Study On The Mechanism Of Cholinergic Anti-inflammatory Pathway Activity In Regulating Axonal Plasticity And Angiogenesis After Cerebral Ischemia/Reperfusion Injury In Rats

The incidence of stroke in the world is increasing year by year,which seriously harms human health and brings heavy social and economic burden,of which about 80% are ischemic stroke.Despite the progress of super-early vascular recanalization,most patients with cerebral infarction are still left with neurological dysfunction,which causes huge economic and mental burden to the society and family.The patients after stroke could slowly recover their physical and neurological functions within several month,which indicates the promoting of corticospinal tracts(CST)plasticity and angiogenesis could facilitate the recovery of neurological functions.Transcutaneous auricular vagus nerve stimulation(ta-VNS)can affect the cholinergic anti-inflammatory pathway(CAP)activity by activating the acetylcholine receptor subunit α7(a7nAchR,a7 nicotinic acetylcholine receptor).In our previous study,we found that ta-VNS could facilitate the recovery of neurological functions whichcould not be explained just by the reaction of anti-inflammatory.So we speculated that CAP may enhance the plasticity of CST,angiogenesis and improve the neurological functional recovery.In this study,MCAO model of rats will be used to develop the animal models and will demonstrate if the regulation of CAP could have an impact on the plasticity of CST,angiogenesis and neurological functions and explore the role of PPAR-γ signaling pathway activity.In addition,ta-VNS was used as an auxiliary treatment for clinical patients with cerebral infarction to observe its efficacy,so as to provide a theoretical basis for the study of supplementary treatment in patients with cerebral infarction at the chronic stage.Part one: Expression characteristics of the key receptor a7 nAchR in the CAP during the chronic stage in rat models of cerebral ischemia/reperfusion injury and the effect of transcutaneous auricular vagus nerve stimulation on itObjective: to observe the expression and localization of a7 nAchR and the effect of transcutaneous auricular vagus nerve stimulation(ta-VNS)on it after cerebral ischemia/reperfusion(I/R)injury in rats,and to investigate the spontaneous changes of cholinergic anti-inflammatory pathway(CAP)activity in the central nervous system after stroke,and whether ta-VNS can promote CAP activity at recoverystage with cerebral I/R injury or not.Methods:1.Sprague-Dawley(SD)rats were subjected to right middle cerebral occlusion/reperfusion(MCAO/R)and subsequently received ta-VNS treatment.2.Western blotting,q-PCR and immunohistochemistry were used to detect the expression changes of a7 nAchR at 14 d and 28 d after the establishment of the MCAO/R model and ta-VNS treatment.3.We used double immunofluorescence labeling technique to detect the cellular localization of a7 nAchR in the ischemic cortex.Results:1.The changes in right regional cerebral blood flow(CBF)were monitored by laser Doppler flowmetry to confirm the successful occlusion of the middle cerebral artery(MCA).The standard I/R model was considered successfully established if the cortical CBF decreased to70–80% of the baseline in the first 30 min after occlusion and > 70% blood flow was recovered during the initial 10 min of reperfusion and bring these successful models into the group.2.After MCAO/R injury in SD rats,the expression of a7 nAchR in the ischemic cortex was significantly higher than that of sham group at 14d(p< 0.05),and returned to normal levels at 28d(p > 0.05).After ta-VNS treatment,the expression of a7 nAchR was significantly higher than that ofthe I/R group at both time points(p < 0.05).3.In the ischemic cortex,α7nAChR colocalized with NeuN,a marker for neurons,and GFAP,a marker for astrocytes.Conclusion:1.After MCAO/R injury,the expression of a7 nAchR in the ischemic cortex was increased at 14 d and returned to normal levels at 28 d,and was mainly expressed in neurons and astrocytes.These results indicate thatα7nAChR may participate in the pathological process of I/R injury.2.Ta-VNS treatment can up-regulate the expression of a7 nAchR and then activate CAP in rats after cerebral I/R injury,suggesting that activation of central CAP activity may be closely related to the neuroprotective effect of ta-VNS treatment at recovery stage after stroke.Part two: Effects of a7 nAchR,a key receptor that regulates central CAP activity(ta-VNS),on axonal plasticity,angiogenesis and motor function recovery in MCAO/R model ratsObjective: To clarify the role of CAP activity(ta-VNS)in regulating cerebral I/R injury,we will assess the effect of CAP on neurofunctional dysfunction caused by cerebral I/R injury.According to recent studies,we speculate that the influence of changes in CAP activity on neurological function may be closely related to the regulation of axonal plasticity and angiogenesis.In this part of the study,we will focus on the regulatory effectof CAP on axonal plasticity and angiogenesis during the recovery period of cerebral I/R injury.Methods:1.We purchased the a7 nAchR antagonist,and then intraperitoneally injected the PBS diluted liquid at a ratio of 6 mg/kg into SD rats to interfere with the function of a7 nAchR.2.The modified