of PSD95-NR2B-nNOS complex induced by ICH,inhibits the activity of MMP2/9,promotes the formation of neurexin-1-nueuroligin-1-PSD95,and finally improves secondary brain injury. |
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