电针对局灶性脑缺血再灌注大鼠脑血管再生及血脑屏障变化影响的实验研究
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摘要
目的：
本课题在导师前期研究成果基础上,采用改良线栓法建立大鼠局灶性脑缺血再灌注模型,以“益气活血,祛瘀生新”为法则,取“百会”、“水沟”、“足三里”穴,观察电针对局灶性脑缺血再灌注大鼠神经行为学,脑TTC染色、HE染色,脑组织中HIF-1α表达,MVD(cD34+细胞)计数,血清白蛋白(Alb)水平及血脑屏障通透性指标——S100β蛋白血清含量的影响,从而探讨电针对大鼠局灶性脑缺血再灌注后血管再生的调节机制,并试图初步探索电针治疗促血管新生与血脑屏障保护之间的相互关系。
方法：
将84只180g～220g清洁级雄性SD大鼠按照数字表随机分为4组,分别为正常对照组12只(不予处理,正常饲养)；假手术组12只(仅分离CCA及IPA至PPA,不插线栓)；模型组30只(改良线栓法制备局灶性脑缺血再灌注模型,按照缺血／再灌注24h、48h、72h又分为3个亚组,每亚组10只)；电针组30只(改良线栓法制备局灶性脑缺血再灌注模型,按照缺血／再灌注24h、48h、72h又分为3个亚组,每亚组10只,加电针治疗)。电针组使用HANS-200A型韩式穴位神经刺激仪,采用疏密波(频率2Hz／15Hz),电流强度约1mA,以引起局部肌肉稍稍颤动为度,刺激“百会”、“水沟”、“足三里”穴,每次持续刺激30min,缺血再灌注2h后行第1次电针刺激,每隔12h再电针刺激1次。分别采用Zea-longa评分法观察实验大鼠神经行为学变化；取脑组织,制片,HE染色,普通光镜下观察脑大体结构变化；TTC染色计算脑梗死体积比例；免疫组化SP法检测HIF-1α表达,MVD(CD34+细胞)计数(Weidner法)；取血清标本,溴甲酚氯(BcG)法测定白蛋白浓度水平,ELISA去测定S-100β蛋白含量；干湿重法计量脑含水量(%)
结果：
(1)脑组织HE染色光镜观察：正常对照组、假手术组无异常。模型组、电针组大鼠脑组织HE染色显示缺血后神经元损伤病理改变随再灌注时间的延长由轻至重。电针组大鼠脑组织缺血坏死区结构较正常对照组、假手术组疏松,但间质水肿不明显,坏死细胞数量较模型组有所减少,排列较模型组规则。
(2) Zea-longa评分：正常对照组、假手术组大鼠均未出现神经功能缺失症状。在我们设定的观察时间窗内(缺血／再灌注24h～72h),模型组大鼠神经功能评分在缺血／再灌注24h最高,处于峰值,随后逐渐下降,至缺血／再灌注72h神经功能评分仍然较高,与正常对照组、假手术组大鼠比较,P<0.01,有非常显著差异性。电针组大鼠神经功能评分在缺血／再灌注24h也是最高,该峰值与模型组峰值比较,P<0.05,有显著差异。电针组大鼠与模型组相比较,缺血／再灌注24h时间点它们的神经功能评分之间出现显著差异,P<0.05；缺血／再灌注48h～72h时间段内它们的神经功能评分之间出现非常显著差异,P<0.01。电针组大鼠在缺血／再灌注72h时间点神经功能评分并未能恢复到正常水平,与正常对照组、假手术组大鼠比较还是有非常显著差异,P<0.01。
(3)HIF-1α的表达：正常对照组、假手术组大鼠脑内出现微量HIF-1α的表达,两者之间无统计差异性,P>0.05。模型组大鼠脑内HIF-1α的表达随缺血／再灌注时间延长不断增加,在我们设定的观察时间窗内至缺血／再灌注72h时出现最高点,并在各个时间点均与正常对照组、假手术组形成非常显著差异,P＜0.01。而电针组大鼠脑内HIF-1α的表达也是随缺血／再灌注时间延长不断增加,且势头较模型组强劲,在我们设定的观察时间窗内至缺血／再灌注72h同样出现最高点,该峰值与模型组峰值比较出现显著差异,P<0.05；同样在各个时间点电针组大鼠脑内HIF-1α的表达也与正常对照组、假手术组形成非常显著差异,P＜0.01。
(4)脑梗死体积比：正常对照组、假手术组大鼠均未出现脑梗死。在我们设定的观察时间窗内(缺血／再灌注24h-72h),模型组和电针组大鼠脑梗死体积比呈现一个基本正态分布,都是在缺血／再灌注48h最大,处于峰值。在缺血／再灌注24h时间点,电针组与模型组比较,大鼠脑梗死体积比之间有显著差异,P<0.05；在缺血/再灌注48h～72h时间段内,电针组与模型组比较,大鼠脑梗死体积比之间有非常显著差异,P＜0.01。与模型组比较,电针组在缺血/再灌注24h时间点大鼠脑梗死体积比减少幅度较小,缺血/再灌注48h～72h时间段大鼠脑梗死体积比减少幅度增大,以缺血/再灌注72h时间点为最大。
