脉冲磁场和补阳还五汤对脑缺血再灌注大鼠IGF-1表达影响的研究
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摘要
研究背景:胰岛素生长因子-1(IGF-1)是一种结构上与胰岛素类似,但在中枢神经系统内有其特异性受体的多肽,它有调节代谢的作用,参与调节多种细胞的分化、增殖和多种损伤后的修复。体内几乎所有组织、细胞都能分泌IGF-1,表达IGF-1受体。IGF-1及其受体在正常脑组织中仅有少量表达,以垂体的IGF-1含量最高,嗅球、脑干上部次之,其后依次为小脑、纹状体、海马和大脑皮质。神经细胞和神经胶质细胞都能分泌IGF-1。从胎鼠到成年鼠发育的各个阶段脑组织中都检测到IGF-1,且胎鼠中IGF-1表达明显高于成年鼠,IGF-1是脑生长、存活和分化必不可少的物质。IGF-1是非选择性的神经营养因子,对各种中枢神经细胞具有营养保护作用,除影响其生长、存活和分化外,局灶性脑缺血发生后,IGF系统被激活上调,表达增加。IGF-1可能在抗凋亡过程中起信使作用,通过磷脂酰肌醇3′-激酶/丝氨酸/苏氨酸蛋白激酶级联通道抑制caspase-3活性而发挥脑保护作用,IGF-1也可能通过改变Bcl家族的蛋白表达而抗凋亡,也有学者认为IGF-1通过提高缺血区γ-氨基丁酸水平,拮抗兴奋性氨基酸的兴奋毒性,降低缺血神经元的代谢率和氧的消耗而发挥脑保护作用。
近年来,对磁场的生物学效应及治疗作用进行了深入的研究,磁场对脑损伤性疾病的影响研究已成为一个热点。动物实验和临床研究均证实磁刺激对脑缺血再灌注损伤具有保护作用,有助于改善急性脑梗死患者神经功能缺损。磁场对脑缺血再灌注损伤的保护机制可能与阻断脑缺血再灌注损伤后诱发的一系列链式瀑布反应如减少自由基产生、改善血液流变学、抑制细胞内钙超载、抑制兴奋性氨基酸产生、抑制细胞凋亡等某一环节或多个环节有关。在临床研究上已证实磁场可以促进脑卒中后神经功能恢复,减少肢体残疾的产生,是一种安全有效的方法。有关磁场与IGF-1关系的研究报道不多。本课题设计利用磁场干预大鼠脑缺血再灌注模型,探讨磁场对脑缺血大鼠脑内IGF-1表达的影响以及对神经功能缺失、脑缺血梗死面积、缺血后病理组织学的影响,为磁场的神经保护作用提供实验室基础。
补阳还五汤(BYHWD)始载于《医林改错》,由清代名医王清任所创,以补气药与活血祛瘀药相配伍,以益气固摄为主,化瘀通络为辅,具有补气活血,通经活络的功效,是治疗气虚血瘀型脑缺血疾病的名方。有关BYHWD对脑缺血再灌注损伤的机制研究报道较多,BYHWD可能通过改善血流动力学、抑制自由基的产生、降低一氧化氮(NO)与一氧化氮合成酶(NOS)的活性、防止钙超载、抑制兴奋性氨基酸的释放、促进星形胶质细胞对损伤脑组织的修复、抑制细胞凋亡等多因素对脑缺血再灌注损伤起修复保护作用。目前关于BYHWD与IGF-1的关系未见报道。基于BYHWD的神经保护作用,我们设想利用BYHWD灌注脑缺血再灌注大鼠,观察BYHWD对IGF-1表达的影响以及BYHWD对神经功能缺失、脑缺血梗死面积、缺血后病理组织学的影响,以期发现BYHWD与IGF-1在神经修复方面有无内在关系,为BYHWD的神经保护作用提供新的实验室基础。
目的:观察脑缺血再灌注后时间与IGF-1表达的关系,研究磁场、BYHWD对脑缺血再灌注大鼠IGF-1表达的影响,为磁场、BYHWD的脑保护作用提供实验室依据。
设计:完全随机设计。
方法:1.40只Sprague Dawley(SD)大鼠分为假手术组、缺血再灌注后2h组、1d组、3d组和7d组,每组8只。2h组、1d组、3d组、7d组用线栓法阻断右侧大脑中动脉供血,2h后拨出线栓恢复大脑中动脉供血制成右侧大脑中动脉闭塞(MCAO)模型,假手术组仅做右侧颈外动脉和颈总动脉结扎,不做右侧大脑中动脉线栓栓塞。参照Zeal Longa 5分法进行评分。0分:无神经功能缺损症状;1分:轻度局灶性神经功能缺损,即提尾悬空不能伸展左侧前爪;2分:中度局灶性神经功能缺损,即行走向左侧转圈;3分:中度局灶性神经功能缺损,即行走困难,并向左侧倾倒;4分:不能自发行走,意识水平下降。评分1～4分为造模成功。32只大鼠造模成功。各组动物分别在造模后0h(假手术组)、2h、1d、3d、7d处死,断头取脑。主要观察指标:用免疫组化法(IHC)观察各组梗死区IGF-1的表达。
2.80只SD大鼠分为假手术组、模型组、脉冲磁场组、BYHWD组、脉冲磁场联合BYHWD组(联合组),每组16只,模型组、脉冲磁场组、BYHWD组和联合组制作右侧MCAO模型,假手术组仅做右侧颈外动脉和颈总动脉结扎,不做右侧大脑中动脉线栓栓塞。脉冲磁场组和联合组于造模结束后开始用脉冲磁疗仪进行干预,异名磁极对置于大鼠头部,磁距为7～10cm,磁场强度为0～0.01T,频率为50HZ,20min/次,每天1次,连续7d;BYHWD组和联合组予灌服BYHWD水煎液,按生药13.3g/kg灌胃,连续给药7d;假手术组和模型组不作磁场处理及BYHWD灌胃处理。7d后处死各组大鼠,断头取脑。主要观察指标:①分别在2h、1d、3d、7d进行神经功能缺失评分;②采用2,3,5—氯化三苯基四氮唑(TTC)染色观察梗死面积大小;③苏木素—伊红染色(HE)观察病理组织学改变;④IHC检测IGF-1的表达。
结果:120只大鼠进入结果分析。
1.脑缺血再灌注大鼠IGF-1的表达
采用单因素方差分析的检验方法,组间多重比较采用LSD检验,组间比较有统计学差异(F=60.867,P=0.000)。经组间多重比较显示:IGF-1在假手术组神经细胞中表达最弱,与假手术组相比,缺血再灌注后2h组、1d组、3d组、7d组梗死区阳性细胞数增多,差异有统计学意义(P=0.000);与2h组、1d组相比,3d组阳性细胞数增多,其差异有统计学意义(P=0.000);与3d组相比,7d组阳性细胞数有所下降,差异有统计学意义(P=0.000);与1d组相比,7d组阳性细胞数无显著性差异(P=0.712),但与假手术组相比,差异有统计学意义(P=0.000)。
2.脉冲磁场、BYHWD对脑缺血再灌注大鼠IGF-1表达的影响
2.1 2h、1d、3d、7d神经功能缺失评分
采用重复测量的检验方法,时间因素不同水平组间多重比较及固定时间因素后不同实验分组间的多重比较均采用LSD方法。
假手术组在2h、1d、3d、7d时神经功能缺失评分均为0分。模型组、脉冲磁场组、BYHWD组和联合组各组间比较存在统计学差异(F=5.479,P=0.032),神经功能缺失评分在不同的时间之间有显著差异(F=106.922,P=0.000),组别与时间有交互效应(F=3.867,P=0.000)。从3d开始,各组神经功能缺失评分随时间的延长呈逐渐下降趋势;从时间点来看,2h和1d时模型组、脉冲磁场组、BYHWD组和联合组的评分无显著差异(F=0.760,P=0.521和F=1.538,P=0.214);3d时模型组、脉冲磁场组、BYHWD组和联合组的评分有显著差异(F=12.612,P=0.000),BYHWD组、脉冲磁场组和联合组的评分低于模型组,联合组的评分低于BYHWD组、脉冲磁场组;7d时模型组、脉冲磁场组、BYHWD组和联合组的评分有显著差异(F=4.609,P=0.002),脉冲磁场组、BYHWD组和联合组评分均低于模型组,脉冲磁场组、BYHWD组和联合组评分无显著性差异
