早期康复训练后大鼠脑缺血半影区生长蛋白Gap-43及突触素P38 mRNA表达的变化(英文)

Expression of growth-associated protein Gap-43 and synaptophysin P38 mRNA in rat ischemic penumbra region after earlier rehabilitative training

背景:近年来证实,成熟大脑在存活期间仍具有再生的可塑性,即结构和功能的重建。发育成熟的中枢神经系统,在脑缺血时Gap-43表达水平是评估轴突损伤和再生反应的一个重要指标。缺血性脑卒中后神经功能的恢复与脑的可塑性有关。目的:探讨早期康复训练对大鼠脑缺血半影区与神经重塑有关的生长蛋白Gap-43和突触素P38mRNA的影响。设计:随机对照实验。单位:青岛大学医学院人体解剖学教研室。材料:实验于1999-12/2002-06在青岛大学医学院神经解剖实验室完成。选取成年健康雌性Wistar大鼠52只,随机分为假手术组4只,康复治疗组24只,自然恢复组24只。方法:康复治疗组和自然恢复组采用线栓法经颈外动脉插入4-0尼龙线建立大鼠大脑中动脉闭塞再灌注模型,假手术组除尼龙线插不到起始部位外,其余步骤相同。动物苏醒后出现左侧Horner征,提尾时右前肢内收屈曲,爬行时向右划圈为手术成功的标志。假手术组于术后24h取材;康复治疗组大脑中动脉闭塞120min再灌注6h后置于MG迷宫中进行康复训练,2次/d,10～20min/次,保证动物不疲劳,于术后6h,1,3,7,14,21d取材;自然恢复组术后120min再灌注6h,1,3,7,14,21d取材,作为自然病程对照。常规制作石蜡切片进行尼氏染色,应用VIDAS21显微图像处理系统测定缺血半影区的反应产物吸光度(A)值,以同一张切片上未受损胼胝A值为背影,减去背影A值,得到校正A值。对缺血中心区的免疫活性未作量化处理。主要观察指标:①主要结局:脑缺血半影区生长蛋白Gap-43及突触素P38mRNA原位杂交检测结果。②次要结局:康复治疗组缺血半影区细胞形态学观察。结果:实验纳入的52只大鼠全部进入结果分析。①脑缺血再灌注后不同时间点各组缺血半影区生长蛋白Gap-43和P38mRNA的表达:假手术组皮质区着色浅,Gap-43和P38mRNA表达的校正A值分别为0.37±0.12,0.70±0.14;自然恢复组Gap-43和P38mRNA表达均于术后6h和3d开始增高,第7天达到高峰,第14天开始降低。康复治疗组Gap-43和P38mRNA的表达在缺血再灌注后6h,1,3,7,14,21d均高于同期自然恢复组,但仅在第14天差异显著(1.19±0.60,0.87±0.18,t=4.13,P<0.05)。②康复治疗组细胞形态学观察结果:低倍镜下可见缺血中心区神经细胞坏死,缺血半影区神经细胞的密度降低,神经元缺血样变性。胞体缩小呈三角形、扇形或长条形,细胞核固缩深染、核仁不清,胞浆呈空泡状。皮质神经元表现为胞浆疏松,核轻度变形、深染。变性神经元与正常神经元共存。手术14d后缺血中心区和半影区可见大量胶质细胞增生。结论:与神经重塑有关的Gap-43和P38mRNA在局部脑缺血再灌注后缺血半影区的表达十分活跃。早期康复训练能够使Gap-43和P38mRNA神经突起出芽并形成新的突触,从而增加脑的可塑性,可能涉及脑缺血后肢体功能的恢复。