(5)缺血脑区MVD计数：正常对照组、假手术组大鼠脑内MVD极小,几乎为零,且它们之间无统计差异,P>0.05。模型组大鼠脑内缺血区MVD随缺血/再灌注时间延长而增加,但是增长幅度有限,其在各个时间点(缺血/再灌注24h～72h)与正常对照组、假手术组比较形成非常显著性差异,P<0.01。电针组大鼠脑内缺血区MVD也随缺血/再灌注时间延长而增加,且增长幅度很大,在各个时间点(尤其是缺血/再灌注72h),与模型组比较形成非常显著性差异,P＜0.01；同时其与正常对照组、假手术组比较也在各个时间点上形成非常显著差异,P<0.01。
(6)血清白蛋白水平：正常对照组大鼠血清白蛋白水平稳定；与正常对照组比较,假手术组大鼠血清白蛋白水平除在缺血/再灌注24h时间点有轻度下降(但无统计学差异,P>0.05)外,其余时间段无明显变化与正常对照组十分接近。模型组和电针组(特别是模型组)大鼠血清白蛋白水平在整个观察时间窗内都呈现明显下降趋势,与正常对照组、假手术组比较,均具有非常显著差异,P＜0.01。在各个时间点电针组大鼠血清白蛋白水平与模型组比较,均具有显著差异,P<0.05。
(7)血清S-100β蛋白浓度：正常对照组、假手术组大鼠在各个时间点血清S-100β蛋白均未被检测到。模型组、电针组大鼠在各个时间点血清中都检测到S-100β蛋白,且与正常对照组、假手术组比较具有非常显著性差异,P<0.01。在我们设定的观察时间窗内(缺血/再灌注24h、48h、72h),模型组、电针组大鼠血清S-100β水平表达规律相似,均从缺血/再灌注24h开始升高,缺血/再灌注48h到达峰值,随后下降,至缺血/再灌注72h仍然维持较高水平,与正常对照组、假手术组大鼠比较,P<0.01。电针组大鼠在各个时间点血清S-100β蛋白水平都较模型组低,P<0.05,表示有显著差异性。
(8)脑含水量：正常对照组、假手术组实验大鼠脑含水量稳定,并保持在正常水平。模型组、电针组实验大鼠脑含水量在缺血再灌注后均明显上升,在各个时间点与正常对照组、假手术组比较,差异性显著,P<0.05甚至P＜0.01。在我们设定的观察时间窗内(缺血／再灌注24h、48h、72h),模型组、电针组实验大鼠脑含水量升高规律相似,均从缺血／再灌注24h开始升高,至缺血／再灌注48h到达峰值,随后下降,缺血／再灌注72h脑含水量仍然维持较高水平,未恢复正常(包括电针组)。电针组大鼠脑含水量在各个时间点都较模型组低,P＜0.05,表示有显著差异性。
结论：
大鼠脑缺血／再灌注后,早期电针治疗介入能改善神经功能缺失症状、提高脑耐缺氧能力、减少脑梗死体积、促进血管新生、一定程度上“内源性”提高血清白蛋白水平、保护血脑屏障、降低脑水肿严重程度,从而发挥积极有效的治疗作用,对抗脑缺血／再灌注损伤。
Objective:
Our research was based on mentor preliminary research results. We setted up MCAO of rats by improved suture method. According to principles of treatment which is "Yiqihuoxue and Quyushengxin", we electro-acupuncture (EA) the piont "Baihui""Shuigou","Zusanli". Then we observed the affect of EA to MCAO rats neurobehavioral score and TTC staining, HE staining, and the expression of HIF-1α, MVD (CD34+) in brain tissure and the level of serum albumin, S-100P (blood-brain barrier permeability index).By doing this, we wanted to investigate the mechanism for the regulation of angiogenesis after focal cerebral ischemia and reperfusion. At the same time, we tried to explain the relationship between the role of angiogenesis and blood-brain barrier permeability by EA.
