Everolimus(RAD001) Ameliorates Vascular Cognitive Impairment By Regulating Microglia Polarization Via The MTORC1 Signaling Pathway

Background and Objective:Vascular dementia(Va D)is a widely prevalent and devastating disease.Va D is a clinical syndrome of cognitive decline caused by ischemic,hemorrhagic,or oligemic injury to the brain as a consequence of cardiovascular disease or cerebrovascular disease.Va D will likely affect an increasing number of patients in the coming decades.Despite the tremendous complexity that limits understanding of the pathophysiology of Va D,microglial dysfunction has been attributed,in part,to immune microenviroment disorder and finally leads to cognitive deficits.Considered the mammalian target of rapamycin complex 1(m TORC1)is a key player in regulation of glial function,our work focused on whether the m TOR inhibitor everolimus(RAD001)could overcome the destructive microglial function,change the phenotype and improve cognitive dysfunctions mediated by cerebral hypoperfusion.This study aimed to assess the underlying role of RAD001 in neuroprotective effects and determine the mechanisms in Va D,all mice were subjected routinely established the bilateral common carotid artery stenosis(BCAS).The Morris water maze(MWM)was used to assess behavioral tests.The expression of the M1 marker CD16/32? M2 marker CD206 and Iba1 cholinergic function and proinflammatory cytokines were measured.Methods: Male,C57BL/6J mice(12-week-old,weighing 30 to 50 grams)mouse were used and subjected to modified BCAS.All the mice were randomly divided into three groups: sham group,BCAS group and the RAD001 treatment group.After 2 weeks of BCAS,we used RAD001 at a dose of mice animals were injected(s.c.)four times with a total of 0.5 mg/kg RAD001 on BCAS dissolved in 200 ?L of PBS.The mice was administrated RAD001 twice injection during the first week.Afterthat,RAD001 was injected total twice eveay 7 days.MWM was used to assess cognitive function,and electrophysiological recordings assessed the synaptic plasticity.Immunofluorescence staining was used to evaluate the central cholinergic activity and Iba-1,CD16/32,CD206 in mice.Western immunoblotting was used to further examine mechanisms mediating the modulation of M1/M2 balance.Real-time PCR was used to detect m RNA expressions of M1/M2.Results: 1.The following results were determined by MWM testing.The longer latency times were compared between BCAS group and sham-operated group: on day 149.2±12s,45.1±12s,P>0.05;on day 2 58.1±11s,40.2±8s,P<0.05,day 3 57.2±7s,29.2±8s,P<0.05,day 4 47.2±6s,25.1±7s,P<0.05 and day 548.3±5s,20.1±6s,P<0.01.The percentage in target quadrant was longer in the sham group compared with he BCAS group 43.52±6.17%,19.85±3.21%,P<0.01.All of this suggested that the mice have significant cognitive impairement induced by the surgery of BCA.However,the mice was dramatically improved in cognitive dysfunctions after the treatment of RAD00.As shown by the lantcey time: on day 3 35.2±5s,P<0.05,day 4 26.3±8s,P<0.05 and day 5 22.2±8s,P<0.01.Meanwhile,the target quadrant time was significantly improved.All these data indicate that RAD001 could improved cognitive impairment.2.The results of H&E staining displayed the change of hippocampal neurons after 5 weeks under the surgery of BCAS.The widely neuronal damage was observed in the hippocampal CA1 area in the BCAS group,whih was manifested by neuronal cell loss,nuclei shrinkage and dark staining of neurons.While these damage were significantly improved after the treatment of RAD001 in the hippocampal CA1 area.3.The results of immunofluorescence showed that the activated microglia determined by Iba-1 and CD16 / 32 positive cell was significantly elevated in the BCAS group compare witn the sham-operated group.The expression of Ch AT and VACh T were declined in the BCAS group.However,all abrove results were reversed by RAD001.On the other hand,the relative expression of ACh E was no change.4.The results of q RT-PCR were used to reflect the expression of inflammatory cytokines.Generally,the expression of these markers increased soon after OGD and peaked after 2 h(i NOS,IL-10,TGF-?)or 4 h(TNF-?,CD16,Arg1).However,in the presence of RAD001,the dramatic increase was blunted from 2 h after OGD,and the effect continued to 6 h.5.Western blots showed the increased expressin of P-Akt in the hippocampus after the RAD001 treatment compared with that in BCAS group.The expression of T-Akt was no change among three groups.However,the expression of P-p70S6 k was decreased under RAD001 treatment.Conclusions The major discovery of the current study is that RAD001 is effective in lessening the cognitive deficits induced by chronic cerebral hypoperfusion and that this beneficial effect is mediated through inhibition of the m TORC1 pathway.These effects stemmed from a balancing adjustment away from the microglial M1 phenotype and toward M2.Our finding suggests that RAD001 is a likely candidate as an adjunct therapy for carotid stenosis and the resulting dementia.