The Mechanism Of Action Of Miro1 On Intracerebral Hemorrhage-induced Secondary Brain Injury In Rats

Part Ⅰ: Time course of the protein levels of Miro1 after ICH in vivoObjective To research the expression level of Miro1(mitochondrial Rho GTPase 1)protein in SBI(secondary brain injury)after Intracerebral hemorrhage(ICH,Intracerebral hemorrhage)in rats.Methods 1.Build ICH model in vivo:The autogenous heart blood was slowly injected into the basal ganglia of the right rat brain by microinjector.2.Group of Experiments:6 rats were randomly selected into the Sham group as the control group,and the 42 rats that survived after ICH were randomly assigned to the groups at specific time points after ICH :3h,6h,12 h,24h,48 h,72h and 7d(6 each).The rats were euthanized at the corresponding time point and brain tissue was retained.3.Western blot(WB)and Immunofluorescence(IF)were used to assess the expression levels of Miro1 in each group of brain tissue samples at different time points.Results 1.Western blot showed that Miro1 expression began to increase significantly at 24 h after ICH compared with Sham group,and reached the peak 48 h after ICH(P < 0.01).2.Immunofluorescence analysis showed that the Miro1 expression level in brain neurons at 48 h after ICH was significantly higher than that in Sham group.Conclusions ICH in rats had an effect on the Miro1 protein which involved in the transport and distribution of mitochondria in neurons of brain tissues,and the expression of Miro1 protein increased in brain tissues around intracranial hematoma.Part Ⅱ: Effect of Miro1 protein expression regulated by gene technologyObjective Study on the influence of MIRO1 protein expression level on the SBI of rats after ICH by using related gene reagents(Recombinant Human His6 Miro1,Miro1 Si RNA).Methods 1.Group of Experiments: 18 rats were randomly selected and assigned to Sham group for control treatment.90 rats were randomly selected and divided into ICH group,ICH + Vehicle group,ICH + rh Miro1(Recombinant Human His6 Miro1)group,ICH + Negative Control si RNA(Si-NC)group and ICH + Miro1 Si RNA(Si-Miro1)group.Each group of 18 rats was treated with corresponding operations.If the rats died in the process,the rats were randomly selected for supplementation.2.Among the 18 rats in each group,6 rats were randomly selected for euthanasia and brain tissue were collected for Western blot,FJB staining and TUNEL staining.3.6 of the remaining 12 rats in each group were randomly selected for assess brain edema.4.The last 6 rats were used to Morris Water Maze,Neurobehavioral Evaluation and Rotarod Test.Results 1.Western blot indicated that 48 h after ICH,the Miro1 levels in ICH group was significantly higher than Sham group(P<0.01).The Miro1 levels in ICH+rh Miro1 group was significantly increase than ICH + Vehicle group(P<0.05).The Miro1 levels in ICH+ SiMiro1 group was significantly lower than Si-NC group(P<0.05).There were no significant changes in ICH + Vector group,and ICH + Si-NC group when compared with the ICH group.2.In FJB staining and TUNEL staining of brain tissue sections,the positive rate of apoptosis and necrosis of ICH+rh Miro1 group was significantly decreased(P<0.05)and that in ICH+ si-miro1 group was remarkably increased(P<0.05).3.Brain edema appeared after ICH in rats,brain edema alleviated in ICH + rh Miro1 group and aggravated in ICH + Si-Miro 1 group.4.After ICH,the neurobehavioral injury of rats was appeared;the neurobehavioral injury was alleviated in ICH + rh Miro1 group and increased in ICH + Si-Miro 1 group.Conclusions Overexpression of Miro1 can reduce the neuronal apoptosis and necrosis,cerebral edema and improve a series of neurobehavioral injuries induced by ICH of SBI in rats.Part Ⅲ: Studying the roles of Miro1 and its possible mechanisms in SBI after ICHObjective Oxy Hb was used to simulate the ICH model in vitro to further explore the effect of Miro1 on neurons and its potential mechanism in SBI after ICH.Methods 1.The primary neurons were cultured and divided into control group and Oxy Hb group at 6h,12 h,24h,48 h and 72 h after stimulation.Protein was extracted and the level of Miro1 in neurons was assessed by Western blot.2.Primary cultured neurons divided into 6 groups: Control group,Oxy Hb group,Oxy Hb + vrctor group,Oxy Hb + Overexpression Miro1(OE-Miro1)group,Oxy Hb + Negative Control si RNA(Si-NC)group and ICH + Miro1 Si RNA(Si-Miro1)group.These cultured neurons were analyzed by Western blot,Immunofluorescence,JC-1 staining,LiveDead Cell Staining and Annexin V and PI staining.Results 1.In vitro,Western blot indicated that the Mior1 levels was remarkably higher at 24 h after Oxy Hb treated(P < 0.05).2.The Immunofluorescence was suggested that the Miro1 and mitochondria were distributed in the soma and axons of neurons in vitro.The Kinesin(Kif5A+5B+5C)was mainly located at soma and distal axonal and the Dynein was clear distribution at soma and proximal axonal.3.The flow cytometry analysis was shown that overexpression of Miro1 significantly increased the percentage of living cells and decreased the rate of dead cells.4.Live-Dead Cell Staining suggested that overexpression of Miro1 could significantly increase the percentage of living cells.5.JC-1 staining was suggested that overexpression of Miro1 improved the mitochondrial membrane potential level of mitochondrial and alleviated the damage of neuronal.Conclusions The Miro1 protein assists the distribution and transportation of mitochondria after ICH.The overexpressed Miro1 assists transports the repaired mitochondria to the distal axon with the help of Kinesin.At the same time,with the help of Dynein the damaged mitochondria are reversely transported to the soma for repair.In this way,mitochondrial activity is increased and neuronal activity is further increased.