BACKGROUND： Recent researches have shown that mature survival brain has been proved possessing regenerative plasticity, namely structural and functional rebuilding. In mature CNS, Gap-43 level can be used as an important index for axonal impairment and regenerative reaction during brain isehemia. Moreover neural functional recovery, is proved closely connected with brain plasticity after ischemic stroke. OBJECTIVE： To investigate the influence of earlier rehabiftative training on the expression of neural plasticity related with growth-associated protein Gap-43 and synaptophysin P38 mRNA in isehemic penumbra region of rat. DESIGN： Randomized controlled study. SETTING： Anatomical Department of Affiliated Hospital of Qingdao University. MATERIALS： This study was conducted at Anatomical Laboratory of Affiliated Hospital of Qingdao University from December 1999 to June 2002. Totally 52 healthy female Wistar rats were randomly divided into sham operation group of 4 rats, rehabilitative training group of 24 rats and nature recovery group of 24 rats. METHODS： Thread bolt methods was used to establish Cerebral Middle Artery IR model on rats in rehabilitative group and nature recovery group by inserting an 4-0 nylon thread from external carotid artery, while rats in sham operation group received the same treatment without thread insertion. The experimental models were believed successfully established, if rat displayed left Homer＇s sign with right forearm flexed and abducted when lifting it up by tails after awakened. Specimen were collected at postoperative 24 hours in sham operative group; while rats in rehabilitative training group received rehabilitative training in MG labyrinth after CMA blocked for 120 minutes and reperfusion for 6 hours, twice a day for 10-20 minutes each lime, in avoid of fatigue. Specimen were collected at postoperative 6 hours, 1, 3, 7, 14, 21 days in operative group; specimen were collected at occlusion 120 minutes reperfusion 6 hours, 1, 3, 7, 14, 21 days in nature recovery group which was used as control of nature course of disease. They were made into wax slices and underwent Nissl＇s staining, meanwhile VIDAS 21 microscopic image analysis system was used to detect the products absorbency in ischemic penumbra region （value A）, unimpaired callus value A in the same slice was considered as background value, therefore adjusted value A could be obtained by removing background value A. The immuneactivity at ischemic center were not quantitatively analyzed. MAIN OUTCOME MEASURES： ① Major outcomes： RT-PCR results of growth-associated protein Gap-43 and synaptophysin P38 mRNA at ischemic penumbra region. ② Secondary. outcomes： Ceil morphological observations at ischemic penumbra region in rehabilitative training group. RESULTS： Totally 52 rats were enrolled in this study and all data were remained in the results analyses. ① The expression of growth-associated protein Gap-43 and synaptophysin P38 mRNA at ischemic penumbra region at various IR time points： Cortical region was lightly stained in sham operation group, and the adjusted value A of Gap-43 and P38 mRNA were 0.37±0.12 and 0.70±0.14 respectively; while the Gap-43 and P38 mRNA expression began increase at postoperative 6 hours and 3 days, reaching to the peak level at 7 days and then gradually descended from onset of 14 days in nature recovery group. The expression of Gap-43 and P38 mRNA in rehabilitative training group were found higher than the corresponding nature recovery group at IR 6 hours, 1, 3, 7, 14 and 21 days, but the difference was proved significantly only at 14 days （1.19±0.60, 0.87±0.18, t =4.13, P 〈 0.05）.② Cell morphological observations in rehabilitative training group： Under low-power microscope, we can observed necrotic nerve cells in isebemie center, with the number of nerve cell decreased and neurons displayed isehemie denaturalization： shrunk cell seemed like triangle, fan and strip with nuclei deeply eolored, the nueleolus was unclear and cytoplasm displayed vacuole denaturalization. Cortical neuronal cytoplasm was loose with nuclei slightly metamorphosed and deep colored. Denatured neurons were co-existed with normal neurons. There was great deal of eollagenocyte that appeared in isehemie center and penumbra region at postoperative 14 days. CONCLUSION： The expression of Gap-43 and P38 mRNA related to neural plasticity becomes active in post IR penumbra region. Since earlier rehabilitative training can stimulate synaptogenesis from Gap-43 and P38 mRNA neurons, thereby improving the brain plasticity and postisehemie limb functional recovery.