MicroRNA-132 Attenuates Neurobehavioral And Neuropathological Changes Associated With Intracerebral Hemorrhage In Mice

Objectives:1,To get three groups of mice with different expression levels of miR-132.2,To establish the intracerebral hemorrhage(ICH)model of mice.3,To observe the neurobehavioral changes and survival rates of mice in different groups after ICH.4,To investigate the effect of miR-132 on brain water content,hematoma volume and blood brain barrier(BBB)permeability after ICH.5,To evaluate the content of Ach E in brains of mice.6,To observe the infiltration of microglia and neuronal apoptosis in mice brains.7,To investigate the effect of miR-132 on expression levels of inflammatory cytokines such as IL-6,IL-1β and TNF-α.8,To analysis the neurobehavioral and neuropathological changes in different groups of mice after ICH and then find the role of miR-132 and the possible mechanism.Method:Male C57BL/6J mice were randomly divided into LV-control group,LV-miR-132 group and LV-anti-miR-132 group,they were accordingly injected with lentivirus two weeks before the second injection of blood to get the ICH model.There were 64 mice in each group.1,Lentivirus was injected into striatum of mice,two weeks later the expression of miR-132 was tested by Immunofluorescence and RT-q PCR.2,Blood was withdrawn from the angular vein of mice and totally 30 ul of blood was injected into striatum of each mouse to generate the mice model of ICH.3,Modified Neurological Severity Score(m NSS),Corner Turning Test and Forelimb Placing(FP)test were performed with each mouse and the 14-day-survival indices were calculated for all three groups.4,At day 3 after the induction of ICH,brains were isolated,the wet weight and the dry weight of the brain were obtained.We also compared the hematoma volumes of each brain after being fixed in 4% paraformaldehyde overnight at 4℃.5,We measured and compared the BBB permeability of all mice in each group,by injecting 2% Evans Blue dye as a tracer at day 3 after ICH into the femoral vein 2 h before sacrifice.We calculated the concentration of Evans Blue(EB)and analysed the expression level of proteins for tight junctions.6,The number of Iba-1 positive cells were counted by Immunofluorescence,three days after ICH,we assessed the extent of cell death by using a Td T-mediated Biotin-d UTP Nick End labeling(TUNEL)kit.7,The relative m RNA expression level of inflammatory cytokines such as IL-6,IL-1βand TNF-α in mice brains of different groups were measured by real-time quantitative PCR.8,We compared the protein level of ACh E in mice brains,then confirmed the role of miR-132 in attenuating changes in mice brains after ICH and found the possible mechanism.Results:1,Two weeks after the injection of lentiviruses,we confirmed by RT-PCR that mice in each of our experimental groups expressed miR-132.Subsequently,the level of miR-132 was up-regulated in the LV-miR-132-treated mice and decreased in the recipients of LV-anti-miR-132.The efficacy of the lentivirus was also observed under fluorescence microscope as the effective expression of e GFP.2,We chose the blood injection model to induce ICH,briefly,blood was withdrawn from the angular vein and totally 30 ul of blood was injected into the each mouse.3,To determine the comprehensive impact of miR-132 on ICH,we performed m NSS,corner turning and forelimb placing tests to evaluate motor,sensory,reflex and balance functions,respectively.Further functional analyses indicated that overexpression of miR-132 significantly attenuated clinical neurodeficits in all three tests on day 1 and day 3 after ICH was introduced into the mice(P < 0.05).The On the contrary the LV-anti-miR-132 treated mice showed severer clinical neurodeficits compaired with the group in control(P < 0.05).The overexpression of miR-132 improved survival after ICH,though not to the level of statistical significance.4,At day 3 after ICH,water content of the brains from all groups was measured.Compared with the control group,mice that overexpressed miR-132 had less water in the ipsilateral brain hemisphere(P < 0.01).Conversely,the group injected with LV-anti-miR-132 showed increased brain water content despite the lack of statistical significance(P > 0.05)(Fig.2A).However,hematoma volumes at day 3 after ICH were not significantly different among these groups(P > 0.05).5,We examined the impact of miR-132 on neurovascular function in ICH by evaluating BBB permeability and the integrity of tight junctions after ICH among the three groups.The group treated with LV-miR-132 showed a significantly reduced extent of EB dye extravasation at day 3 after ICH(P < 0.05),which reflected a decrease in BBB permeability.In