Methods:
According to the number table, we divided84SD rats (180g-220g, male)of clean grade into four groups randomly including normal control group, sham operation group, model group and EA group. The normal control group haved12rats, which not be processed, the normal feeding. The sham operation group haved12rats, which haved only separation of the CCA and IPA to the PPA, do not plug wire tied. The model group haved30MCAO rats, which is divided into3subgroups and each subgroup of10according to ischemia/reperfusion24h,48h,72h. EA group also haved30MCAO rats, which is divided into3subgroups and each subgroup of10according to ischemia/reperfusion24h,48h,72h, but coupled with electro-acupuncture treatment based on of the model group. We use the HANS-200A type acupoint nerve stimulation instrument, the use of density wave (frequency2Hz/15Hz), about1mA current intensity, a little quiver in order to stimulate local muscle for the degree, to stimulate "Baihui","Shuigou" and "Zusanli" of the EA group rats, each time to stimulate30mins. The1st electro-acupuncture stimulation happened at2h after ischemia and reperfusion. And then, the rats in EA group accepted an electro-acupuncture stimulation every12h.According to the literature method, we have done the following research. We observed changes in the experimental rat neural behavior Zea-longa score. We observed the changes of the brain in general structural by ordinary optical microscope through HE staining way. We calculated the ratio of cerebral infarct volume by Weidner's methods through TTC staining way. We determined the expression of HIF-1a, MVD (CD34+) in brain tissure by immunohistochemistry SP way. We determined the level of serum albumin by BCG and the level of serum S-100β by EILSA way. We measured the brain water content of wet/dry weight method.
Result:
(1) HE staining observed by optical microscope:Normal control group, sham operation group have no abnormal result. The HE staining of model group showed that pathological change to the neuronal damage with the extension of the time points from small to big after ischemia. Loose ischemia necrosis structure of the EA group compared with the sham group, but interstitial edema was not obvious, the number of necrotic cells were reduced compared with the model group, arranged compared with the model group rules.
(2) Zea-longa score:Normal group and sham-operated rats did not appear the symptoms of neurological deficit. Neurological deficit scores of the model group were most obvious in the I/R24hours, at the peak, and then decreased gradually to I/R72h, but neurological deficit scores were still higher than normal control group and sham-operated group (P<0.01).The neurological deficit scores of the EA group were most obvious in the I/R24h, but it is still difference from model group (P<0.05). The neurological deficit score of EA group and model group compared with I/R24h showed a significant difference (P<0.05); In I/R48h and72h, the neurological deficit score still showed a particularly significant difference (P<0.01) compared with the model group. The neurological deficit scores of EA group did not fully recover to normal levels. Compared with normal control group and sham operation, it haved a special significant difference (P<0.01)
(3) The expression of HIF-1a:Micro-expression of HIF-1α appeared in the rat brain of normal group and sham-operated group. The expression of HIF-1α in the rat brain of model group increased with I/R time. It arrived at the highest point in I/R72h.It constituted a particularly significant difference with the normal group and sham-operated group (P<0.01). The stronger expression of HIF-1a in the rat brain of EA group increased with I/R time compared with model group (P<0.05).It also arrived at the highest point in I/R72h.
(4) The ratio of cerebral infarct volume:Normal group and sham-operated rats did not appear to cerebral infarction. In our observation time window, the volume of cerebral infarction in rats brain of model group and EA group presented a basic normal distribution. The two peak appeared at I/R48h both. In the I/R24h time window, the ratio of cerebral infarct volume between the EA group and model group showed significant difference (P<0.05) Special significant difference (P<0.01) between the EA group and model group appeared in the I/R48h,72h time window. The reducing of ratio of cerebral infarct volume in rats of EA group was bigger than that of model group with I/R time increasing. The peak appeared at I/R72h.