2.2脑组织TTC染色结果
脑梗死面积百分数的比较采用单因素方差分析,组间多重比较采用LSD检验。假手术组脑组织切片均红染,无白色梗死灶。模型组大鼠梗死灶位于缺血侧脑组织额顶部外侧皮层,脑梗死灶明显,梗死区内坏死组织呈苍白色,未坏死组织呈玫瑰红色。各组间比较差异有统计学意义(F=30.383,P=0.000)。经组间多重比较显示:与模型组相比,脉冲磁场组、BYHWD组、联合组梗死面积均减小,差异有统计学意义(P=0.000)。与脉冲磁场组相比,BYHWD组梗死面积差异没有显著性意义(P=0.552)。联合组与脉冲磁场组和BYHWD组相比,梗死面积减小,其差异有统计学意义(P=0.009,P=0.035)。
2.3脑组织切片HE染色结果
假手术组:神经细胞数目多,形态结构清楚,细胞排列规整。核深染,核膜清晰,核仁明显。高倍镜下可见神经元细胞胞浆丰富,呈灰蓝色斑块状,表明尼氏体丰富。
模型组:坏死区结构紊乱,组织染色变浅,组织结构疏松,胶质细胞增生,部分神经元细胞核固缩,胞体缩小变形,残留的神经元细胞周围间隙增宽,神经元细胞正常形态消失,胞质红染、均匀,可见鬼影细胞。边缘区低倍镜下可见较多形态大致正常的神经元细胞,胶质细胞无明显增生,组织颜色较深。在高倍镜下可见边缘区部分神经元细胞浆染色呈淡染,尼氏小体消失,并可见神经元细胞核固缩。
脉冲磁场组:坏死区胶质细胞较模型组增生明显,形成胶质结节,出现噬神经现象,仍可见较完整的红色神经元细胞,个别神经元细胞核固缩,胞体缩小变形。边缘区在高倍镜下可见边缘区神经元细胞胞浆蓝染,呈颗粒状,尼氏体丰富。神经元形态较规整,核深染,核仁清。
BYHWD组:坏死区胶质细胞较模型组增生明显,有胶质结节形成,可见较完整的红色神经元细胞,个别神经元细胞核固缩,胞体缩小变形。高倍镜下可见边缘区神经元细胞胞浆蓝染,颗粒状,尼氏体较丰富,神经元形态较规整,核深染,核仁清。
联合组:坏死区胶质细胞较模型组增生明显,可见完整的红色神经元细胞。高倍镜下可见边缘区神经元细胞形态规整,核深染,核仁清,神经元细胞胞浆蓝染明显,呈颗粒状,尼氏体丰富。。
2.4脉冲磁场和BYHWD干预后IGF-1的表达结果
脉冲磁场与BYHWD干预后IGF—1阳性细胞数比较采用单因素方差分析,组间多重比较采用LSD检验。各组间比较,差异有统计学意义(F=592.714,P=0.000)。经组间多重比较显示,IGF-1阳性细胞数在假手术组最少;与假手术组相比,模型组缺血再灌注7d后缺血梗死侧大脑皮层IGF-1阳性细胞数增多,差异有显著性意义(P=0.007);与假手术组和模型组相比,BYHWD组缺血再灌注7d后IGF-1阳性细胞数增多,差异有显著性意义(P=0.000);与假手术组、模型组和BYHWD组相比,脉冲磁场组阳性细胞数增多,差异有统计学意义;与假手术组、模型组、脉冲磁场组和BYHWD组相比,联合组再灌注7d后IGF-1阳性细胞数增多,差异有显著性意义(P=0.000)。
结论:正常情况下脑组织中仅有少量IGF-1表达,脑缺血再灌注发生后,脑中IGF-1的自分泌即启动,再灌注3d后IGF-1的表达达到高峰,7d缺血侧大脑皮层IGF-1的表达有所下降,但仍明显高于假手术组。这表明脑缺血再灌注可促进内源性IGF-1的分泌,有助于缺血状态下神经组织的自我保护和修复,但这种促分泌作用维持时间不长。脉冲磁场和BYHWD干预后缺血再灌注大鼠神经功能评分改善明显、脑梗死面积减小,神经系统的保护和修复作用明显增强,且脉冲磁场、BYHWD可以促进IGF-1的表达,增强脑损伤后的内源性保护机制,二者联合使用在促进神经功能恢复、减小脑梗死面积,及促进IGF-1表达方面作用更强,说明脉冲磁场和BYHWD有协同作用。
Insulin-like growth factor 1 (IGF-1)is a kind of polypeptide and involved in many physiological functions, such as cell proliferation,differentiation and maturation of organic tissues. It is named as IGF because its structure has high homogeneity with insulin prosome. Almost all the tissue can excrete IGF-1 and express IGF-1 receptor. In the physiological condition there are extensive IGF-1 immunoreactivities and IGF-1 mRNA expression in brain. It was found that IGF-1 and IGF-1R extensively existed in central nervous system, especially there were higher expression in hypophysis, then in olfactory bulb and superior brain stem, and followed by cerebrum, striatum, hippocampus and cerebral cortex in order. The normal brain in mature can express lower IGF-1 protein and both neurons and neuroglial cells can express IGF-1 protein. It has been demonstrated that IGF-1 is a kind of neurotrophic factor and has various functions. It can not only promote the differentiation and proliferation of neurons and neuroglial cells in brain, but also accelerate the growth and development of brain. It also protects cerebrum from ischemia reperfusion injury. It is believed that IGF-1 play an important role in the pathophysiological process of cerebral infarction. After cerebral ischemia, the IGF-1 system was activated, its expression and distribution in brain were