Study On The Inhibitory Effect Of Miro1 On Neuron Apoptosis And Its Mechanism

Part ?:Establishment and Evaluation of Spinal Cord Injury Animal ModelObjective:1.To establish a suitable rat model of spinal cord injury(SCI);2.To observe and verify the morphological changes and apoptosis of neurons after SCI.Methods:1.54 male SD rats(weight 273-282g)were randomly divided into Blank Control group(Blank,3rats),Sham operation group(Sham,18 rats),Spinal Cord injury Group(SCI,33 rats).2.The method of aneurysm clamp was used in SCI modeling(the target segment was T10,the calibration force of aneurysm clamp was 70g,and the clamp lasted for 30s).Sham group rats were only exposed T10 spinal cord segment,then opened and sutured for 4 minutes.Blank rats were not operated.3.For T10 and peripheral spinal cord tissues:three rats in Sham group were randomly selected at72h after Sham operation;18 rats in SCI group were randomly selected at 6 time points after modeling(12h,24h,48h,72h,7d,14d);blank rats were randomly selected at weight=278±2g,and the tissues were made into frozen sections.4.Neurobehavioral score of spinal cord:BBB score of SCI group(n=15)and Sham group(n=15)were recorded at 7 time points,including before operation and 12 h,24 h,48 h,72 h,7 d and 14 d after operation.5.The frozen sections of spinal cord of different rats were treated with Nissl staining to observe the morphology of neurons and the changes of Nissl bodies.The apoptosis of neurons was observed by TUNEL staining.Results:The rats developed complete paralysis(BBB score=0)after modeling.The BBB score fluctuated slightly(0.43±0.40)after 72 hours,and increased significantly(8.03±0.53)after 7 days.The BBB score of the rats in the Sham group decreased slightly(20.33±0.61)after 24 hours,and returned to normal 48 hours later(BBB score=21).Nissl staining showed that Nissl body of neurons decreased gradually after spinal cord injury,and the least Nissl body was found at 72h,and then recovered slightly.TUNEL staining showed that the number of neuron apoptosis in 72 hours model rats was the largest.The sections of Sham group and Blank group were similar in both staining conditions.Conclusion:The model of acute spinal cord injury in rats can be successfully made by the compression of aneurysm clip,which is a reliable and economic modeling method.In this study,we found that 72 hours after SCI,neuron apoptosis was the peak.Part ?:Changes of Miro1 in Different Time Periods of Rats'Spine After Spinal Cord InjuryObjective:1.To observe the change of Miro1 after SCI;2.To clarify the relationship between the change of Miro1 and neuronal apoptosis.Methods:1.54 male SD rats(weight 276-285g)were randomly divided into Blank group(6 rats),Sham operation group(9 rats)and Spinal Cord Injury group(39 rats).SCI modeling,Sham group and Blank group were treated the same as Part I.2.6 rats in Sham group and 36 rats in SCI group were randomly selected.The The Sham group rats were collected at 72 hours after Sham operation,the SCI rats were collected at 6 time points(12h,24h,48h,72h,7d,14d)n=6×6,Blank rats were collected at weight=278±2g;RNA and spinal cord protein were extracted respectively.3.qRT-PCR was used to detect Miro1 gene expression in T10 segment of Blank?Sham and SCI at6 different time points(n=3);Western blot was used to detect Miro1 expression(n=3).4.Three rats in Sham group and SCI group were taken 72h after operation to make frozen sections of spinal cord(n=3).The neurons and astrocytes with Miro1 positive expression were observed and counted by immunofluorescence.Results:qRT-PCR:after SCI,Miro1 gene expression in T10 segment increased gradually,peaked at 72h,and then decreased to 14d,which was still higher than that in the control group.Western-blot:after SCI,the expression of Miro1 in T10 segment also increased,reaching the maximum value at 72 h.Immunofluorescence staining:positive expression of Miro1 was found in neurons and astrocytes;the ratio of Miro1 positive neurons to all neurons in SCI-72h was significantly higher than that in the control group(P<0.01);astrocytes increased,and Miro1 co-staining positive cells also increased(P<0.01).Conclusion:Miro1 showed increased reactivity after spinal cord injury,and its change trend was consistent with the trend of neuron apoptosis,Miro1 may be involved in the regulation of neuronal apoptosis after SIC.Part ?:Miro1's Inhibition of Neuron Apoptosis in Spinal Cord Injury and its MechanismObjective:1.To inhibit Miro1 expression in the spinal cord after SCI;2.To observe the neuronal apoptosis and function in spinal cord;3.To observe the expression of mitochondrial transport-related molecules,and to explore the inhibitory effect Miro1 on neuronal apoptosis after SCI and its'mechanism.Methods:1.60 male SD rats(weight 273-282g)were randomly divided into four groups:Blank control group(Sham),Saline control group(NS),Solvent control group(MOCK)and Experimental group(Si RNA).Sham group received Sham operation;the other three groups exposed T10 segment,NS group injected 8?l saline with micro injection pump in T10 segment;MOCK group injected 8?ltransfection reagent;experimental group injected 8?ltransfection complex containing Si RNA.The SCI injury was caused by aneurysm clamp in NS group,MOCK group and Si RNA group.2.Six rats in each group were selected to record the BBB scores at seven time points:before operation,12h,24h,48h,72h,7d and 14d after operation.3.All the samples were collected 72 hours after the operation.RNA and spinal cord protein were extracted and frozen sections were made.4.qRT-PCR was used to detect Miro1 gene expression in spinal