(5) The MVD in ischemia brain regions:The MVD in the rats brain of normal control group and sham group were almost zero. The limited growth of MVD in rats ischemia brain regions of model group constituted a particularly significant difference with the normal control group and sham group (P<0.01). The MVD in rats ischemia brain regions of EA group increased with time especially in the I/R72h time point. Compared with the model group, it constituted a particularly significant difference (P<0.01)
(6) The level of serum albumin:The level of serum albumin of normal control group keeped stability.Compared with the normal control group, the level of serum albumin of sham group rat was almost unchangde except the slight decrease in I/R24h which was no significant difference(P>0.05). The level of serum albumin of model group and EA group showed a clear downward trend with particularly significant difference (P<0.01), in particular the model group, compared with normal control group and sham group during the entire observation period. The level of serum albumin of EA group rat compared with model group showed significant difference (P<0.05). But the level of serum albumin of of EA group rat compared with normal control group and sham-operation group showed no difference (P>0.05).
(7) The level of serum S100β:We could not detected any S-100β protein in normal control group and sham-operated rats serum at each time point. The S-100β protein could be detected in the serum of model group and EA group rats at each time piont, which haved a very significant difference from the normal control group and sham-operation group (P<0.01). Both model group and EA group haved the similar expression pattern of the level of serum S100β.The level of serum S100β increased from I/R24h began, reached the peak at I/R48h and then decreased. It still maintained a high level at I/R72h during the observation period we setted. The level of serum S100β of EA group indicaed significant differences from the model group at each time point (P<0.05)
(8) Brain water content:Thebrain water content in bothnormal control group and sham-operation group rats keeped normal levels.Compared with normal control group and sham-operation group, the brain water content in both model group and EA group rats showed significantly increased after ischemia and reperfusion. The difference was obvious. Both model group and EA group haved the similar expression pattern of the brain water content.The brain water content increased from I/R24h began, reached the peak at I/R48h and then decreased. It still maintained a high level at I/R72h during the observation period we setted. The brain water content of EA group indicated significant difference from the model group at each time point (P<0.05)
Conclusion:
Early EA intervention can play as a positive and effective treatment against brain ischemia and reperfusion injury by improving the symptoms of neurological deficit, improving cerebral hypoxia tolerance, reducing the volume of cerebral infarction, promoting angiogenesis, increasing serum albumin levels to a certain extent endogenous, protecting the blood-brain barrier and reducing the severity of cerebral edema.
