针刺“百会透曲鬓”穴对脑缺血再灌注大鼠脑微血管内皮细胞黏附分子1表达的影响

Effect of acupuncture at Baihui towards Qubin acupoint on the expression of microvascular endothelial intercellular adhesion molecule-1 in rats with cerebral ischemia reperfusion

目的:观察针刺“百会透曲鬓”穴位对脑缺血再灌注后大鼠脑微血管内皮细胞黏附分子1表达变化的影响。方法:实验于2001-09/2002-05在青岛大学医学院脑血管病研究所完成。选用清洁级SD大鼠15只,随机分为假手术组、针刺组和对照组,5只/组。①分组及造模:针刺组和对照组建立局灶性脑缺血再灌注模型,假手术组手术步骤同对照组,但不阻塞大脑中动脉。针刺组在缺血后10min给予针刺治疗,在大鼠头顶部相当于人体百会、风府穴常规剪毛消毒,用0.35mm×40mm毫针快速平刺入皮下左前下方(相当于人体曲鬓穴),进针深度约0.8cm。另于风府穴直刺一针,深度约0.5cm。连接电麻仪,百会穴接阳极,风府穴接阴极,电针频率7Hz,强度6mA,时间30min。②脑片制备及内皮细胞黏附分子1免疫组化检测:各组大鼠麻醉后,左心室插管经升主动脉快速灌注生理盐水200mL,再持续灌注质量浓度40g/L多聚甲醛300mL,断头取脑,相同固定液后固定1h。自视交叉处开始向后取冠状切片,片厚约7μm。400倍显微镜下观察各组切片微血管阳性反应物沉积情况,随机选取缺血区内3个不重复视野,进行内皮细胞黏附分子1阳性微血管计数。结果:15只大鼠全部进入结果分析。①内皮细胞黏附分子1免疫组化结果:阴性对照组微血管无阳性反应物出现。假手术组、对照组、针刺组非缺血侧微血管均未见明确阳性反应物沉积。对照组缺血侧和针刺组缺血侧可见棕黄色阳性反应物沉积的微血管,主要分布于梗死区局部。②内皮细胞黏附分子1的表达:假手术组几乎不表达,阳性微血管数为0个;对照组呈过表达,阳性微血管数为(9.20±1.30)个;针刺组阳性微血管数为(6.20±0.84)个,较对照组表达降低(t=4.33,P=0.003)。结论:脑缺血再灌注后脑微血管内皮细胞黏附分子1表达增强,针刺“百会透曲鬓”穴位可明显抑制其表达,从而减轻白细胞向周围组织的浸润,减少了大量炎性递质对脑组织的损伤,发挥脑保护作用。

AIM:To observe the effect of acupuncture at Baihui towards Qubin acupoint on the expression of microvascular endothelial intercellular adhesion molecule1. METHODS:The experiment was carried out in the Institute of Cerebrovascular Disease, Medical College of Qingdao University from September 2001 to May 2002.Fifteen SD rats of clean grade were randomly divided into shamoperated group(n=5),acupuncture group(n=5) and control group (n=5).①Grouping and model establishment:Rats in the acupuncture group and control group were made into models of focal cerebral ischemia reperfusion,the operative procedure in the shamoperated group was the same as that in the control group,but the middle cerebral artery was not occluded. Ten minutes after ischemia,rats in the acupuncture group were treated with acupuncture:The hair on the head acupoints,where were equal to the Baihui and Fengfu acupoints of human,was cut and then disinfected,filiform needle of 0.35 mm×40 mm was quickly and horizontally inserted into left anterior region subcutaneously(equal to the acupoint of qubin in human body),the inserted depth was 0.8 cm; Another needle was vertically inserted at the acupoint of Fengfu to the depth of about 0.5 cm;Electroanesthetic instrument was connected, the acupoints of Baihui and Fengfu were connected to the positive and negative poles respectively, the frequency of electroneedle was 7 Hz,the intensity was 6 mA,and the time was 30 minutes.②Preparation of brain section and immunohistochemical detection of endothelial intercellular adhesion molecule1: After rats in each group were anesthetized, left ventricle was intubated and rapidly perfused with 200 mL saline via ascending aorta, and then continuously perfused with 300 mL citromint with the concentration of 40 g/L;The heads were cut down and brain was taken, and then fixed with the same fix liquid for 1 hour;Coronal section was taken backward from optic chiasma, the thickness was about 7 μm.The deposition of the microvascular positive reactant in the section was observed under microscope of 400 resolution, 3 not repeated visual fields in the ischemic region were selected to count the positive microvessels of intercellular adhesion molecule1.RESULTS:All the 15 rats were involved in the analysis of results. ①Immunohistochemical results of intercellular adhesion molecule1: No positive reactant in microvessels was observed in the negative control group;There was no deposition of positive reactant in microvessels in the shamoperated group and on the nonischemic sides in the control group and acupuncture group. Microvessels with deposition of light brown positive reactant,which mainly located in the infarcted regions,could be observed on the ischemic sides in the control group and acupuncture group.②Expression of intercellular adhesion molecule1:Almost no expression was observed in the shamoperated group,and the number of positive microvessel was 0; There was overexpression in the control group,and (9.20±1.30) microvessels in the control group;The number of positive microvessels was 6.20±0.84 in the acupuncture group,and the expression was decreased as compared with that in the control group(t=4.33,P=0.003).CONCLUSION:The expression of microvascular intercellular adhesion molecule1 after ischemia reperfusion is enhanced,and acupuncture at Baihui towards Qubin acupoint can obviously inhibit the expression,so that it can relieve the infiltration of leucocyte to the tissue around it,and reduce the damage of brain tissue induced by plenty of inflammatory transmitters.