obviously changed, and its protective actions were activated at same time. In clinical reports, it has been reported that IGF expression is correlated with the attack of ischemic stroke.IGF-1 dripped via nasal cavity or intravenous injection could decrease the expression of caspase-1 in brain after hypoxic ischemic injury, and could protect the brain from damage. The mechanism of protecting brain from damage of IGF-1 is not very clear. IGF-1 could adjust forkhead like1 through the phosphatidylinositol-3-kinase/serine/threonine kinase pathway, inhibit caspase-3 activity, restrain apoptosis of neurons and neuroglial cells or change the expression of Bcl protien or increase the level ofγ-aminobutyric acid, restrain aminobutyric acid and reduce metabolic rate of neuron in ischemic infarcs area and protect the brain.
Magnetic therapy has a long history and is used in the treatment of various diseases. It possesses certain function of protecting brain ischemia reperfusion injury through holding up the pathophysiological development of brain ischemia injury, but the mechanism are not very clear. In animal experiment, magnetic field exposure can improve rat hemodynamic property, increase antioxidase activity, improve organic anti-oxidation capability, prevente free radicals, adjust the intra-cellular calcium content, inhibit the cell apoptosis and reduce neural damage. In clinical research, Magnetic therapy may reduce disability resulting from stroke, and promote neurological and functional improvement. Magnetic therapy can be recognized as a safe method and worth making popular in clinical practice. The experiment is aimed to study the effect of pulsed magnetic field in inducing IGF-1 expressing and the influence on cerebral infarct volume, pathological changes in focal cerebral ischemica reperfusion rat, for providing new theoretic foundation for magnetic therapy.
Buyanghuanwu decoction(BYHWD) is the classical prescriptions for hemiplagia differentiated as qi deficiency and blood stasis created by Wang Qingren,the famous doctor in Qing dynasty in "Yi LingGai Chuo". It has the ability of invigorating qi and activating blood circulation. In all the ingredients of BYHWD, the ingredients invigorating qi act as host, and the ingredients activating blood circulation act as supplement. Many scientists have studied the mechanism of BYHWD and its decomposed formulas on cerebral ischemia. They have demonstrated that BYHWD can improve hemodynamic property, increase antioxidase activity, resist free radicals, decrease the intra-cellular calcium overload, etc. inhibit the cell apoptosis and reduce neural damage. There were no reports about the relationship between BYHWD and IGF-1. In this experiment, cerebral ischemia reperfusion model will be prepared for the observation of the effect of BYHWD on the expresion of IGF-1 in brain tissue, and of the influence of BYHWD on neurologic deficit, cerebral infarct area, pathological changes of brain tissue, to provide new breadboard foundation for BYHWD.
