Effect Of Activating The SHH Pathway On Expression Synaptic Regeneration In Rats With Acute Cerebral Ischemia

Background and ObjectiveAs the social and lifestyle changes, the morbidity of cerebrovascular disease areon the rise every year, and tend to be younger, not only lead to a heavy burden to thecommunity and families, but also the psychological burden to patients. Ischemic isthe most common type of the cerebrovascular disease, which features in its highmorbidity, mortality and teratogenic rate. With regard to the countermeasure, earlydiagnosis and aggressive treatment, rehabilitation exercises to strengthen and preventrecurrence are essential. With the continuous research of neural prosthetics, nerveregeneration, especially synaptic regeneration,is of great significance to the ischemiccerebrovascular disease. Recent studies indicate, Shh signal transduction pathwayand ischemic cerebrovascular disease most likely has significant correlations on thesynaptic regeneration performance. Shh signal path includes three principal parts-asignal peptide Shh, the film factor Ptch、Smo and downstream transcription factor Gli,the cascade acts or the mutations of factors can activate this pathway, and effects on downstream target genes through the Gli finally. Activation of Shh signalingpathway has a protective effect on cerebral ischemia, can promote the regenerationand reconstruction of synapses and encourage the restoration of neurological function.Thence, the present study take method of Longa to make permanent ischemia modelin rats, to observe the expression of Gli1-downstream molecular of Sonic Hedgehogsignaling pathway and SYN, GAP-43--synaptic associated synthesis proteins inischemic penumbra at different time points after apply with the specific activatorPurmorphamine,thus, to further explore the effects and mechanism of Shh signalingpathway influenced on the synaptic regeneration of the ischemic cortex.Materials and MethodsPermanent middle cerebral aretery occlusion models were produced by sutureemboli technique in rats.76healthy male SD rats (weighing about280g~330g) weredivided randomly into three groups: sham operation group, model group and drugtreatment group.The drug treatment group was given immediately after PMCAO by0.69mg/kg purmorphamine solution by intraperitoneal injection, while the same doseDMF solution was given in sham operation group and model group in the same way.Model group and drug group took out4rats which were anesthetized and quicklytook out the tissue of the ischemic brain cortex and stored in liquid nitrogen for theinvestigation of the expression of Gli1mRNA at Different time points. The remaining4rats was perfused and fixated through heart,then the tissue of the ischemic braincortex were removed quickly to paraffin section.The expression of Gli1mRNA inischemic penumbra of rat brain cortex was investigated by real-time PCR,and theexpression of SYN and GAP-43was investigated by immunohistochemistry.Results(1) The determination results of SYN in ischemic cortex area From the resultsof Immunohistochemical staining,we found tan particles in the cytoplasm,Which isthe positive expression of SYN. the expression of SYN in model group began todecrease in6h,72h most significant, began to rise after7d, comparing with the shamgroup was significantly lower at each time point, and the difference was statisticallysignificant(P<0.05). The expression of SYN in drug treatment group was significantlyhigher compared with model group after ischemia6h,24h,72h,7d, the difference was statistically significant(P<0.05).(2)The determination results of GAP-43in the ischemic cortex From the resultsof Immunohistochemical staining, we found granular deposition in ischemic cortex,Which is the positive expression of GAP-43.and we could see trace amounts ofGAP-43in sham operation group. the expression of GAP-43in model group began toincrease in6h,72h most significant, began to decline after7d, comparing with thesham group was significantly lower at each time point, and the difference wasstatistically significant(P<0.05). The expression of GAP-43in drug treatment groupwas significantly higher compared with model group after ischemia6h,24h,72h,7d,the difference was statistically significant(P<0.05).(3)The determination results of SYN mRNA、GAP-43mRNA、Gli1mRNA inthe ischemic cortex The expression of SYN mRNA in model group began to declineat6h,72h is the most significant,and began to rise at7d. The expression of SYNmRNA was decreased compared with the sham operation group at different timepoints, and the difference was statistically significant(P<0.05)；The expression ofSYNmRNA in drug group was significantly increased compared with the modelgroup at6h,24h,72h,7d after ischemia,and the difference was statisticallysignificant(P<0.05). The expression of GAP-43mRNA in model group began toincrease at6h,72h is the most significant,and began to decline at7d. The expressionof GAP-43mRNA was increased compared with the sham operation group at differenttime points, and the difference was statistically significant(P<0.05)；The expression ofGAP-43mRNA in drug group was significantly increased compared with the modelgroup at6h,24h,72h,7d after ischemia,and the difference was statisticallysignificant(P<0.05). The expression of Gli1mRNA in drug group were increasedcompared with the sham group and model group. the difference was statisticallysignificant(P<0.05). The expression of Gli1mRNA in model group began to increaseat6h, peaked at24h, After a decline, but at a higher level.The expression ofGli1mRNA in drug group was significantly increased compared with the model group,and the difference was statistically significant(P<0.05).Conclusions1.The expression of the signaling pathway downstream effectors Gli1can be activated by purmorphamine,an agonists of Shh signaling pathway.2. Shh signaling pathway can upregulate the expression of GAP-43, SYN inischemic brain tissue.3.Shh signaling pathway may participate focal cerebral ischemia nerve regulationmechanism, which can promote synapse regeneration.