Mechanism Of Transcutaneous Electrical Acupoint Stimulation Through SHH-ptch-smo-gli1 Signal Pathways Facilitated Synaptic Plasticity Improve Cognition In VD Rats

Objective:Part 1:The changes of cerebral blood flow and activation of brain regions in patients with vascular dementia(VD)and healthy elder(ELD)during different cognitive tasks were investigated based on functional near-infrared spectroscopy(FNIRS).To explore whether transcutaneous electrical acupoint stimulation(TEAS)improves the cognitive function of VD rats through SHH-Ptch-Smo-Gli1 Signal Pathways Facilitated Synaptic Plasticity.The aim is to develop a safe,effective and affordable rehabilitation technique and provide a neuroscientific basis for thetreatment of VD.Methods:Part 1:In this study,20 patients with post-stroke cognitive dysfunction(VD group)who met the inclusion criteria were enrolled in Ningbo Hospital(July 2022 to December 2022),and 20age-matched healthy elderly people(ELD group)were recruited.Subjects in both groups were evaluated by mini-mental state examination(MMSE).Meanwhile,FNIRS was used to monitor blood flow and brain activation in the two groups during Verbal Fluency Task(VFT),continuous subtract-7 and Stroop cognitive tasks.By recording the concentration of oxygenated hemoglobin(Oxy-Hb)and activation of 3D visualized brain regions in pre-frontal cortex(PFC)and hemoglobin in two groups under different cognitive paradigms-hemoglobin,the neurovascular coupling between VD patients and ELD subjects during cognitive tasks was compared to explorethe pathogenesis of VD.Part 2:Atotal of 80 healthy male Sprague Dawley(SD)rats(250-300g)were divided into: Sham group(Sham),Model group(Model),TEAS group(TEAS),TEAS+SHH inhibitor group(TEAS+cyc),and EAgroup(EA).Except the Sham,all the other groups adopted the improved two-vesselocclusion(2-VO)method to preparethe VD rat model.After successful modeling,TEAS rats were treated with TEAS stimulation at Baihui and Zusanli point for 9 days.TEAS +cyc group rats were given TEAS treatment and injection of the SHH-specific inhibitor cipropylamine solution(10mg/kg),and the other group were injected with PBS solution.The rats in EA were stimulated by EAat Baihui point and Zusanli point for 9 days.The rats in Sham and Model were held captive every day without treatment.After treatment,the novel object recognition test(NORT)and morris water maze(MWM)experiments were used to detect the changes in cognitive behavior of rats.Hematoxylin-eosin staining(HE)was used to observe the morphologic changes of neurons in CA1 region of hippocampus.The changes of synaptic structure and morphology in hippocampal CA1 region were observed by transmission electron microscopy.The protein expressions of SHH,Ptch,Smo,Gli1,SYN,PSD-95 and GAP-43 in CA1 region of the hippocampus of VD rats were detected by Western Blot assay after TEAS and EAintervention.Results:Part 1:1.When performing the VFT,continuous subtract-7 and Stroop cognitive paradigm,compared with the resting state,the Oxy-Hb in the PFC region in the task state of ELD subjects increased,while the Oxy-Hb inthe PFC region inthe task state of VD patients decreased.2.When performing the VFT,continuous subtract-7 and Stroop cognitive paradigm,the relative changes of Oxy-Hb(△Hb O)in VD group during the rest and task phases were significantly reduced compared with ELD group.3.The results of 3D visualization showed that the PFC region in ELD group was significantly activated during VFT,subtract-7 and Stroop cognitive paradigm,and theactivation was most obvious during Stroop task,while the PFC region was not significantly activated in VDpatients.Part 2:1.Neurobehavioral experiments showed that compared with Sham group,discrimination index(DI)and residence time in the target quadrant of rats in Model group decreased,and escape latency was prolonged.After TEAS and EA treatment,DI and residence time in the target quadrant were increased and escape latency was shortened in VD rats.In the TEAS+cyc group,DI and residence time in the target quadrant were decreased and the escape latency was prolonged compared with TEAS group.After TEAS and EAtreatment,the spatial learning and memory function of VD rats improved.2.HE showed that the hippocampal neurons in the TEAS and EA groups were more orderly arranged,the number of neurons increased,and the cell structure was more complete than Model,while the neurons in the TEAS+cyc group were disordered and the cell structure was less complete than TEAS.TEAS can improve the morphological structure of neurons in thehippocampus of VDrats.3.TEM results showed that compared with Model,hippocampus synapses in TEAS and EA groups were more densely distributed,with clear synaptic structure,distinct anterior and posterior boundaries,abundant presynaptic vesicles and deep staining.TEAS+cyc had fewer synapses than TEAS.TEAS canimprove synaptic morphology inhippocampus of VD rats.4.Western Blot results showed that compared with Sham group,the expressions of SHH,Ptch,Smo,Gli1,SYN,PSD-95 and GAP-43 in Model group decreased.Compared with the Model group,the expressions of Smo,Gli1,SYN and GAP-43 increased in the TEAS group,the expressions of SHH,Ptch,Smo,Gli1,SYN,PSD-95 and GAP-43 were increased in EA group.Compared with the TEAS group,the proteins of SHH,Ptch,Smo,Gli1,SYN,PSD-95 and GAP-43 decreased inthe TEAS+cyc group.Conclusion:1.Reduced blood flowand dysfunction in theprefrontal cortex inpatients with VD.2.TEAS improve cognitive function in VD rats by promoting synaptic plasticity through SHH-Ptch-Smo-Gli1.