Objective:To observe the effect of pulsed magnetic field and BYHWD in inducing IGF-1 expressing and influence on neurologic deficit, cerebral infarct area, pathological changes in focal cerebral ischemica reperfusion rat, providing new breadboard foundation for magnetic therapy and BYHWD.
Design: Randomized controlled animal experiment.
Methods: 1. Forty SD rats were randomized into 5 groups, namely, sham-operation group, 2h group, 1d group, 3d group and 7d group with 8 in each group. MCAO method was employed to establish focal cerebral ischemia reperfusion model in 2h group, 1d group, 3d group and 7d group, they were occluded for 2h at the right middle cerebral artery, and then they were reperfused, but 8 rats was only tied up without occlusion in sham-operation group. The rats in the experimental group were assessed according to Zeal Longa standard after 2h reperfusion. Without neurologic deficit was marked as 0 point, flexion of left anterior claws when hanging tail as 1 point, circling to left while walking as 2 points, difficulty in walking and falling down to left as 3 points, unconscious mind as 4 points. The rats assessed 1 to 4 point were successful MCAO rats. 32Rats were successfully MCAO. After that, brain tissues were harvested at Oh (sham-operation group), 2h, 1d, 3d and 7d after cerebral ischemic reperfusion injury, 40 rats at each time point. Main outcome measure: IGF-1 expression in the brain tissue was observed by immunohistochemical method.
2. Eighty SD rats were randomized into 5 groups, namely, sham-operation group, model group, pulsed magnetic field group, BYHWD group and pulsed magnetic field combined BYHWD group (combination group) with 16 in each group. Also MCAO method was employed to establish cerebral ischemia reperfusion model in model group, pulsed magnetic field group, BYHWD group and combination group. Rats was only tied up without occlusion in sham-operation group. BYHWD was applied by gastric perfusion at the dosages of 13.3g/kg.d for 7d in BYHWD group and combination group. Rats in pulsed magnetic field group and combination group were exposed to 0 to 0.01T intensity pulsed magnetic field of 50 Hz for 20 minutes per day for 7d. Rats in sham-operation group and model group only eat food and drink freely. The rats in the experimental group were assessed according to Zeal Longa standard after 2h ischemia and 1, 3, 7 d reperfusion respectively. Main outcome measure:①Neurologic deficit test.②Infarct area was detected by TTC staining.③Pathological changes were observed microscopically in HE stained sections.④IGF-1 expression in the brain tissue was observed by immunohistochemical method. Statistical management was carried out with SPSS13.0 software. Data were presented as Mean±SD, Repeated Measures and one-way ANOVE were used.
Results: 120 rats were involved in the final analysis without deletion.
1.Expression of IGF-1 in cerebral ischemica reperfusion rats
One-way ANOVE were used. There were a few IGF-1 positive cells stained lightly in the sham-operation group. The IGF-1 positive cells of 2h group were more than sham-operation group (P=0.000) . The IGF-1 positive cells in 1d group were more than sham-operation group and 2h group (P=0.000) . The IGF-1 positive cells of 3d group were significantly more than all other group (P=0.000) . There were no significant difference between 7d group and 1d group (P=0.712) , but the IGF-1 positive cells of 7d group were more than sham-operation group and 2h group (P = 0.000) .
2. The effect of pulsed magnetic field and BYHWD in inducing IGF-1 expressing and the influence on neurologic deficit, cerebral infarct area, pathological changes in focal cerebral ischemica reperfusion rats
2.1 The neurologic deficit
The scores of neurologic deficit of sham-operation group were 0 point all the time. In othe four groups, repeated measures were used. There were significant difference in the scores of neurologic deficit at 2h, 1d, 3d and 7d (F=106.922, P=0.000) . The scores of neurologic deficit of model group, pulsed magnetic field group and BYHWD group were keeping decreasing all the time. There were significant difference in model group, pulsed magnetic field group , BYHWD group and combination group (F=5.479, P=0.032) .At 2h and 1d there were no significant difference in all the groups(F=0.760, P=0.521; F=1.538, P=0.214). After 3 to 7day reperfusion the scores of neurologic deficit of pulsed magnetic field group and BYHWD group were lower than that of model group ((F=12.612, P=0.000; F=4.609, P=0.002).
2.2 Cerebral infarct area
One-way ANOVE were used. There were no infarcts in sham-operation group. The infarct area of pulsed magnetic field group and BYHWD group were significant less than that of model group (P = 0.000) . There were no significant difference between pulsed magnetic field group and BYHWD group (P=0.552). The infarct area of combination group were significant less than that of pulsed magnetic field group and BYHWD group (P=0.009, P=0.035) .
2.3 Pathological changes
In the sham-operation group, the morphology of neurons were ovale or multi-angles in shapes, Nissl bodies were stained as dark blue. So there were no obviously changes in sham-operation group. In model group, many neurons disappeared in the center of the infarcts, the nuclei of the remaining neurons were pyknotic and the neural fibers became vacuolar. There were no Nissl bodies. Many neurons became pyknotic and deep-stained around the infarcs. Neuron defects in the infarcted center of pulsed magnetic field group, BYHWD group and combination group were significantly slighter than that of model group. The morphology of many neurons were also ovale or multi-angles in shapes, Nissl bodies still existed and were stained as blue. A few neurons became pyknotic and deep-stained around the infarcs.
