| Part Ⅰ Investigate the expression of MIC60 in brain tissue after intracerebral hemorrhage in ratsObjective:1.By establishing intracerebral hemorrhage(ICH)model in adult Sprague-Dawley(SD)rats,to protein expression of MIC60 were measured in brain tissue at different time points after ICH.2.Explore the expression and positioning of MIC60 in brain tissue cells 3 Observe if the mitochondria are damaged after intracerebral hemorrhage.Methods:1.42 adult male SD rats(250g-350g,8-12w)were randomly divided into 7 groups:sham operation group(Sham),3h after ICH group,6h after ICH group,12h after ICH group,24h after ICH group,48h after ICH group and 72h after ICH group,6 rats in each group.Establishment of rat model in Sham group:the rats in Sham group were anesthetized by intraperitoneal injection of 4%chloral hydrate,and then 100μl sterile normal saline was slowly injected into the right basal ganglia with a microinjector at the speed of 20μl/min.Establishment of rat model in ICH group:the rats in ICH group were anesthetized by intraperitoneal injection of 4%chloral hydrate solution,and the blood samples were taken from the femoral artery of rats with a microinjector.During the operation,100μl autologous arterial blood was slowly injected into the right basal ganglia at the speed of 20μl/min.In case of death during anesthesia or modeling,the corresponding number of rats from the same batch of purchased rats were selected for the same treatment to supplement the sample number of rats in each group.2.3h,6h,12h,24h,48h and 72h after modeling,the rats were anesthetized by intraperitoneal injection of 4%chloral hydrate at the dose of lml/100g.The rats were sacrificed by decapitation according to the guidance of euthanasia.The brain tissue of the hematoma side of the cerebral hemisphere was taken out,the protein was grinded and extracted for Western blot.The changes of MIC60 protein expression in Sham group and 3 h,6h,12h,24h,48h and 72h after ICH were detected.Paraffin sections were made to observe the distribution of MIC60 protein in neurons by immunofluorescence assay.The morphology and damage of mitochondria in Sham group and ICH group were observed by electron microscope.Results:1.The results of Western blot showed that the expression level of MIC60 protein in rat brain tissue fluctuated to a certain extent within 3-12 hours after ICH,but compared with sham group,the degree of change was smaller,and there was no significant difference in the level of change.The expression level of mic60 protein decreased sharply 24 hours after ICH,less than half of that in sham group,48 hours after ICH and 7 hours after ICH The protein expression level of 2hmic60 increased gradually.2.The results of immunofluorescence staining showed that MIC60 was mainly expressed in neurons.3.The morphology of mitochondria in neurons was observed under electron microscope.We found that mitochondria in neurons of ICH group were damaged,mitochondria swelled and deformed,cristae junction was broken.Conclusion:1.The expression level of MIC60 protein was the lowest at 24h after ICH.2.MIC60 was mainly expressed in neurons.3.There was mitochondrial damage in neurons after ICH.Part Ⅱ Investigate the effects of regulating MIC60 expression on cell death and ethology of rats after ICHObjective:By regulating the protein expression of MIC60 with siRNA and plasmid,effects on the expression of downstream proteins Pink1 and Parkin proteins,the changes of cell death in CNS and ethology of rats after ICH were observed.Methods:1.72 adult male SD(Sprague Dawley)rats were randomly divided into 6 groups:sham group,24h after ICH group(ICH),siRNA negative control group(Si-NC),MIC60-siRNA group(Si-RNA),vector group(Vector)and overexpression MIC60 group(Over-MIC60),12 rats in each group.The sterile saline was inject into the right basal ganglia at the speed of 20ml/min with a microinjector.The rats in Si-NC group,Si-RNA group,Vector group and Over-MIC60 group were anesthetized by intraperitoneal injection of 4%chloral hydrate solution(1ml/100g).After anesthesia,siRNA and its control reagent,overexpression MIC60 plasmid and its control reagent were injected into the lateral ventricle of rats in each group.48 hours after the injection of related reagents,100μl autologous arterial blood was slowly injected into the right basal ganglia at the speed of 20μl/min to establish ICH model.In case of death during anesthesia,intracerebroventricular injection of intervention reagent or ICH modeling,the corresponding number of rats were selected for the same treatment to supplement the rats in each group.2.6 rats in each group were randomly selected and anesthetized by intraperitoneal injection of 4%chloral hydrate at the dose of lml/100g.Then the rats were sacrificed by decapitation.The brain tissue of the hematoma side of the cerebral hemisphere was taken and the protein was grinded and extracted.Western blot was used to test the protein expression level of MIC60 after transfection of siRNA and overexpression plasmid.The brain tissue was fixed with formalin and paraffin sections were made.The FJB experiment and TUNEL experiment were carried out respectively.The cell death in brain tissue after the change of MIC60 protein expression level was observed by fluorescence microscope.3.The remaining 6 rats in each group were taken for neurological function score,rotarod test,adhesive removal test and morris water maze to detect the effect of MIC60 protein expression on the ethology of rats.Results:1.The result of Western blot shows that compared with sham group,the protein level of MIC60 in ICH group was the same as part I.Compared with Vector group,the protein level of MIC60 increased significant after intracerebro ventricular injection the plasmid of MIC60.While the protein level of MIC60 in ICH group,Si-NC group and Vector group showed no significant difference.2.The result of western blot showed that Pink1 expression was significantly reduced at 24 h after ICH.Furthermore,we found that bidirectional changes in protein levels of Pink 1 following MIC60 overexpression/knockdown were similar to those of MIC60 levels.Additionally,as a marker of mitochondrial mitophagy,we also measured the protein levels of Parkin.The results showed that there were no significant