Remote Ischemic Conditioning Improves Cognitive Function During Cerebral Vascular Injury Through The Induction Of Autophagy

Research purposesThe aging population in the world increases as well as the chronic diseases,e.g.hypertension,diabetes and stroke.The impact of vascular factors on dementia has become a recent research focus,which received more and more attention.Vascular cognitive impairment(VCI)is a cognitive dysfunction caused by vascular or vascular related factors.VCI can occur alone or in combination with Alzheimer’s disease,including various degree and types of cognitive dysfunction,e.g.non-dementia vascular cognitive functions obstructive,vascular dementia and mixed dementia.One of the major causes of the mechanisms is the repeated cerebral ischemia-reperfusion injury,which includes vascular factors,neurodegeneration,neurotransmitters and genes.There is no effective treatment currently.So far,the drugs used to improve the cognitive function of VCI are only the therapeutic drugs for the treatment of AD.Therefore,seeking effective treatment methods have become a hot spot in the medical field at present.Remote ischemic conditioning(RIC)gives a brief,non-lethal mild ischemic treatment of the organ,thereby attenuating lethal ischemic injury to the distant organ.By the transient,non-fatal ischemia-reperfusion(IR)of distant organs and tissues,the protected organs can sustain the IR damage for a longer period of time.Thus,The RIC is applied to the non-invasive pressure double limbs or both limbs clinically,and the blood flow is temporarily blocked.RIC is a noninvasive,simple and cost-effective treatment method.Many previous literatures have proved that RIC may exert protective effect on distant organs by complex biological cascade such as nerve,body fluid,immune and gene.RIC can then protect heart,lung and brain,spinal cord and other important organs.The protective effects have been verified by both a variety of organ preservation surgeries and related clinical,basic tests.Potential neuroprotective mechanisms of RIC involve excitatory or inhibitory neurotransmitters,inflammatory cytokines,adenosine,cellular energy metabolism and activation of autophagy lysosomes,inhibition of cell death and apoptosis,activation of DNA repair and self-repair/remodeling mechanism,vascular remodeling and protection of the blood-brain barrier,nitric oxide-related signaling pathways.RIC can motivate endogenous self-protection mechanisms and effectively reduce cerebral ischemic injury.However,no studies have reported whether its mechanism of action is related with the regulation of autophagy.In our study,we established the model of vascular cognitive impairment treated by remote ischemia condition.We studied the expression of autophagy in the VCI model after remote ischemic conditioning.Autophagy is involved in the protective mechanism of RIC.Autophagy is a lysosome-mediated cell self-processing system.It is also a cells degration process to degrade longevity proteins or functionally impaired organelles for recycling and maintain cell homeostasis.Repeated cerebral ischemia-reperfusion induces mitochondrial damage and produces a large number of reactive oxygen free radicals.It can cause the accumulation of abnormal functional proteins and damaged organelles,thereby activating autophagy and causing autophagic death.Although autophagy induces the type II programmed death,the protective role of autophagy has been already elucidated in recent ischemia studies.In the case of mild ischemia,hypoxia and/or ischemia and pre-hypoxic preconditioning,the autophagy is mostly moderatly activated and has neuroprotective function,which can be modeled by the inducers of autophagy.The microtubule-associated protein 1 light chain(MAP1-LC3)is a mammalian autophagosome-associated protein.When autophagy occurs in mammalian cells,both the content of LC3 and the conversion of LC3-Ⅰ to LC3-Ⅱ are significantly increased.Mammalian mammalian target of rapamycin(mTOR)is a nutrient,energy receptor and a negative regulator of autophagy.Recent studies have shown that,glycogen synthase kinase-3 β(GSK-3β)plays a crucial role in the survival of cells by regulating the phosphorylation of mTOR.Before hypoxia and ischemia,it is treated by ischemic preconditioning and simvastatin preconditioning.After 24 hours,it can be observed the Beclin1 upregulation and brain damage reduction.Autophagy plays a neuroprotective role in the IPC process,probably by activating the PI3K-Akt-mTOR pathway.Treated by transient ischemic preconditioning,the rat suffers the permanent focal cerebral ischemia for 24 h.It is found that ischem:ic preconditioning increased the level of LC3-Ⅱ and the number of autophagosomes after permanent focal cerebral ischemia.It prolongs the duration of autophagy after ischemia and reduces the volume of cerebral infarction.Regulation of autophagy may be one of the pathological mechanisms of VCI for RIC.Domestic and foreign scholars have carried out some researches on the treatment of cardiovascular and cerebrovascular diseases by RIC.Animal studies have found that RIC can improve cerebral blood flow in VCI rat animal models,reduce inflammatory reaction and degeneration and necrosis of nerve cells,improve cognitive function and reduce white matter demyelination.However,the clinical effect of RIC on VCI and its pathological mechanism have not been reported yet.We found that with the treatment of RIC,the cognitive function improves for the patients with cerebral infarction,including those in acute and convalescent stages of cerebral infarction.Therefore,we propose the idea that RIC can effectively improve the cognitive dysfunction of VCI.The mechanism may be the regulation of pathways,e.g.PI3K/Akt,MAPK,autophagy and vascular endothelial growth factor.In conclusion,we intend to treat VCI patients and rat model by RIC to explore its possible pathological mechanism.We aim to detect the expression of autophagy associated protein in RIC,and to study the relationship between RIC and autophagy in cerebral ischemia injury,and to detect whether RIC can improve brain function