| [Objective] Demyelination of white matter is one of the main causes of cognitive impairment such as spatial memory impairment.In this study,lpc-induced demyelination was used as the entry point to verify whether the Hv1 deficiency could reduce the damage to the structural integrity of myelin sheath and the cognitive impairment such as spatial memory ability caused by local demyelination injury.To investigate whether Hv1 deficiency can improve demyelination through ROS mediated microglia phenotype to m2-type transformation pathway.[Methods]LPC demyelinating mouse model was established to simulate the white matter damage caused by demyelination alone.It was divided into Sham group of Hv1 deficiency mice and wild-type mice,and lpc-induced demyelination group of Hv1 deficiency mice and wild-type mice.(1)MWM was used to detect whether the spatial memory impairment at 10 and 28 days after modeling was alleviated in the Hv1 deficiency group.(2)the rats were sacrificed at 5,10 and 28 days after modeling,and their brains were taken and stained with LFB to observe the damage of the myelin sheath structure in the focal area of the corpus callosum in each group,and the scores were statistically analyzed.(3)the expression of myelin protein was observed by immunofluorescence staining and Western Blott.(4)immunofluorescence was used to detect the distribution and expression of mature oligodendrocytes,oxidative stress ROS and M1 and M2 microglia.(5)after the number of microglia was activated by immunofluorescence technology,the steps and Image J plug-in for converting the photos of the fluorescence microscope into representative binomial and skeletonized images were performed,and the changes in cell body morphology of each group of mice were analyzed using Analyze Skeleton(3D)and Graph Pad software.[Results]1.Hv1 deficiency has a certain improvement in cognitive function caused by lpc-induced demyelination.After the model of local demyelination caused by LPC injection in corpus callosum was successfully established,mice in each group were continued to be fed,and MWM behavioral test was conducted.The results showed that at the time points of day 10 and day 28,the cognitive impairment of spatial memory ability of Hv1 deficiency mice was less than that of the wild-type group,and all of them were statistically significant.This suggests that Hv1 deficiency has a certain improvement effect on cognitive impairment caused by demyelination.2.Hv1 deficiency showed certain improvement in white matter damage,myelin structure of MBP and myelin cells of OLG.At 5,10,and 28 days of the lpc-induced demyelination model,the mice were killed and brains taken :(1)LFB staining results hint at various time points myelin has its the change of myelin damage degree,PM demyelination in 5 days time,ten days time to take off the pith of peak and 28 days started to repair itself to take off the medullary lesions began to recover,the Hv1 deficiency group compared with wild type LPC in 10 days time points to improve more,and at three time points of the improvement of the myelin damage degree have statistical significance.(2)immunofluorescent-labeled MBP results showed that in LPC mice with Hv1 deficiency,the area of myelin injury in the focal area of the corpus callosum at 3 time points was smaller than that in the wild-type LPC group,and reached the highest at 10 days.Western Blot was used to detect the expression of MBP.The results showed that the MBP expression in the Hv1 deficiency group was higher than that in the wild-type LPC group at the time point of28 days,suggesting that in the myelin sheath structural layer,Hv1 deficiency had a certain protective effect on the myelin sheath structure.(3)mature oligodrocytes promoted myelin formation,and APC/Olig2 was labeled by immunofluorescence technique.The results showed that the expression of Hv1 deficiency LPC group was higher than that of wild type LPC group at 5 and 10 days after APC/Olig2 ratio,suggesting that Hv1 deficiency also had a protective effect on myelin cell level.3.Hv1 deficiency mediated the transformation of microglia phenotype to M2 type by inhibiting ROS expression,and improved demyelination.At 5,10 and 28 days after the establishment of the lpc-induced demyelination model,the mice were sacrificed and brains were taken:(1)immunofluorescence staining(Iba1)showed the aggregation and activation of microglia in the lesion area,but the aggregation of microglia in the Hv1 deficiency group was less than that in the wild-type LPC group,with statistical significance at 5days.After 28 days of microglia activation,the Hv1 knockout group was smaller than the wild type group.(2)in terms of the proportion of m1-type microglia(cd16/32)and m2-type microglia(CD206)transformed in microglia(Iba1)by immunofluorescence labeling,m2-type transformation was activated and m1-type transformation was inhibited in the Hv1 deficiency model group at the time point of 10 days.(3)after immunofluorescence staining of ROS(8-ohg marker)in the lesion area,the proportion of ROS in the Hv1 model group was less than that in the wild model group.The above results suggested that after LPC modeling,the Hv1 deficiency group reduced microglia aggregation and inhibited activation through ROS mediated microglia,and promoted the transformation of microglia phenotype into M2 type.[Conclusion] Hv1 deficiency can improve lpc-induced demyelination,which may be caused by ROS inhibition after Hv1 deficiency,which mediates microglia phenotyp... |
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