2.4 Expression of IGF-1
One-way ANOVE were used.There were a few IGF-1 positive cells stained lightly in the sham-operation group. The IGF-1 positive cells of model group were more than sham-operation group (P = 0.007) .The IGF-1 positive cells of pulsed magnetic field group and BYHWD group were more than model group (P = 0.000) . The IGF-1 positive cells in combination group were more than pulsed magnetic field group and BYHWD group (P = 0.000) .
Conclusion: The expression of IGF-1 was very low in normal brain tissues. The positive cells of IGF-1 higher expressions maintained from 2h to 7d after cerebral ischemia reperfusion, and reached the peak value at 3d, then decreased at 7d, but maintained a higher level than normal brain tissues. It suggests that cerebral ischemia reperfusion injury can enhanced expression of endogenous IGF-1, but the expression are limited and last for shorter durition. After cerebral ischemia reperfusion the neurological function is damaged, and it has a trend of self-recovery, but self-recovery are very weak. Pulsed magnetic field and BYHWD can promote recovery of neurological function deficit and decrease the infarct area. Pulsed magnetic field and BYHWD can protect the brain tissue after cerebral ischemia reperfusion, and promote IGF-1 expressing. The effect of pulsed magnetic field combined BYHWD on protecting the brain tissue after cerebral ischemia reperfusion, and promoting IGF-1 expressing were more than pulsed magnetic field and BYHWD.
近年来,对磁场的生物学效应及治疗作用进行了深入的研究,磁场对脑损伤性疾病的影响研究已成为一个热点。动物实验和临床研究均证实磁刺激对脑缺血再灌注损伤具有保护作用,有助于改善急性脑梗死患者神经功能缺损。磁场对脑缺血再灌注损伤的保护机制可能与阻断脑缺血再灌注损伤后诱发的一系列链式瀑布反应如减少自由基产生、改善血液流变学、抑制细胞内钙超载、抑制兴奋性氨基酸产生、抑制细胞凋亡等某一环节或多个环节有关。在临床研究上已证实磁场可以促进脑卒中后神经功能恢复,减少肢体残疾的产生,是一种安全有效的方法。有关磁场与IGF-1关系的研究报道不多。本课题设计利用磁场干预大鼠脑缺血再灌注模型,探讨磁场对脑缺血大鼠脑内IGF-1表达的影响以及对神经功能缺失、脑缺血梗死面积、缺血后病理组织学的影响,为磁场的神经保护作用提供实验室基础。
补阳还五汤(BYHWD)始载于《医林改错》,由清代名医王清任所创,以补气药与活血祛瘀药相配伍,以益气固摄为主,化瘀通络为辅,具有补气活血,通经活络的功效,是治疗气虚血瘀型脑缺血疾病的名方。有关BYHWD对脑缺血再灌注损伤的机制研究报道较多,BYHWD可能通过改善血流动力学、抑制自由基的产生、降低一氧化氮(NO)与一氧化氮合成酶(NOS)的活性、防止钙超载、抑制兴奋性氨基酸的释放、促进星形胶质细胞对损伤脑组织的修复、抑制细胞凋亡等多因素对脑缺血再灌注损伤起修复保护作用。目前关于BYHWD与IGF-1的关系未见报道。基于BYHWD的神经保护作用,我们设想利用BYHWD灌注脑缺血再灌注大鼠,观察BYHWD对IGF-1表达的影响以及BYHWD对神经功能缺失、脑缺血梗死面积、缺血后病理组织学的影响,以期发现BYHWD与IGF-1在神经修复方面有无内在关系,为BYHWD的神经保护作用提供新的实验室基础。
目的:观察脑缺血再灌注后时间与IGF-1表达的关系,研究磁场、BYHWD对脑缺血再灌注大鼠IGF-1表达的影响,为磁场、BYHWD的脑保护作用提供实验室依据。
设计:完全随机设计。
方法:1.40只Sprague Dawley(SD)大鼠分为假手术组、缺血再灌注后2h组、1d组、3d组和7d组,每组8只。2h组、1d组、3d组、7d组用线栓法阻断右侧大脑中动脉供血,2h后拨出线栓恢复大脑中动脉供血制成右侧大脑中动脉闭塞(MCAO)模型,假手术组仅做右侧颈外动脉和颈总动脉结扎,不做右侧大脑中动脉线栓栓塞。参照Zeal Longa 5分法进行评分。0分:无神经功能缺损症状;1分:轻度局灶性神经功能缺损,即提尾悬空不能伸展左侧前爪;2分:中度局灶性神经功能缺损,即行走向左侧转圈;3分:中度局灶性神经功能缺损,即行走困难,并向左侧倾倒;4分:不能自发行走,意识水平下降。评分1～4分为造模成功。32只大鼠造模成功。各组动物分别在造模后0h(假手术组)、2h、1d、3d、7d处死,断头取脑。主要观察指标:用免疫组化法(IHC)观察各组梗死区IGF-1的表达。
2.80只SD大鼠分为假手术组、模型组、脉冲磁场组、BYHWD组、脉冲磁场联合BYHWD组(联合组),每组16只,模型组、脉冲磁场组、BYHWD组和联合组制作右侧MCAO模型,假手术组仅做右侧颈外动脉和颈总动脉结扎,不做右侧大脑中动脉线栓栓塞。脉冲磁场组和联合组于造模结束后开始用脉冲磁疗仪进行干预,异名磁极对置于大鼠头部,磁距为7～10cm,磁场强度为0～0.01T,频率为50HZ,20min/次,每天1次,连续7d;BYHWD组和联合组予灌服BYHWD水煎液,按生药13.3g/kg灌胃,连续给药7d;假手术组和模型组不作磁场处理及BYHWD灌胃处理。7d后处死各组大鼠,断头取脑。主要观察指标:①分别在2h、1d、3d、7d进行神经功能缺失评分;②采用2,3,5—氯化三苯基四氮唑(TTC)染色观察梗死面积大小;③苏木素—伊红染色(HE)观察病理组织学改变;④IHC检测IGF-1的表达。
结果:120只大鼠进入结果分析。
1.脑缺血再灌注大鼠IGF-1的表达
采用单因素方差分析的检验方法,组间多重比较采用LSD检验,组间比较有统计学差异(F=60.867,P=0.000)。经组间多重比较显示:IGF-1在假手术组神经细胞中表达最弱,与假手术组相比,缺血再灌注后2h组、1d组、3d组、7d组梗死区阳性细胞数增多,差异有统计学意义(P=0.000);与2h组、1d组相比,3d组阳性细胞数增多,其差异有统计学意义(P=0.000);与3d组相比,7d组阳性细胞数有所下降,差异有统计学意义(P=0.000);与1d组相比,7d组阳性细胞数无显著性差异(P=0.712),但与假手术组相比,差异有统计学意义(P=0.000)。