differences in the protein levels of Parkin across the various groups.3.FJB staining and TUNEL staining showed that compared with sham group,the number of cell death in ICH group was significantly increased.When the expression level of MIC60 decreased,the number of dead cells increased compared with Si-NC group.When the expression level of MIC60 increased,the number of dead cells decreased compared with Vector group.The number of cell death in ICH group,Si-NC group and vector group showed no significant difference.4.After ICH,the spontaneous activity,body proprioception,vibrissae touch,limb symmetry,lateral turning,forelimb outstretching and climbing of rats were impaired.The behavioral performance of rats in Over-MIC60 group was better than Vector group.5.Compared with sham group,the motor,sensory,learning and memory abilities of rats in ICH group were impaired.When the expression level of MIC60 increased,the recovery speed of motor,sensory,learning and memory abilities of rats was faster than Vector group.Conclusion:1.After transfection of MIC60 siRNA,the expression level of MIC60 decreased,while the protein level of MIC60 increased after transfection of MIC60 overexpression plasmid.2.The expression level of PINK1 protein changed with the expression of MIC60,while the expression level of parkin did not change with the expression of MIC60.3.Over-expression of MIC60 can inhibit nerve cell death caused by ICH and protect brain tissue.4.The performance of rats in Over-MIC60 group was better than Vector group in spontaneous activity,body proprioception,vibrissae touch,limb symmetry,lateral turning,forelimb outstretching and climbing.5.The recovery speed of motor ability,limb sensation,spatial learning,and memory in Over-MIC60 group was faster than Vector group.Part Ⅲ Potential mechanism of MIC60 in brain protection after ICHObjective:By establish the vitro ICH model with primary neurons to explore the mechanism of neuroprotective effect and the related signal molecules of MIC60.Methods:1.The cortical tissues from fetal rats at gestational day 18 were isolated to establish primary neuronal cultures.First,the cerebral hemispheres(without meninges or blood vessels)of these fetal rats were collected.Next,the cerebral hemispheres were digested for 5 min at 37℃ with 0.25%trypsin(Gibco,Grand Island,NY,USA)and were then washed three times with PBS.Then,the suspension was centrifuged at 1,000 g for 5 minutes,and the pellet was then resuspended in complete medium(neurobasal medium containing 0.5 mM of Glut Amax TM-I,2%B27,50 U/mL of streptomycin,and 50 U/mL of penicillin;all from Gibco).Finally,the isolated cells were seeded into six-well plates precoated with 0.1 mg/mL of poly-D-lysine(Gibco)at a density of 30,000 cells/cm2.The seeded cells were cultured in complete medium and maintained at 37℃ and 5%CO2 under humidified conditions.Half of the volume of the complete medium was exchanged every 2 d with fresh complete medium.At 7-10 d after isolation,these cultured neurons were treated with 5 μM of OxyHb at 37℃ for 24 h in order to mimic ICH in vitro.2.The cultured neurons were divided into 7 groups:control group,3h group,6h group,12h group,24h group,48h group and 72h group.The expression of MIC60 protein was detected by Western blot.3.Primary-cultured cortical neurons were divided into the following six groups for western blot analysis,immunofluorescent analysis,live-dead cell staining,JC-1 staining,and Annexin-V and PI staining:a control group;oxygen hemoglobin(OxyHb)group;OxyHb+Si-NC group;OxyHb+Si-RNA group;OxyHb+Vector group;and OxyHb+Over-MIC60 group.4.Primary-cultured cortical neurons were divided into the following six groups:control group;oxygen hemoglobin(OxyHb)group;OxyHb+Si-NC group;OxyHb+SiRNA group;OxyHb+Vector group;and OxyHb+Over-MIC60 group.Western blot was used to detect the expression of pinkl in primary cultured neurons.Then CCCP was used to induce neuronal mitochondrial autophagy.The autophagy of neurons in each group was observed to determine whether pink1 was involved in mitochondrial autophagy.Results:1.The protein level of MIC60 in cultured neurons was the lowest after 12 hours of stimulation with OxyHb.The protein level of MIC60 in cultured neurons decreased after SiRNA transfection,while the protein level of MIC60 in cultured neurons increased after overexpression plasmid transfection.2.When the expression level of MIC60 protein increased,the number of apoptotic cells decreased.When the expression level of MIC60 protein decreased,the number of apoptotic neurons increased.3.A decrease in red-fluorescent intensity and an increase in green-fluorescent intensity were observed in the OxyHb group compared with these signals in the control group,which indicated a reduction in the MMP and an opening in the mitochondrial-permeability transition pore(MPTP).In the OxyHb+Over-MIC60 group,this effect was reversed;in contrast,this effect was exacerbated in the OxyHb+siRNA group.4.Mt-Keima plasmids contain a mitochondrial-mapping sequence,and the excitation light of the plasmid under normal physiological states is 458 nm.However,when mitochondrial autophagy occurs,the excitation light of the plasmid located on lysosomalencapsulated mitochondria changes from 458 nm to 543 nm.Therefore,the fluorescent ratio of these two kinds of excitation light reflects the level of mitophagy within cells.After CCCP treatments,our results showed that the mitophagy levels in the OxyHb+vector group were inhibited compared with those in the control group,and MIC60 overexpression reversed this effect5.The changes in protein levels of Pink1 in vitro were similar to those in vivo.Next,we determined the localization of Parkin in neurons after CCCP treatments in vitro.In the control group,Parkin was recruited to mitochondria labeled by mitotracker red to induce mitophagy,thereby clearing damaged mitochondria.Compared with that of the control group,Parkin recruitment was inhibited following OxyHb treatment but was recovered following MIC60 overexpression.Conclusion:Mitochondrial autophagy was inhibited after OxyHb stimulation,and overexpression of MIC60 contributed to mitochondrial autophagy,which was related to the recruitment of parkin to damaged mitochondria. |
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