cord segments of different groups(n=3);Western blot was used to detect Miro1 expression in different groups(n=3).5.Western-blot was used to detect the expression of Kinesin Heavy Chain(KHC),Kinesin Light Chain 1(KLC-1),Dynein Intermediate Chain(DIC),Mitochondrial anchoring protein(Syntaphilin,SNPH),apoptosis related gene protein BAX and B-Cell lymphama-2(Bcl-2).6.TUNEL staining was used to observe the apoptosis of spinal cord in different groups(n=3).Result:1.qRT-PCR and Western-blot showed that Miro1 gene expression or protein expression in Si RNA group was lower than that in MOCK group and NS group at SCI-72h.2.In Si RNA group,the expression of KHC and SNPH decreased significantly,but DIC and KLC-1did not change significantly.In Si RNA group,BAX expression increased,Bcl-2 expression decreased,BAX/Bcl-2 increased.3.TUNEL staining:the number of positive cells in Si RNA group was significantly higher than that in MOCK group and NS group.4.BBB score:After SCI,BBB score of Si RNA group(7d:6.5±0.45;14d:8.58±0.80)was significantly lower than that of MOCK(7d:7.67±0.75;14d:9.67±0.75)on 7d and 14d(7d:P<0.01;14d:P<0.05).Conclusion:Miro1,combined with Kinesin and Syntaphilin,can regulate mitochondrial movement,participate in the process of neuronal apoptosis in SCI rats,and protect the neurons to a certain extent.And this regulation process is most likely achieved by regulating the amount of BAX/Bcl-2 protein.The main site of interaction with kinesin is Kinesin Heavy Chain(KHC).Part ?:Miro1 Over-expression Induced by Lentiviral Vector Injection Improves the Prognosis of Spinal Cord InjuryObjective:1.To overexpress Miro1 in spinal cord segments with Lentivirus vector;2.To observe the changes of Miro1 expression in spinal cord segments after SCI;3.To observe neuronal apoptosis and function changes in spinal cord;4.To further verify the mechanism of Miro1's effect on neuronal apoptosis after SCI.Methods:1.Construction and production of GV492-Miro1 Lentivirus vector according to the design of Miro1 gene(RHOT1);2.Transfer the empty Lentivirus(gv492)and the overexpression Lentivirus Vector(GV492-Miro1)into suspension of 1×10~9TU/ml.6 male SD rats(weight:271-276g)were divided into two groups.8?l of no-load lentivirus suspension and lentivirus with Miro1 gene suspension were pushed into the T10spinal cord of the rats respectively.On the 7th day after operation,the rats in the two groups were killed,T10 spinal cord was collected and made into frozen section,DAPI sealed,Confocal Microscopy was used to observe and count statistics.Confirm the infection efficiency.3.60 male SD rats(weight:278-287g)were randomly divided into four groups:Sham group,NS group,Lenti-Ctrl group and Lenti-Miro1 group.The rats in each group were treated with two operation.a.Injection operation:exposed rats T10,injected rats in NS group,Lenti-Ctrl group and Lenti-Miro1 group,slowly pushed 8?l saline,no-load Lentivirus suspension and Lentivirus wtih Miro1 gene suspension into the spinal cord.The operation of Sham group was the same as that of Sham group.The rats were fed 7 days after the first operation for the next stage.b.SCI surgery:rats in NS group,Lenti-Ctrl group and Lenti-Miro1 group exposed T10 spinal cord segment and used make SCI model as Part I.rats in Sham group received two operations,sutured the wound after 120 s.4.6 rats were randomly selected from four groups for BBB score.BBB score was observed before injection operation,12h,24h,48h,72h,7d after injection operation,12h,24h,48h,72h,7d and 14d after spinal cord injury operation(secondary operation).5.The rats in each group were killed 72 hours after the second operation to extract RNA or spinal cord protein.6.qRT-PCR was used to detect Miro1 gene expression among different groups(n=3);Western-blot was used to detect Miro1 expression among different groups(n=3).7.Western-blot was used to detect the expression of KHC,KLC-1,DIC,SNPH,BAX and Bcl-2(n=3).8.TUNEL staining was used to observe the apoptosis of spinal cord segments in different groups(n=3).Results:1.7 days after the injection of GV492 and GV492-Miro1,the positive neurons were detected under Confocal Microscope,and there was no significant difference in the number of positive cells between the two groups.2.qRT-PCR and Western-blot showed that Miro1 gene expression or protein expression in Lenti-Miro1 group was higher than that in Lenti-Ctrl group and NS group at SCI 72h;3.Compared with Lenti-Ctrl group,the expression of KHC and SNPH in Lenti-Miro1 group was also significantly increased,DIC and KLC-1 had no significant change;the expression of BAX in Lenti-Miro1 group was similar than the other,Bcl-2 was increased,BAX/Bcl-2 was decreased significantly.4.TUNEL staining:the number of positive cells in Lenti-Miro1 group was lower than that in Lenti-Ctrl group and NS group.5.BBB score:BBB score of Lenti-Miro1 group(7 days after SCI:8±0.45;14 days:11.75±1.17)and that of Lenti-Ctrl group(7 days:7.67±0.75;14 days:9.75±0.52).There was no significant difference between the two groups at 7 days(P>0.05);it was significantly higher at 14 days(P<0.01).Conclusion:The overexpression of Miro1 in the spinal cord can decrease the apoptosis of neurons and protect the function of neurons.It is further confirmed that Miro1 plays a protective role in spinal cord injury by regulating mitochondrial transport and inhibiting neuron apoptosis through its interaction with Kinesin and Syntaphilin.Local injection of lentivirus vector can overexpress Miro1 in spinal cord,which provides a theoretical basis for biotherapy to improve the damage of spinal cord injury to spinal cord function.