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【中图分类号】R852.22【文献标识码】A
【文章编号】0529-1356(2000)02-124
PLASTIC CHANGE IN DENSITY OF PERIVASCULAR PEPTIDERGIC NERVE FIBERS AROUND THE CEREBRAL ARTERIES OF RATS DURING SIMULATED WEIGHTLESSNESS AND ITS REVERSAL
MAO Qin-wen,ZHANG Li-fan,MA Jin,
（Departments of Aerospace Physiology,and Histology and Embryology,）
MAO Qin-wen，ZHANG Yuan-qiang,HUANG Wei-quan
（The Fourth Military Medical University, Xi'an 710032,China）
【Abstract】Objective The aim of the present study was to observe the plastic change of the innervation state of peptidergic nerve fibers around the cerebral arteries of rats during both simulated weightlessness and its reversal. MethodsThe tail-suspension rat model was used to simulate the hemodynamic effect of weightlessness. The innervation state of peptidergic nerve fibers supplying the cerebral arteries of control(CON),4-wk suspended(SUS-4), and 1-wk recovered(RE-1) rats were examined by the free-floating ABC-GDN immunostaining technique in whole mount preparations. ResultsAll these nerve fibers around the cerebral arteries were clear and dark in staining in rats from SUS-4 group, and became dim and discontinuous in appearance in REC-1 rats as compared with that in CON rats.Quant
itatively, in SUS-4 group, the density of the three kinds of nerve fibers around the main cerebral arteries were greater than that in CON group(P＜0.01)， for example, the density of NPY-, CGRP-,and SP-containing fibers in the cerebral arteries in SUS-4 were 70%, 78% and 111% higher than that of CON group (P＜0.01)， respectively. In REC-1, the fiber densities showed a general tendency of decrease in these cerebral arteries as compared with that in CON group, but the differences were not significant(P＞0.05).ConclusionThese data have provided the evidence that the perivascular peptidergic innervation of the cerebral arteries is quite sensitive to local hemodynamic changes induced by simulated weightlessness and the adaptive change can occur rapidly.
【Key words】Simulated weightlessness; Cerebral artery; Perivascular nerve fiber; Neuropeptide Y; Calcitonin gene-related peptide; Substance P; Rat
我们在前一工作已观察到长期头低位可引起大鼠后身动脉血管收缩反应性降低，管壁出现萎缩性变化及肾上腺素能神经支配减弱现象，并提出这可能是导致航天员飞行后立位耐力不良的重要机制之一［1,2］。上述一系列变化显然是动脉血管系统对局部血液动力学条件变化的适应性表现。失重时，由于血液头向转移，航天员还有面部水肿、头痛、鼻充血等症状，可能与头部血流量增大、血管跨壁压力升高有关。我们的工作已证明，这种局部血液动力学变化可引起脑部动脉血管肥厚性变化，收缩反应性增强及肾上腺素能神经支配增强［1～5］。至于是否可引起脑血管肽能神经的支配状态［6］也发生相应改变，尚不清楚。本文报道我们在这方面的观察。
材料和方法
1. 动物模型
雄性SD大鼠(体重250～300g)18只。动物被随机分为3组(每组n=6)，即悬吊4周(SUS-4)，悬吊4周后、恢复1周(REC-1)及同步对照组(CON)。尾部悬吊模拟失重大鼠模型参照陈杰等［7］的方法进行。CON组有3只动物的取材与SUS-4组同步，其余3只对照大鼠与REC-1组取材同时进行。
2.标本制备
腹腔内注射戊巴比妥钠(45 mg/kg)将动物麻醉后，经主动脉插管灌注固定。在3.4 kPa下用Krebs平衡液(含硝普钠10 mg/L，37℃)以1 ml/(min·100g)(体重)的流量灌流15min。然后在相同压力下以4%多聚甲醛250ml灌注固定。开颅，小心取出整脑，浸入相同固定液中进行后固定，过夜。在立体显微镜下，分离包括大脑前动脉(ACA)、大脑中动脉(MCA)、劲内动脉(ICA)、大脑后动脉(PCA)、小脑上动脉(SCA)与基底动脉(BA)在内的完整脑底动脉环；并在镜下仔细剔除血管周围的蛛网膜。再将上述分离的血管组织浸入磷酸缓冲液(PBS，0.01 mol/L,pH 7.4)漂洗备用。
3. 染色程序
采用ABC免疫组织化学技术结合硫酸镍胺增强显色法［8,9］。血管标本均先经3%甲醇双氧水及1%正常猪血清封闭各1h，然后分别入兔抗CGRP、兔抗SP血清(INC公司产品，均为1∶5 000)或兔抗NPY血清(本实验室自制，1∶2 000)中孵育48h，再依次进入生物素标记的猪抗兔IgG血清(DAKO公司产品，1∶500)中孵育12h，卵白素-生物素复合物(avidin biotin complex,ABC;DAKO公司产品，1∶200)中孵育1h。以上孵育均在室温下进行。再入葡萄糖氧化酶-DAB-硫酸镍胺(glucose oxidase-diaminobenzidine-nickel sulfate,GDN)中呈色20 min。以上各步骤之间均用0.01 mol/L PBS (pH7.4)漂洗(10 min×3)。最后在解剖镜下将血管展开，自然干燥，透明，封片。3组标本的免疫组织化学染色过程均系在同一时间、相同条件下进行。
对照试验：用正常猪血清取代第一抗体进行孵育；用第一抗体经过量相应抗原(0.1 mg/L)吸收后进行孵育。
4.血管周围神经纤维密度测量
用交点计数法［10］测量3组大鼠脑动脉血管ACA、MCA、ICA、PCA、SCA和BA血管上的神经纤维密度。在×40物镜与×10目镜(内装测微网格)下，分别计数与血管纵轴／横轴相平行的0.1mm目镜网格线上环行／纵行神经纤维的交点数。在每段血管上均任选3个视野，取平均值，作为本段血管纵行或环行神经纤维的密度。纵行与环行纤维密度之和为血管周围神经纤维的总密度。记录3组大鼠的神经纤维密度。组间数据用t检验进行统计学处理。
结果
大鼠脑动脉经ABC-GDN法染色后，神经纤维呈深蓝色，纤维和膨体清晰完整，背景浅淡，阴性对照未见特异性着色。大鼠脑动脉含NPY神经纤维在各主要脑动脉上分布密集，多呈螺旋状走行。含CGRP与含SP神经纤维较纤细，在脑底动脉环吻侧呈网状分布，在尾侧较稀少且多沿血管纵向走行。图1～9分别表示模拟失重及恢复期含NPY、CGRP及SP神经纤维密度的变化。由图可见：与CON组相比，脑动脉上述几种神经纤维在SUS-4组变得更加清晰、染色更深；而在REC-1组，纤维变得模糊且不连续。
