The Role Of CB1 Receptors On Glutamatergic And Gamma-aminobutyric Acid Neurons In Cerebral Ischemia

Stroke is a major reason of death and disability in China,and cerebral ischemia is the most common type of stroke clinically.At present,the treatment methods of cerebral ischemia on the one hand,the use of drug thrombolysis or surgical thrombolysis to dredge the blocked blood vessels,but this is only applicable to the early stage of cerebral ischemia,the later thrombolysis will lead to ischemia and reperfusion injury;On the other hand,the use of neuroprotective agents,which not only helps to establish lateral branch circulation,but also can inhibit ischemia reperfusion injury and nerve cell death.At present,there are few truly effective neuroprotective agents for cerebral ischemia,and most of them are forced to terminate their use due to clinical failure or side effects.Many studies have found ECS with regulatory effects on cerebral ischemia,focusing mainly on CB1R,but the specific role of CB1R in the brain deficiency is not determined,some studies believe that the activation of CB1R plays an active protective role in the cerebral ischemia,and some studies have reached the contrary.This study focuses on the role of Glutamatergic neurons and GABAergic neurons CB1R in cerebral ischemia.First detecting brain CB1R expression after cerebral ischemia;then phenotypic analysis,including infraction volume,cerebral edema,nerve function damage and cell death,Finally explore the molecular mechanism of CB1R in cerebral ischemia based on phenotypic results.The results are as follows:（1）There was no statistical difference in CB1R expression in the brain on the ischemic side than on the non-ischemic side,but there was an upward trend.We respectively tested 2 h,4 h,6 h,12 h,24 h CB1R expression in hippocampus and cortex of the CTR mice after cerebral ischemia by Western blot.At each test point in cortex and hippocampus regions,ischemia side and non-ischemia side CB1R expression had no significant difference,the ischemic side had increasing trend compared with the non-ischemic side during 2-6 hours after cerebral ischemia.（2）In cerebral ischemia models,there was a significant increase in infarction volume of CNR1CMVKO and CNR1GlutKO mice,while there was no significant difference in the infarction volume of CNR1GABAKO and CTR mice.CB1R have no regulation on brain edema.The tissue level includes measurements of infarction volume and brain water content.In the statistics of infarction volume.The infarction volume of CNRlCMVKO and CNR1GlutKO mice was significantly higher than that of CTR mice,but the infarction volume of CNR1GABAKO mice was not significantly difference from that of CTR mice.CNR1CMVKO、CNR1GlutKO and CNR1GABAKO mice had no differences in the same brain regions on Cerebral edema,However,in different brain regions,the ischemic hemisphere has significantly higher water content than the non-ischemic hemisphere and cerebellum.（3）Nerural function damage increased significantly after the systemic CB1R knockout,and there was no statistical difference between CB1R knockout mice in Glutamatergic neurons and GABAergic neurons and CTR mice,but after the CB1R knockout of Glutamatergic neurons,nerural function damage showed increasing tendency.Nerural function damage assessment included Longa score and mNSS score.In Longa score,CNR1CMVKO mice was significantly higher than that of CTR mice,while CNR1GABAKO and CNR1GlutKO mice were not statistical difference compared with CTR mice.In mNSS score,CNR1CMVKO mice were significantly higher than CTR mice,and there was no statistical difference between CNR1GABAKO、CNR1GlutKO and CTR mice.According to mNSS score,1-6 is divided into mild injuries,7-12 is divided into moderate injuries,and 13-18 is divided into serious injuries.CNR1CMVKO mice were classified as severe injury,CNR1GlutKO mice were classified as moderate injury,and CNR1GABAKO and CTR mice were classified as mild injury.（4）CNR1CMVKO mice significantly increased cell damage in the hippocampus CA 2 region after cerebral ischemia,CNR1GlutKO mice significantly increased cell damage in the hippocampus CA 1 region after cerebral ischemia,CNR1GABAKO were not different from CTR mice in cell death.Glutamatergic and systemic CB1R have positive protective effects on cell damage in hippocampus CA 1 and CA 2 regions after cerebral ischemia respectively,while CB1R of GABAergic neurons currently does not show protective effects on cell damage after cerebral ischemia.On the ischemic side of hippocampus and cortex,nissl staining show that CNR1CMVKO,CNR1GlutKO and CTR have no significant difference,the same nissl bodies is reduced or disappeared.Compared with CTR control mice,the cell damage of CNR1GABAKO mice is slightly improved,only a small amount of nissl bodies are deformed,and the morphological character of neurons is relatively clear.In the statistics of the cell number of cortex,the CNR1CMVKO,CNR1GlutKO and CNR1GABAKO mice had no significant differences compared with the CTR mice.In the cell count statistics of hippocampus CA 1 area,the CNR1GlutKO mice were significantly lower than the CTR mice.In the CA 2 area,the CNR1CMVKO mice was significantly lower than that the CTR mice.There was no difference between all groups of mice in CA 3 and DG areas.（5）To further explore the ischemic mechanism of Glutamatergic neurons CB1R,based on Western blot results,blocked NMDA receptors can significantly reduce infarction volume and Longa score in CNR1GlutKO mice.MK 801 was injected at 4 h and 6 h after cerebral ischemia in CNR1GlutKO and CTR mice,the infarction volume and Longa scores were statistics after 24 h.For CNR1GlutKO mice,6 h drug group compared with solvent group had significantly reduction in infarct volume,4 h drug group compared with solvent no statistical difference in infarction volume;but in Longa score,4 h drug group had significantly reduction compared with solvent group,There was no statistical difference in longa score between the 6 h drug group and the solvent group.And there was no statistical difference in infarct volume and Longa score between others groups of mice.In conclusion,the phenotypic results show that CB1R is protective against tissue,behavioral and cell damage after ischemia,where CB1R plays a main role on Glutamatergic neurons,There was no significant difference between CNR1GABAKO and CTR mice.Combining the mechanism results,CNR1GlutKO mice blocking the NMDA receptor significantly reduced the infarction volume and Longa score,speculated that the activation of gluaminergic neuron CB1R may inhibit the release of excitatory neurotransmitter glutamate and weaken the excitatory amino acid toxicity to cerebral ischemia to the protection effect.Therefore,CB1R is a new target for the future development of drugs for cerebral ischemia.