2.脉冲磁场、BYHWD对脑缺血再灌注大鼠IGF-1表达的影响
2.1 2h、1d、3d、7d神经功能缺失评分
采用重复测量的检验方法,时间因素不同水平组间多重比较及固定时间因素后不同实验分组间的多重比较均采用LSD方法。
假手术组在2h、1d、3d、7d时神经功能缺失评分均为0分。模型组、脉冲磁场组、BYHWD组和联合组各组间比较存在统计学差异(F=5.479,P=0.032),神经功能缺失评分在不同的时间之间有显著差异(F=106.922,P=0.000),组别与时间有交互效应(F=3.867,P=0.000)。从3d开始,各组神经功能缺失评分随时间的延长呈逐渐下降趋势;从时间点来看,2h和1d时模型组、脉冲磁场组、BYHWD组和联合组的评分无显著差异(F=0.760,P=0.521和F=1.538,P=0.214);3d时模型组、脉冲磁场组、BYHWD组和联合组的评分有显著差异(F=12.612,P=0.000),BYHWD组、脉冲磁场组和联合组的评分低于模型组,联合组的评分低于BYHWD组、脉冲磁场组;7d时模型组、脉冲磁场组、BYHWD组和联合组的评分有显著差异(F=4.609,P=0.002),脉冲磁场组、BYHWD组和联合组评分均低于模型组,脉冲磁场组、BYHWD组和联合组评分无显著性差异
2.2脑组织TTC染色结果
脑梗死面积百分数的比较采用单因素方差分析,组间多重比较采用LSD检验。假手术组脑组织切片均红染,无白色梗死灶。模型组大鼠梗死灶位于缺血侧脑组织额顶部外侧皮层,脑梗死灶明显,梗死区内坏死组织呈苍白色,未坏死组织呈玫瑰红色。各组间比较差异有统计学意义(F=30.383,P=0.000)。经组间多重比较显示:与模型组相比,脉冲磁场组、BYHWD组、联合组梗死面积均减小,差异有统计学意义(P=0.000)。与脉冲磁场组相比,BYHWD组梗死面积差异没有显著性意义(P=0.552)。联合组与脉冲磁场组和BYHWD组相比,梗死面积减小,其差异有统计学意义(P=0.009,P=0.035)。
2.3脑组织切片HE染色结果
假手术组:神经细胞数目多,形态结构清楚,细胞排列规整。核深染,核膜清晰,核仁明显。高倍镜下可见神经元细胞胞浆丰富,呈灰蓝色斑块状,表明尼氏体丰富。
模型组:坏死区结构紊乱,组织染色变浅,组织结构疏松,胶质细胞增生,部分神经元细胞核固缩,胞体缩小变形,残留的神经元细胞周围间隙增宽,神经元细胞正常形态消失,胞质红染、均匀,可见鬼影细胞。边缘区低倍镜下可见较多形态大致正常的神经元细胞,胶质细胞无明显增生,组织颜色较深。在高倍镜下可见边缘区部分神经元细胞浆染色呈淡染,尼氏小体消失,并可见神经元细胞核固缩。
脉冲磁场组:坏死区胶质细胞较模型组增生明显,形成胶质结节,出现噬神经现象,仍可见较完整的红色神经元细胞,个别神经元细胞核固缩,胞体缩小变形。边缘区在高倍镜下可见边缘区神经元细胞胞浆蓝染,呈颗粒状,尼氏体丰富。神经元形态较规整,核深染,核仁清。
BYHWD组:坏死区胶质细胞较模型组增生明显,有胶质结节形成,可见较完整的红色神经元细胞,个别神经元细胞核固缩,胞体缩小变形。高倍镜下可见边缘区神经元细胞胞浆蓝染,颗粒状,尼氏体较丰富,神经元形态较规整,核深染,核仁清。
联合组:坏死区胶质细胞较模型组增生明显,可见完整的红色神经元细胞。高倍镜下可见边缘区神经元细胞形态规整,核深染,核仁清,神经元细胞胞浆蓝染明显,呈颗粒状,尼氏体丰富。。
2.4脉冲磁场和BYHWD干预后IGF-1的表达结果
脉冲磁场与BYHWD干预后IGF—1阳性细胞数比较采用单因素方差分析,组间多重比较采用LSD检验。各组间比较,差异有统计学意义(F=592.714,P=0.000)。经组间多重比较显示,IGF-1阳性细胞数在假手术组最少;与假手术组相比,模型组缺血再灌注7d后缺血梗死侧大脑皮层IGF-1阳性细胞数增多,差异有显著性意义(P=0.007);与假手术组和模型组相比,BYHWD组缺血再灌注7d后IGF-1阳性细胞数增多,差异有显著性意义(P=0.000);与假手术组、模型组和BYHWD组相比,脉冲磁场组阳性细胞数增多,差异有统计学意义;与假手术组、模型组、脉冲磁场组和BYHWD组相比,联合组再灌注7d后IGF-1阳性细胞数增多,差异有显著性意义(P=0.000)。
结论:正常情况下脑组织中仅有少量IGF-1表达,脑缺血再灌注发生后,脑中IGF-1的自分泌即启动,再灌注3d后IGF-1的表达达到高峰,7d缺血侧大脑皮层IGF-1的表达有所下降,但仍明显高于假手术组。这表明脑缺血再灌注可促进内源性IGF-1的分泌,有助于缺血状态下神经组织的自我保护和修复,但这种促分泌作用维持时间不长。脉冲磁场和BYHWD干预后缺血再灌注大鼠神经功能评分改善明显、脑梗死面积减小,神经系统的保护和修复作用明显增强,且脉冲磁场、BYHWD可以促进IGF-1的表达,增强脑损伤后的内源性保护机制,二者联合使用在促进神经功能恢复、减小脑梗死面积,及促进IGF-1表达方面作用更强,说明脉冲磁场和BYHWD有协同作用。
Insulin-like growth factor 1 (IGF-1)is a kind of polypeptide and involved in many physiological functions, such as cell proliferation,differentiation and maturation of organic tissues. It is named as IGF because its structure has high homogeneity with insulin prosome. Almost all the tissue can excrete IGF-1 and express IGF-1 receptor. In the physiological condition there are extensive IGF-1 immunoreactivities and IGF-1 mRNA expression in brain. It was found that IGF-1 and IGF-1R extensively existed in central nervous system, especially there were higher expression in hypophysis, then in olfactory bulb and superior brain stem, and followed by cerebrum, striatum, hippocampus and cerebral cortex in order. The normal brain in mature can express lower IGF-1 protein and both neurons and neuroglial cells can express IGF-1 protein. It has been demonstrated that IGF-1 is a kind of neurotrophic factor and has various functions. It can not only promote the differentiation and proliferation of neurons and neuroglial cells in brain, but also accelerate the growth