本课题在导师前期研究成果基础上,采用改良线栓法建立大鼠局灶性脑缺血再灌注模型,以“益气活血,祛瘀生新”为法则,取“百会”、“水沟”、“足三里”穴,观察电针对局灶性脑缺血再灌注大鼠神经行为学,脑TTC染色、HE染色,脑组织中HIF-1α表达,MVD(cD34+细胞)计数,血清白蛋白(Alb)水平及血脑屏障通透性指标——S100β蛋白血清含量的影响,从而探讨电针对大鼠局灶性脑缺血再灌注后血管再生的调节机制,并试图初步探索电针治疗促血管新生与血脑屏障保护之间的相互关系。
方法：
将84只180g～220g清洁级雄性SD大鼠按照数字表随机分为4组,分别为正常对照组12只(不予处理,正常饲养)；假手术组12只(仅分离CCA及IPA至PPA,不插线栓)；模型组30只(改良线栓法制备局灶性脑缺血再灌注模型,按照缺血／再灌注24h、48h、72h又分为3个亚组,每亚组10只)；电针组30只(改良线栓法制备局灶性脑缺血再灌注模型,按照缺血／再灌注24h、48h、72h又分为3个亚组,每亚组10只,加电针治疗)。电针组使用HANS-200A型韩式穴位神经刺激仪,采用疏密波(频率2Hz／15Hz),电流强度约1mA,以引起局部肌肉稍稍颤动为度,刺激“百会”、“水沟”、“足三里”穴,每次持续刺激30min,缺血再灌注2h后行第1次电针刺激,每隔12h再电针刺激1次。分别采用Zea-longa评分法观察实验大鼠神经行为学变化；取脑组织,制片,HE染色,普通光镜下观察脑大体结构变化；TTC染色计算脑梗死体积比例；免疫组化SP法检测HIF-1α表达,MVD(CD34+细胞)计数(Weidner法)；取血清标本,溴甲酚氯(BcG)法测定白蛋白浓度水平,ELISA去测定S-100β蛋白含量；干湿重法计量脑含水量(%)
结果：
(1)脑组织HE染色光镜观察：正常对照组、假手术组无异常。模型组、电针组大鼠脑组织HE染色显示缺血后神经元损伤病理改变随再灌注时间的延长由轻至重。电针组大鼠脑组织缺血坏死区结构较正常对照组、假手术组疏松,但间质水肿不明显,坏死细胞数量较模型组有所减少,排列较模型组规则。
(2) Zea-longa评分：正常对照组、假手术组大鼠均未出现神经功能缺失症状。在我们设定的观察时间窗内(缺血／再灌注24h～72h),模型组大鼠神经功能评分在缺血／再灌注24h最高,处于峰值,随后逐渐下降,至缺血／再灌注72h神经功能评分仍然较高,与正常对照组、假手术组大鼠比较,P<0.01,有非常显著差异性。电针组大鼠神经功能评分在缺血／再灌注24h也是最高,该峰值与模型组峰值比较,P<0.05,有显著差异。电针组大鼠与模型组相比较,缺血／再灌注24h时间点它们的神经功能评分之间出现显著差异,P<0.05；缺血／再灌注48h～72h时间段内它们的神经功能评分之间出现非常显著差异,P<0.01。电针组大鼠在缺血／再灌注72h时间点神经功能评分并未能恢复到正常水平,与正常对照组、假手术组大鼠比较还是有非常显著差异,P<0.01。
(3)HIF-1α的表达：正常对照组、假手术组大鼠脑内出现微量HIF-1α的表达,两者之间无统计差异性,P>0.05。模型组大鼠脑内HIF-1α的表达随缺血／再灌注时间延长不断增加,在我们设定的观察时间窗内至缺血／再灌注72h时出现最高点,并在各个时间点均与正常对照组、假手术组形成非常显著差异,P＜0.01。而电针组大鼠脑内HIF-1α的表达也是随缺血／再灌注时间延长不断增加,且势头较模型组强劲,在我们设定的观察时间窗内至缺血／再灌注72h同样出现最高点,该峰值与模型组峰值比较出现显著差异,P<0.05；同样在各个时间点电针组大鼠脑内HIF-1α的表达也与正常对照组、假手术组形成非常显著差异,P＜0.01。
(4)脑梗死体积比：正常对照组、假手术组大鼠均未出现脑梗死。在我们设定的观察时间窗内(缺血／再灌注24h-72h),模型组和电针组大鼠脑梗死体积比呈现一个基本正态分布,都是在缺血／再灌注48h最大,处于峰值。在缺血／再灌注24h时间点,电针组与模型组比较,大鼠脑梗死体积比之间有显著差异,P<0.05；在缺血/再灌注48h～72h时间段内,电针组与模型组比较,大鼠脑梗死体积比之间有非常显著差异,P＜0.01。与模型组比较,电针组在缺血/再灌注24h时间点大鼠脑梗死体积比减少幅度较小,缺血/再灌注48h～72h时间段大鼠脑梗死体积比减少幅度增大,以缺血/再灌注72h时间点为最大。
(5)缺血脑区MVD计数：正常对照组、假手术组大鼠脑内MVD极小,几乎为零,且它们之间无统计差异,P>0.05。模型组大鼠脑内缺血区MVD随缺血/再灌注时间延长而增加,但是增长幅度有限,其在各个时间点(缺血/再灌注24h～72h)与正常对照组、假手术组比较形成非常显著性差异,P<0.01。电针组大鼠脑内缺血区MVD也随缺血/再灌注时间延长而增加,且增长幅度很大,在各个时间点(尤其是缺血/再灌注72h),与模型组比较形成非常显著性差异,P＜0.01；同时其与正常对照组、假手术组比较也在各个时间点上形成非常显著差异,P<0.01。
(6)血清白蛋白水平：正常对照组大鼠血清白蛋白水平稳定；与正常对照组比较,假手术组大鼠血清白蛋白水平除在缺血/再灌注24h时间点有轻度下降(但无统计学差异,P>0.05)外,其余时间段无明显变化与正常对照组十分接近。模型组和电针组(特别是模型组)大鼠血清白蛋白水平在整个观察时间窗内都呈现明显下降趋势,与正常对照组、假手术组比较,均具有非常显著差异,P＜0.01。在各个时间点电针组大鼠血清白蛋白水平与模型组比较,均具有显著差异,P<0.05。
(7)血清S-100β蛋白浓度：正常对照组、假手术组大鼠在各个时间点血清S-100β蛋白均未被检测到。模型组、电针组大鼠在各个时间点血清中都检测到S-100β蛋白,且与正常对照组、假手术组比较具有非常显著性差异,P<0.01。在我们设定的观察时间窗内(缺血/再灌注24h、48h、72h),模型组、电针组大鼠血清S-100β水平表达规律相似,均从缺血/再灌注24h开始升高,缺血/再灌注48h到达峰值,随后下降,至缺血/再灌注72h仍然维持较高水平,与正常对照组、假手术组大鼠比较,P<0.01。电针组大鼠在各个时间点血清S-100β蛋白水平都较模型组低,P<0.05,表示有显著差异性。
(8)脑含水量：正常对照组、假手术组实验大鼠脑含水量稳定,并保持在正常水平。模型组、电针组实验大鼠脑含水量在缺血再灌注后均明显上升,在各个时间点与正常对照组、假手术组比较,差异性显著,P<0.05甚至P＜0.01。在我们设定的观察时间窗内(缺血／再灌注24h、48h、72h),模型组、电针组实验大鼠脑含水量升高规律相似,均从缺血／再灌注24h开始升高,至缺血／再灌注48h到达峰值,随后下降,缺血／再灌注72h脑含水量仍然维持较高水平,未恢复正常(包括电针组)。电针组大鼠脑含水量在各个时间点都较模型组低,P＜0.05,表示有显著差异性。
结论：
大鼠脑缺血／再灌注后,早期电针治疗介入能改善神经功能缺失症状、提高脑耐缺氧能力、减少脑梗死体积、促进血管新生、一定程度上“内源性”提高血清白蛋白水平、保护血脑屏障、降低脑水肿严重程度,从而发挥积极有效的治疗作用,对抗脑缺血／再灌注损伤。
