The Role And Mechanism Of MiR-29b/miR-124-mediated MCT1 Loss In White Matter Injury After Subarachnoid Hemorrhage In Rats

Part ? Time course and cellular localization of MCT1 and its roles in white matter injury after SAHObjective:To investigate the change and distribution of protein MCT1 in brain tissues at different time points after SAH in rats,and to observe its effect on acute white matter injury and long-term neurobehavioral outcomes after increasing the level of MCT1 by overexpression plasmid transfection technique.Methods:Part I had two experiments.In experiment 1,36 rats were randomly divided into six groups of six rats each,a sham group and five experimental groups ordered by time points as follows:3,6,12,24,and 48 h after SAH.At the indicated time point after SAH,rats were euthanized by chloral hydrate,and the cortex and corpus callosum(CC)tissues were separated and taken for Western blot and immunofluorescence analysis after transcardial perfusion with PBS.In experiment 2,72 rats were randomly assigned to four groups of 18 rats each:the sham group,the SAH group,the SAH+Vector group,and the SAH+MCT1 overexpression plasmid(Over-MCT1)group.Using a table of random numbers,a technician who did not participate in this study randomly assigned the 18 rats in each group into two subgroups(of six and 12 rats).For six rats,the cortex and CC tissues were separated and taken for Western blot and immunofluorescence analysis 48 h after SAH.For the other 12 rats,three behavior tests were conducted until 35 d after SAH,and after that,the rats were euthanized by chloral hydrate.Results:Experiment 1:1.The expression level of MCT1 was not significantly downregulated until 48 h after SAH induction.2.MCT1 is localized to the oligodendrocyte in the CC region,and the reduction in the expressions of MCT1 48 h after SAH induction was further verified in this area.Experiment 2:1.The transfection of overexpression plasmid can significantly increase the protein level of MCT1 and its distribution in CC region.2.Compared with the sham group,the protein level of MBP decreased significantly while the protein level of SMI32 increased significantly,and the SMI32/MBP ratio in CC increased significantly in the SAH group.However,the ratio in the MCT1 overexpression group was significantly lower than Vector group.3.The accumulation of ?-APP in SAH group was significantly higher than sham group,and the overexpression of MCT1 could effectively reduce the accumulation of ?-APP in CC.4.Compared with Vector group,overexpression of MCT1 could significantly prolong the latency to fall in rotarod test,shorten the time to remove front paw stickers in adhesive removal test and effectively improve the spatial learning and memory ability of rats in Morris water maze test.Conclusions:The level of MCT1 in rat brain decreased significantly at 48 hours after SAH,and MCT1 could co-locate with oligodendrocytes in the CC region.The overexpression of MCT1 could effectively attenuate the acute white matter injury in the CC region after SAH and further improved the long-term outcomes of neurobehavioral function in rats.Part ? The effect of post-SAH rotarod training on oligodendrocyte phenotype and MCT1Objective:To explore the effect of post-SAH motor training on MCT1 and phenotypic changes of oligodendrocytes and their correlation with sensorimotor ability of rats after SAH.Methods:80 rats were randomly divided into following four groups of 20 rats each:sham group,sham+rotarod training group,SAH group,and SAH+rotarod training group.Using a table of random numbers,a technician who did not participate in this study randomly assigned the 20 rats in each group into three subgroups(of four,six,and 10 rats).Four rats received two-day rotarod training after SAH,and their cortex and CC tissues were then separated and taken for immunofluorescence analysis.The other 16 rats received seven-day rotarod training after SAH.Six of them were euthanized after the last training session,and their cortex and CC tissues were separated and taken for Western blot and immunofluorescence analysis.The remaining 10 rats were sacrificed after undergoing the rotarod and adhesive removal tests at 35 d after SAH.For the two non-training groups,the rats were euthanized at the corresponding times point after SAH,and their cortex and CC tissues were separated and taken for analysis together with the training groups.Results:1.In both the sham and SAH groups,the training rats exhibited more ITPR2+SOX10+cells in the CC than no-training rats.Additionally,no significant difference was found between the sham and SAH groups in the number of ITPR2+SOX10+cells.2.In both the sham and SAH groups,the training rats exhibited an increased protein level of MCT1 and more MCTM+SOX10+cells in the CC than the no-training rats.3.The number of MCT1+SOX10+cells in the CC was positively correlated with the latency to fall in the rotarod test and was inversely correlated with the time to remove the adhesive tapes in the adhesive removal test.Conclusions:Post-SAH motor training could increase the level of MCT1 on the exercise-related phenotypic oligodendrocytes and thus accelerate the long-term neurobehavioral recovery after SAHPart ? The roles of miRNA-29b and miRNA-124 in regulating MCT1Objective:To explore the changes of miRNA-29b and miRNA-124 expression and their negative regulation on MCT1 protein level after SAH.Methods:Firstly,CSF samples of 12 patients from The First Affiliated Hospital of Soochow University were collected to detect the differences in the expression levels of miRNA-29b and miRNA-124 between the non-SAH and SAH groups.Then,30 rats were randomly divided into five groups of six rats each:sham group,SAH group,SAH+antagomir-negative control(antagomir-NC)group,SAH+antagomir-29b-3p group,and SAH+antagomir-124-3p group.After the indicated treatments,rats were euthanized 48 h after SAH,and their cortex and CC tissues were separated and taken for Western blot and immunofluorescence analysis.Results:1.The results of PCR showed that both miR-29b-3p and miR-124-3p were significantly upregulated in the SAH patients compared with the non-SAH patients.2.The rats injected with antagomir-29b-3p and antagomir-124-3p exhibited a higher protein level than the rats injected with antagomir-NC at 48 h after SAH.Moreover,the MCT1 signal in the CC was increased with injection of antagomir-29b-3p and antagomir-124-3p.No significant differences were observed between the antagomir-29b-3p and antagomir-124-3p groups in the level of MCT1.Conclusions:The upregulation of miR-29b and miR-124 after SAH might be the main reason for the acute reduction in MCT1 expression.