and development of brain. It also protects cerebrum from ischemia reperfusion injury. It is believed that IGF-1 play an important role in the pathophysiological process of cerebral infarction. After cerebral ischemia, the IGF-1 system was activated, its expression and distribution in brain were obviously changed, and its protective actions were activated at same time. In clinical reports, it has been reported that IGF expression is correlated with the attack of ischemic stroke.IGF-1 dripped via nasal cavity or intravenous injection could decrease the expression of caspase-1 in brain after hypoxic ischemic injury, and could protect the brain from damage. The mechanism of protecting brain from damage of IGF-1 is not very clear. IGF-1 could adjust forkhead like1 through the phosphatidylinositol-3-kinase/serine/threonine kinase pathway, inhibit caspase-3 activity, restrain apoptosis of neurons and neuroglial cells or change the expression of Bcl protien or increase the level ofγ-aminobutyric acid, restrain aminobutyric acid and reduce metabolic rate of neuron in ischemic infarcs area and protect the brain.
Magnetic therapy has a long history and is used in the treatment of various diseases. It possesses certain function of protecting brain ischemia reperfusion injury through holding up the pathophysiological development of brain ischemia injury, but the mechanism are not very clear. In animal experiment, magnetic field exposure can improve rat hemodynamic property, increase antioxidase activity, improve organic anti-oxidation capability, prevente free radicals, adjust the intra-cellular calcium content, inhibit the cell apoptosis and reduce neural damage. In clinical research, Magnetic therapy may reduce disability resulting from stroke, and promote neurological and functional improvement. Magnetic therapy can be recognized as a safe method and worth making popular in clinical practice. The experiment is aimed to study the effect of pulsed magnetic field in inducing IGF-1 expressing and the influence on cerebral infarct volume, pathological changes in focal cerebral ischemica reperfusion rat, for providing new theoretic foundation for magnetic therapy.
Buyanghuanwu decoction(BYHWD) is the classical prescriptions for hemiplagia differentiated as qi deficiency and blood stasis created by Wang Qingren,the famous doctor in Qing dynasty in "Yi LingGai Chuo". It has the ability of invigorating qi and activating blood circulation. In all the ingredients of BYHWD, the ingredients invigorating qi act as host, and the ingredients activating blood circulation act as supplement. Many scientists have studied the mechanism of BYHWD and its decomposed formulas on cerebral ischemia. They have demonstrated that BYHWD can improve hemodynamic property, increase antioxidase activity, resist free radicals, decrease the intra-cellular calcium overload, etc. inhibit the cell apoptosis and reduce neural damage. There were no reports about the relationship between BYHWD and IGF-1. In this experiment, cerebral ischemia reperfusion model will be prepared for the observation of the effect of BYHWD on the expresion of IGF-1 in brain tissue, and of the influence of BYHWD on neurologic deficit, cerebral infarct area, pathological changes of brain tissue, to provide new breadboard foundation for BYHWD.