Objective:
Our research was based on mentor preliminary research results. We setted up MCAO of rats by improved suture method. According to principles of treatment which is "Yiqihuoxue and Quyushengxin", we electro-acupuncture (EA) the piont "Baihui""Shuigou","Zusanli". Then we observed the affect of EA to MCAO rats neurobehavioral score and TTC staining, HE staining, and the expression of HIF-1α, MVD (CD34+) in brain tissure and the level of serum albumin, S-100P (blood-brain barrier permeability index).By doing this, we wanted to investigate the mechanism for the regulation of angiogenesis after focal cerebral ischemia and reperfusion. At the same time, we tried to explain the relationship between the role of angiogenesis and blood-brain barrier permeability by EA.
Methods:
According to the number table, we divided84SD rats (180g-220g, male)of clean grade into four groups randomly including normal control group, sham operation group, model group and EA group. The normal control group haved12rats, which not be processed, the normal feeding. The sham operation group haved12rats, which haved only separation of the CCA and IPA to the PPA, do not plug wire tied. The model group haved30MCAO rats, which is divided into3subgroups and each subgroup of10according to ischemia/reperfusion24h,48h,72h. EA group also haved30MCAO rats, which is divided into3subgroups and each subgroup of10according to ischemia/reperfusion24h,48h,72h, but coupled with electro-acupuncture treatment based on of the model group. We use the HANS-200A type acupoint nerve stimulation instrument, the use of density wave (frequency2Hz/15Hz), about1mA current intensity, a little quiver in order to stimulate local muscle for the degree, to stimulate "Baihui","Shuigou" and "Zusanli" of the EA group rats, each time to stimulate30mins. The1st electro-acupuncture stimulation happened at2h after ischemia and reperfusion. And then, the rats in EA group accepted an electro-acupuncture stimulation every12h.According to the literature method, we have done the following research. We observed changes in the experimental rat neural behavior Zea-longa score. We observed the changes of the brain in general structural by ordinary optical microscope through HE staining way. We calculated the ratio of cerebral infarct volume by Weidner's methods through TTC staining way. We determined the expression of HIF-1a, MVD (CD34+) in brain tissure by immunohistochemistry SP way. We determined the level of serum albumin by BCG and the level of serum S-100β by EILSA way. We measured the brain water content of wet/dry weight method.
Result:
(1) HE staining observed by optical microscope:Normal control group, sham operation group have no abnormal result. The HE staining of model group showed that pathological change to the neuronal damage with the extension of the time points from small to big after ischemia. Loose ischemia necrosis structure of the EA group compared with the sham group, but interstitial edema was not obvious, the number of necrotic cells were reduced compared with the model group, arranged compared with the model group rules.