neurological severity score(mNSS)and the adhesive-removal somatosensory tests(ARST)were used to evaluate the neurofunctional changes.3.The co-localization relationship between a7 nAchR and Nestin(neural stem cell marker)or DCX(immature neuron marker)were measured by double immunofluorescence labeling technique,so as to explore the effects of CAP on neurogenesis after cerebral I/R injury.4.Changes on the expression of growth associated protein-43(GAP43)and neurofilament-200(NF-200)with a7 nAchR blockade and ta-VNS treatment were detected to reveal the role of CAP regulation on axonal growth after cerebral I/R injury.5.Stereotaxic injection of biotinylated dextran amine(BDA)was performed in the contralateral motor sensory cortical region of the brain caused by I/R injury in rats.In order to reveal the role of CAP regulation on axonal reorganization after cerebral I/R injury,the BDA-positive fibers were evaluated by counting all fibers that crossed the midline in thecorpus callosum and red nuclear plane.6.We used the endothelial cell marker CD31 for immunofluorescence staining to reflect the role of CAP in angiogenesis after cerebral I/R injury.7.We used western blot technology to detect the expression of BDNF and VEGF in each group to reflect the molecular basis for the regulation of CAP on axonal plasticity and angiogenesis after cerebral I/R injury.Results:1.Both mNSS and ARST neurological function assessments suggested that the neurological function was significantly worse at 14 and 28 d after I/R injury(p < 0.05).Ta-VNS treatment can significantly activate the CAP to promote the neurofunctional recovery(p < 0.05).Strikingly,the beneficial effects of ta-VNS were diminished after α7nAChR blockade.2.The results of double immunofluorescence labeling showed that a7 nAchR was co-expressed with Nestin and DCX in the peri-infarct cortex in rats.3.The results of WB showed that the activation of CAP(ta-VNS)at28d after cerebral I/R injury significantly promoted the expression of GAP43(p <0.05),while antagonist of a7 nAchR function significantly inhibited the promotion of GAP43 expression by ta-VNS treatment(p<0.05).The immunohistochemical results indicated that after cerebral I/R injury,NF-200 positive fibers were significantly less and shorter than the sham group,and their arrangement was disordered(p <0.05).After 28 consecutive days of ta-VNS treatment,the NF-200 positive fiber damage in the ischemic cortex was significantly reduced,while the protective effects of ta-VNS treatment on nerve fibers disappeared after the use of a7 nAchR antagonist(p<0.05).4.The BDA nerve tracer showed that activation of CAP(ta-VNS)significantly promoted the number of axonal fibers that crossed the midline of the corpus callosum and red nucleus plane(p < 0.05).a7 nAchR antagonist can significantly eliminate the promotive effect of ta-VNS treatment(p < 0.05).5.The immunofluorescence results indicated that the ischemia group had partial angiogenesis compared with the sham group(p < 0.05),and the angiogenesis after activation of CAP(ta-VNS)was significantly improved compared with the I/R group(p < 0.05),while the angiogenesis promoting effect of ta-VNS treatment disappeared after the use of a7 nAchR antagonist(p < 0.05).6.The results of WB indicated that compared with the sham group,the expression of BDNF and VEGF in the I/R group was significantly increased at 28d(p < 0.05),and the expression of BDNF and VEGF was further increased after CAP(ta-VNS)activation compared with that in the ischemic group(p < 0.05).However,when a7 nAchR antagonist was used,the up-regulation effects of ta-VNS on BDNF and VEGF expression diminished(p < 0.05).Conclusion:1.Cerebral ischemia itself can trigger axonal reorganizasion and angiogenesis to a certain extent,which is the anatomical basis for the recovery of neurological function after cerebral ischemia.2.The regulation of CAP may be closely related to axonal plasticity and angiogenesis after cerebral I/R injury,and its activation can promote axonal plasticity and angiogenesis after injury.CAP activation(ta-VNS)may promote axonal plasticity and angiogenesis after cerebral I/R injury by upregulating the expression of BDNF and VEGF growth factor,thus improving the neurofunctional recovery at the chronic stage of ischemic stroke.Part three: CAP promotes axonal plasticity and angiogenesis after cerebral I/R injury by activating PPAR-γObjective: As an important nuclear transcription factor,PPAR-γ plays a crucial role in vivo.Our previous studies have found that the vagus nerve electrical stimulation can increase PPAR-γ expression.According to recent research,PPAR-γ can regulate the expression and secretion of BDNF and VEGF growth factor,and based on the above,we can infer that ta-VNS activated CAP,and in turn,activated the downstream PPAR-γ,which promoted the expression of BDNF and VEGF,and then advanced axonal plasticity and angiogenesis,eventually improving neurofunctionalrecovery.In this part of our study,we will focus on the role of PPAR-γ as an important molecule in the downstream of CAP in the regulation of axonal plasticity and angiogenesis after cerebral I/R injury.Methods:1.WB,q-PCR and immunofluorescence methods were used to measure the expression changes of PPAR-γ after MCAO/R and ta-VNS treatment at 14 and 28 d,as well