addition,the expression of claudin-5 and ZO-1increased significantly(P < 0.01)By contrast,the group treated with LV-anti-miR-132 exhibited increased EB extravasation(P < 0.05),but decreased expression of claudin-5 and ZO-1 compared to the controls(P < 0.01).6,To evaluate inflammatory responses after ICH,we compared the amounts of microglia in the peri-hematomal areas among the three groups of mice.In the group treated with LV-miR-132,the number of Iba-1+ cells dropped below those of the comparative groups(P < 0.01),denoting a decreased amount of activated microglia compared with the LV-control group.This outcome was consistent with the result of Western blot assays.Three days after ICH,we evaluated cell death among the three experimental groups,According to those counts,less apoptosis occurred in the group treated with LV-miR-132.In contrast,significantly increased cell apoptosis was seen in the grouptreated with LV-anti-miR-132 compared with the control group(P < 0.01).7,We also evaluated the content of inflammatory cytokines among the three groups of mice.Expression levels of the cytokines IL-6,IL-1β and TNF-α in the ipsilateral hemisphere were all significantly reduced(P < 0.01),presumably due to the overexpression of miR-132,while the LV-anti-miR-132 treated group showed increased expression levels of these inflammatory cytokines compared to the group in control(P< 0.01).8,Finally,we examined the mechanism that enabled miR-132,to exert these anti-inflammatory and neuroprotective effects.The overexpression of miR-132 reduced the level of ACh E and,thereby,enhanced the protective effect of ACh on the brain after ICH.We furthered that assumption by using Western blots to evaluate the content of ACh E in the brains of ICH mice.Compared with the control group,the recipients of LV-miR-132 had a significantly decreased content of ACh E(P < 0.01).Conclusions:1,Three groups of mice were successfully obtained with different expression levels of miR-132,we confirmed by RT-PCR that mice in each of our experimental groups expressed miR-132.the level of miR-132 was up-regulated in the LV-miR-132-treated mice and decreased in the recipients of LV-anti-miR-132 compared with the control group.2,Mice were characterized by neurologic impairments after ICH,which could be obviously alleviated by the overexpression of miR-132 and be worsen once the expression of miR-132 were inhibited.According to the comparasion of survival rates among three groups,we came to the conclusion that the overexpression of miR-132 improved survival after ICH,though not to the level of statistical significance.3,Brain edema was an essential evaluation index after ICH,according to our test,LV-miR-132 reduced brain edema in ipsilateral hemispheres at day 3 after ICH,without obvious effects on the contralateral part and cerebellum,in addition,LV-anti-miR-132 did not yield a significant difference in brain water level.Hematoma volume was also assessed at day 3 after ICH,but no protective effect of miR-132 on hematoma volume was obvious among the three groups.4,Damage to the blood brain barrier(BBB)as well as the tight junctions was the direct and dramatic change in brain after ICH,which furtherly led to brain edema.We chose Evans Blue dye(EB)as a tracer to evaluate its leakage in the brain tissue.We also measured the content of proteins for tight junctions among the three groups.Compared with the group in control,the overexpression of miR-132 decreased the leakage of EB and increased the expression of claudin-5 and ZO-1.These data suggest that miR-132 provides a protective effect on BBB permeability.5,The activated microglia played important role in the process of inflammation after ICH,which could worsen the cell apoptosis.We found less activated microglia and obviously decresed apoptotic cells in the brain after ICH with miR-132 overexpressed by the immunofluorescent staining.While the group with the expression of miR-132 inhibited showed aggravation of the inflammation and cell apoptosis.6,We assumed that miR-132 exerted its effect in the brain tissue after ICH by reducing the expression of inflammatory cytokines.It was confirmed by RT-q PCR that mice treated with LV-miR-132 had decreased inflammatory cytokines.7,ACh E is the target of miR-132,while the acetyl choline had been considered to attenuate inflammation.By comparing the content of ACh E in the mice brain after ICH among the three groups,we confirmed that miR-132 could inhibit the expression of ACh E and strengthen the anti-inflammtion effect of acetyl choline.All these results supported the assumption that miR-132 exerted its effect in brain after ICH through the cholinergic anti-inflammatory pathway.