by activating autophagy.The study will provide the theoretical and practical basis for RIC treatment of VCI.It can be imagined that,the clinical application will not be too long if we can achieve the desired results.It has important theoretical significance and potential clinical application value.MethodsThe rat were randomly divided into sham operation group(sham group),model control group(BCAS group)and model-far ischemic group(BCAS+RIC).1.VCI rat model is established by the ligation of bilateral common carotid artery.Meanwhile,it is observed whether ischemic treatment can improve rat cognitive function.We observe memory and orientation behavior cognitive function by positioning navigation experiment,space exploration experiment,trajectory of motion in Morris water maze.2.When RIC is given discontinuously after cerebral ischemia,it is observed the rat infarct volume and neurological function after 7 d and 28 d as well as the neuronal apoptosis.We aim to observe the pathological changes and neuron apoptosis of brain white matter and hippocampus by HE staining,3.During the influence process of RCI on the VCI,the effect of autophagy at different times is detected.In the VCI pathogenesis process after remote ischemic treatment,the dynamic changes of protein ATG7,Beclin-1,LC3 and Atg5-Atgl2 can be got by western blot method.The expression of autophagy-related protein genes including Beclinl,Atg5,Atg7 can also detected by RT-PCR.ResultsFor the experimental animal behavior,the rat latency and spatial exploration ability of sham group was higher than that of both BCAS group and BCAS + RIC group.In the positioning navigation experiment,the shorter the incubation period and the more time to traverse the platforms,the learning and cognitive ability of rat became stronger.As the training days increased,the mice in each group avoided the incubation period.In the BCAS group and the BSAS+RIC group,the latency of the incubation period was longer than that in the sham group,and BSAS+RIC group was better than the BCAS group,but the difference between the two groups was not statistically significant(P>0.05).In the 7th day space exploration experiment,the number of times of crossing the platform was reduced with the sham group,BCAS group and BCAS+RIC group,and BSAS+RIC group was better than the BCAS group,but the difference between the two groups was not statistically significant(P>0.05).In the 28th day space exploration experiment,the number of times of crossing the platform was decreased with the sham group,BCAS group and BCAS+RIC group.The results of BCAS+RIC group were better than that of the BCAS group.In the third quadrant(target quadrant),the percentage of total time,sham group(33.8%),BCAS group(17.34%),BCAS+RIC group(20.56%).In the swimming trajectory,compared with the sham group,the mice in the BCAS group presented a chaotic platform search trajectory,and the BCAS+RIC group showed certain regularity compared with the BCAS group.According to the mean value analysis,it can be seen the trend of learning and cognitive abilities of rat in each group(Bilateral carotid stenosis severely affected the cognitive ability of rat;post-ischemic postconditioning had a certain improvement on BCAS-induced injury).However,there was no significant difference between BCAS and BCAS + RIC groups.This result may be related to the individual differences between animals and the lack of experimental samples.Western blot analysis turned out that:According to the experimental results,bilateral carotid artery stenosis(BCAS)can up-regulate the expression of ATG7,Beclin-1,LC3,Atg5-Atg12 in white matter and hippocampus of rat compared with sham group.Post-ischemic postconditioning(BCAS+RIC)can further promote the changes of the corresponding proteins in white matter and hippocampus of rat.ATG7,Beclin-1,LC3,Atg5-Atg12 are the key proteins in the process of autophagy.The up-regulation of these proteins suggests the increase of the autophagy;p62 is an autophagy substrate protein whose expression level has negative correlation to autophagy intensity.According to above results,it can be seen that bilateral common carotid artery stenosis can activate the autophagy in the white matter and hippocampus of rat.The autophagy in rat brain tissue is further improved after the ischemic postconditioning treatment.Real-time PCR results showed that,the levels of BECN1,Atg5 and Atg7 in the white matter and hippocampus of BCAS rat were significantly higher than those in sham group.Post-ischemic postconditioning further enhances the expression of mRNA levels of these genes in brain tissue.ConclusionIn our study,remote ischemic conditioning can improve the cognitive function of VCI mice and reduce nerve damage.By both gene and protein levels detection,the autophagy level increases in brain tissue of rat with bilateral common carotid artery stenosis;The changes of autophagy related proteins in the brain white matter and hippocampal tissue were more significant.RIC can further enhance autophagy.It suggests that autophagy plays a role in the mechanism of protection in bilateral common carotid artery stenosis process.It is suggested that RIC can induce autophagy in cells,and it may be possible to activate autophagy to perform the neuroprotective effect of VCI and improve cognitive impairment.However,it is still necessary to further study the specific mechanism of remote ischemic conditioning to activate autophagy.The animal experiment found that RIC could improve the cerebral blood flow in the rat model of VCI rats,reduce the inflammatory response and degeneration and necrosis of nerve cells,improve cognitive function,and reduce the loss of white matter myelin.Ischemic treatment is applied in clinical practice by means of the temporary blockage of blood flow in both upper and lower limbs.This therapy is highly translatable to humans.If successful,this no-additional cost therapy using BP cuff will be an "exercise equivalent" which will be highly convenient for elderly patients as well as caretakers to perform,and therefore,may change the current therapeutic paradigm of VCI in humans.