as the tissue cell localization of PPAR-γ.2.We used wb and q-PCR to detect the effects of activation and antagonism of CAP on PPAR-γ expression in MCAO/R model rats.3.We constructed Lentivirus(LV)with PPAR-γ-RNAi fragment,and then injected LV-siPPAR-γ stereotaxically into the cortex of ipsilateral sensory-motor area with microsyringe to inhibit PPAR-γ expression.The expression level of PPAR-γ in brain tissue of the virus injection area was detected by WB at 28 d after MCAO/R,so as to verify the intervention efficiency of LV.4.Two weeks before the modeling,LV-siPPAR-γ was stereotaxicall y injected into the ipsilateral sensory-motor cortex to inhibit PPAR-γ e xpression.The rats were treated with percutaneous vagal nerve electri cal stimulation 30 min after MCAO(twice a day),and the expressions of GAP43 and NF-200 in the brain tissues of each group were detec ted by immunohistochemistry at 28 d after MCAO/R.Thus,it reflects t he regulatory effect of PPAR-γ as a downstream molecule of CAP onaxonal plasticity after cerebral I/R injury.5.We used the endothelial cell marker CD31 and the cell prolifer ation marker Ki67 to reflect the regulatory effect of PPAR-γ as a do wnstream molecule of CAP on angiogenesis after cerebral I/R injury by immunofluorescence staining.6.Wb and qPCR were used to detect the expression of BDNF and VEGF in MCAO/R model rats by ta-VNS treatment combined with inhibition of PPAR-γ expression.Results:1.After MCAO/R injury in SD rats,the expression of PPAR-γ in the ischemic cortex was significantly higher than that of sham group at 14d(p< 0.05),and returned to normal levels at 28d(p>0.05).After ta-VNS treatment,the expression of PPAR-γ was significantly higher than that of the I/R group at both time points(p < 0.05).In the ischemic cortex,PPAR-γcolocalized with NeuN,a marker for neurons and GFAP,a marker for astrocytes.2.The results of WB and qPCR indicated that CAP activation(ta-VNS)at 14 and 28 d after ischemic stroke could significantly promote the PPAR-γexpression(p < 0.05),while blockade of a7 nAchR could significantly inhibit the promoting effect of ta-VNS on PPAR-γ expression(p < 0.05).3.The results of WB suggested that LV-siPPAR-γ can effectively reduce PPAR-γ expression.4.The immunohistochemical results showed that activation of CAP(ta-VNS)at 28 d after cerebral I/R injury could significantly promote the expression of GAP43(p < 0.05),while inhibition of PPAR-γ expression with corresponding LV significantly inhibited the expression of GAP43(p<0.05).The immunohistochemical results indicated that after cerebral I/R injury,NF200 positive fibers were significantly less and shorter than sham group,and their arrangement was disordered(p<0.05).After 28 consecutive days of ta-VNS treatment,the NF200 positive fiber damage was significantly reduced,while the protective effect of ta-VNS treatment on nerve fibers disappeared after the inhibition of PPAR-γ expression with LV(p < 0.05).5.The immunofluorescence results indicated that,compared with the normal group,partial angiogenesis was observed in the ischemic group at28d(p < 0.05),and the angiogenesis after activation of CAP(ta-VNS)was significantly improved compared with that of the ischemic group(p < 0.05),while the promoting effect of ta-VNS treatment on angiogenesis disappeared after inhibition of PPAR-γ expression by LV(p < 0.05).6.The results of WB suggested that compared with the sham group,the expression of BDNF and VEGF at 28 d after cerebral I/R injury were significantly increased(p < 0.05)and activation of the CAP(ta-VNS)further increased BDNFand VEGF expression in the ischemic cortex(p <0.05),and when the corresponding LV inhibit PPAR-γ expression,thepromoting effect of BDNF and VEGF expression by ta-VNS treatment were diminished(p < 0.05).Conclusion:After cerebral I/R injury in rats,activation of CAP can affect the expression and secretion of BDNF and VEGF molecules by regulating the activity of downstream PPAR-γ,thus effectively promoting axonal plasticity and angiogenesis.Part four: transcutaneous auricular vagus nerve stimulation can improve the neurofunctional recovery in patients with clinical strokeObjective: to investigate the role of transcutaneous auricular vagus nerve stimulation on neurofunctional recovery in patients with clinical stroke.Methods:From April 2018 to April 2019,according to the inclusion criteria,patients with cerebral infarction were randomly divided into the conventional treatment group(n = 40)and the vagus nerve stimulation group(n = 40).All patients of the two groups had received routine rehabilitation training and basic medication.The patients in the vagus nerve stimulation group received ta-VNS therapy for 28 consecutive days,and the patients in the routine treatment group received no energy output.Fugl-meyer motor function rating scale(FMA)and functionalindependence rating scale(FIM)were used to assess related neurofunction before and after treatment.Results:There were no significant differences in gender,age,disease course and NIHSS score between the two groups.The improvement of FMA score and independent function FIM score in the ta-VNS group was significantly better than that in the conventional treatment group(P<0.05).Conclusion:Ta-VNS treatment can improve the neurofunctional recovery in patients with clinical stroke.