| Objective: To explore the effect of moxibustion and electroacupuncture(EA)of Baihui”(DU20)on the glymphatic system and the expression of aquaporin 4(AQP4),as well as the polarity distribution of AQP4 in the perivascular space of penetrating arteries in normal mice.Methods: 1.Group and intervention: Eighteen healthy male C57BL/6 mice at 7-9weeks were selected and randomly divided into control group,EA group and moxibustion group,with 6 mice in each group.The heads of the mice in each group were shaved to let DU20 acupoint be exposed.In moxibustion group,moxibustion was applied to DU20 for 30 min.In EA group,an intradermal needle was inserted into DU20 acupoint,and another needle was shallowly inserted into the side of DU20 acupoint as an auxiliary needle.Connect the positive and negative ends of Han’s electroacupuncture instrument to the needle handles of the two acupoints,and the EA parameters were 0.5 m A,100 Hz,30min.Mice in control group were tied for 30 min.2.Vitro fluorescence tracer method to measure the function of glymphatic system of mice: After the intervention,the fluorescent tracer was injected in to the cisterna magna of mice.10 μL of the mixture of OA-45 and FITC-d3(5 μL each)was injected into cisterna magna of the mouse at a rate of 1μL /min for 10 min.After the injection,the mouse was stand for 20 min,and then the mice were subjected to cardiac perfusion.The brain was sliced on a vibratome.Observe and take pictures under a laser scanning microscope,use Image J software to quantify the influx of fluorescent tracers into the brain parenchyma.Analyze the cerebrospinal fluid in the subarachnoid space of each group into the brain to evaluate the influence of moxibustion and EA on glymphatic system.3.Expression of AQP4 and its polarity distribution of AQP4 in the perivascular space of penetrating arteries in normal mice: After the intervention,the fluorescent tracer was injected in to the cisterna magna of mice.After 30 minutes,take cardiac perfusion and vibratory slices of whole brain.Select the most clear and representative forebrain slice(third slice)and hindbrain slice(sixth slice)for AQP4 immunofluorescence.Observe and take pictures under a laser scanning microscope,use Image J software to quantify the expression of AQP4 and its polarity distribution in the perivascular space of penetrating arteries,so as to explain the mechanism of moxibustion and EA on glymphatic system.Results:(1)Both fluorescent tracers accumulated in the perivascular space of penetrating arteries.Small-molecule fluorescent tracers were widely distributed in the brain parenchyma,while large-molecule fluorescent tracers only accumulated in the penetrating arterial paravascular space.(2)Compared with OA-45,the mean fluorescence intensity of FITC-d3 was significantly increased(P<0.001).Compared with control group,the mean fluorescence intensity of FITC-d3 of each brain slice in moxibustion group was significantly increased(P<0.05,P<0.01),and the mean fluorescence intensity of FITC-d3 of each brain slice in EA group increased,but there was no statistical difference.(3)Compared with control group,the mean fluorescence intensity of FITC-d3 of VC and MS of anterior brain slices in moxibustion group was significantly increased(P<0.05,P<0.01).The mean fluorescence intensity of FITC-d3 of DC,LC,VC,HIP,HYP,and THA of posterior brain slices in moxibustion group was increased(P<0.05).(4)Compared with control group,the mean fluorescence intensity of AQP4 in moxibustion group increased significantly(P<0.001),and the polarity distribution of AQP4 in penetrating arterial perivascular space was significantly increased(P<0.05,P<0.01).Conclusion: Moxibustion and EA on DU20 can regulate the glymphatic system,but the increase of the inflow of cerebrospinal fluid of moxibustion on DU20 was stronger than EA on DU20.The mechanism may be related to the up-regulation of AQP4 expression and the increased polarity distribution in penetrating arterial perivascular space of AQP4. |
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