(2) Zea-longa score:Normal group and sham-operated rats did not appear the symptoms of neurological deficit. Neurological deficit scores of the model group were most obvious in the I/R24hours, at the peak, and then decreased gradually to I/R72h, but neurological deficit scores were still higher than normal control group and sham-operated group (P<0.01).The neurological deficit scores of the EA group were most obvious in the I/R24h, but it is still difference from model group (P<0.05). The neurological deficit score of EA group and model group compared with I/R24h showed a significant difference (P<0.05); In I/R48h and72h, the neurological deficit score still showed a particularly significant difference (P<0.01) compared with the model group. The neurological deficit scores of EA group did not fully recover to normal levels. Compared with normal control group and sham operation, it haved a special significant difference (P<0.01)
(3) The expression of HIF-1a:Micro-expression of HIF-1α appeared in the rat brain of normal group and sham-operated group. The expression of HIF-1α in the rat brain of model group increased with I/R time. It arrived at the highest point in I/R72h.It constituted a particularly significant difference with the normal group and sham-operated group (P<0.01). The stronger expression of HIF-1a in the rat brain of EA group increased with I/R time compared with model group (P<0.05).It also arrived at the highest point in I/R72h.
(4) The ratio of cerebral infarct volume:Normal group and sham-operated rats did not appear to cerebral infarction. In our observation time window, the volume of cerebral infarction in rats brain of model group and EA group presented a basic normal distribution. The two peak appeared at I/R48h both. In the I/R24h time window, the ratio of cerebral infarct volume between the EA group and model group showed significant difference (P<0.05) Special significant difference (P<0.01) between the EA group and model group appeared in the I/R48h,72h time window. The reducing of ratio of cerebral infarct volume in rats of EA group was bigger than that of model group with I/R time increasing. The peak appeared at I/R72h.
(5) The MVD in ischemia brain regions:The MVD in the rats brain of normal control group and sham group were almost zero. The limited growth of MVD in rats ischemia brain regions of model group constituted a particularly significant difference with the normal control group and sham group (P<0.01). The MVD in rats ischemia brain regions of EA group increased with time especially in the I/R72h time point. Compared with the model group, it constituted a particularly significant difference (P<0.01)
(6) The level of serum albumin:The level of serum albumin of normal control group keeped stability.Compared with the normal control group, the level of serum albumin of sham group rat was almost unchangde except the slight decrease in I/R24h which was no significant difference(P>0.05). The level of serum albumin of model group and EA group showed a clear downward trend with particularly significant difference (P<0.01), in particular the model group, compared with normal control group and sham group during the entire observation period. The level of serum albumin of EA group rat compared with model group showed significant difference (P<0.05). But the level of serum albumin of of EA group rat compared with normal control group and sham-operation group showed no difference (P>0.05).
(7) The level of serum S100β:We could not detected any S-100β protein in normal control group and sham-operated rats serum at each time point. The S-100β protein could be detected in the serum of model group and EA group rats at each time piont, which haved a very significant difference from the normal control group and sham-operation group (P<0.01). Both model group and EA group haved the similar expression pattern of the level of serum S100β.The level of serum S100β increased from I/R24h began, reached the peak at I/R48h and then decreased. It still maintained a high level at I/R72h during the observation period we setted. The level of serum S100β of EA group indicaed significant differences from the model group at each time point (P<0.05)
(8) Brain water content:Thebrain water content in bothnormal control group and sham-operation group rats keeped normal levels.Compared with normal control group and sham-operation group, the brain water content in both model group and EA group rats showed significantly increased after ischemia and reperfusion. The difference was obvious. Both model group and EA group haved the similar expression pattern of the brain water content.The brain water content increased from I/R24h began, reached the peak at I/R48h and then decreased. It still maintained a high level at I/R72h during the observation period we setted. The brain water content of EA group indicated significant difference from the model group at each time point (P<0.05)
Conclusion:
Early EA intervention can play as a positive and effective treatment against brain ischemia and reperfusion injury by improving the symptoms of neurological deficit, improving cerebral hypoxia tolerance, reducing the volume of cerebral infarction, promoting angiogenesis, increasing serum albumin levels to a certain extent endogenous, protecting the blood-brain barrier and reducing the severity of cerebral edema.
引文
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