Objective:To observe the effect of pulsed magnetic field and BYHWD in inducing IGF-1 expressing and influence on neurologic deficit, cerebral infarct area, pathological changes in focal cerebral ischemica reperfusion rat, providing new breadboard foundation for magnetic therapy and BYHWD.
Design: Randomized controlled animal experiment.
Methods: 1. Forty SD rats were randomized into 5 groups, namely, sham-operation group, 2h group, 1d group, 3d group and 7d group with 8 in each group. MCAO method was employed to establish focal cerebral ischemia reperfusion model in 2h group, 1d group, 3d group and 7d group, they were occluded for 2h at the right middle cerebral artery, and then they were reperfused, but 8 rats was only tied up without occlusion in sham-operation group. The rats in the experimental group were assessed according to Zeal Longa standard after 2h reperfusion. Without neurologic deficit was marked as 0 point, flexion of left anterior claws when hanging tail as 1 point, circling to left while walking as 2 points, difficulty in walking and falling down to left as 3 points, unconscious mind as 4 points. The rats assessed 1 to 4 point were successful MCAO rats. 32Rats were successfully MCAO. After that, brain tissues were harvested at Oh (sham-operation group), 2h, 1d, 3d and 7d after cerebral ischemic reperfusion injury, 40 rats at each time point. Main outcome measure: IGF-1 expression in the brain tissue was observed by immunohistochemical method.
2. Eighty SD rats were randomized into 5 groups, namely, sham-operation group, model group, pulsed magnetic field group, BYHWD group and pulsed magnetic field combined BYHWD group (combination group) with 16 in each group. Also MCAO method was employed to establish cerebral ischemia reperfusion model in model group, pulsed magnetic field group, BYHWD group and combination group. Rats was only tied up without occlusion in sham-operation group. BYHWD was applied by gastric perfusion at the dosages of 13.3g/kg.d for 7d in BYHWD group and combination group. Rats in pulsed magnetic field group and combination group were exposed to 0 to 0.01T intensity pulsed magnetic field of 50 Hz for 20 minutes per day for 7d. Rats in sham-operation group and model group only eat food and drink freely. The rats in the experimental group were assessed according to Zeal Longa standard after 2h ischemia and 1, 3, 7 d reperfusion respectively. Main outcome measure:①Neurologic deficit test.②Infarct area was detected by TTC staining.③Pathological changes were observed microscopically in HE stained sections.④IGF-1 expression in the brain tissue was observed by immunohistochemical method. Statistical management was carried out with SPSS13.0 software. Data were presented as Mean±SD, Repeated Measures and one-way ANOVE were used.
Results: 120 rats were involved in the final analysis without deletion.
1.Expression of IGF-1 in cerebral ischemica reperfusion rats
One-way ANOVE were used. There were a few IGF-1 positive cells stained lightly in the sham-operation group. The IGF-1 positive cells of 2h group were more than sham-operation group (P=0.000) . The IGF-1 positive cells in 1d group were more than sham-operation group and 2h group (P=0.000) . The IGF-1 positive cells of 3d group were significantly more than all other group (P=0.000) . There were no significant difference between 7d group and 1d group (P=0.712) , but the IGF-1 positive cells of 7d group were more than sham-operation group and 2h group (P = 0.000) .
2. The effect of pulsed magnetic field and BYHWD in inducing IGF-1 expressing and the influence on neurologic deficit, cerebral infarct area, pathological changes in focal cerebral ischemica reperfusion rats
2.1 The neurologic deficit
The scores of neurologic deficit of sham-operation group were 0 point all the time. In othe four groups, repeated measures were used. There were significant difference in the scores of neurologic deficit at 2h, 1d, 3d and 7d (F=106.922, P=0.000) . The scores of neurologic deficit of model group, pulsed magnetic field group and BYHWD group were keeping decreasing all the time. There were significant difference in model group, pulsed magnetic field group , BYHWD group and combination group (F=5.479, P=0.032) .At 2h and 1d there were no significant difference in all the groups(F=0.760, P=0.521; F=1.538, P=0.214). After 3 to 7day reperfusion the scores of neurologic deficit of pulsed magnetic field group and BYHWD group were lower than that of model group ((F=12.612, P=0.000; F=4.609, P=0.002).
2.2 Cerebral infarct area
One-way ANOVE were used. There were no infarcts in sham-operation group. The infarct area of pulsed magnetic field group and BYHWD group were significant less than that of model group (P = 0.000) . There were no significant difference between pulsed magnetic field group and BYHWD group (P=0.552). The infarct area of combination group were significant less than that of pulsed magnetic field group and BYHWD group (P=0.009, P=0.035) .
2.3 Pathological changes
In the sham-operation group, the morphology of neurons were ovale or multi-angles in shapes, Nissl bodies were stained as dark blue. So there were no obviously changes in sham-operation group. In model group, many neurons disappeared in the center of the infarcts, the nuclei of the remaining neurons were pyknotic and the neural fibers became vacuolar. There were no Nissl bodies. Many neurons became pyknotic and deep-stained around the infarcs. Neuron defects in the infarcted center of pulsed magnetic field group, BYHWD group and combination group were significantly slighter than that of model group. The morphology of many neurons were also ovale or multi-angles in shapes, Nissl bodies still existed and were stained as blue. A few neurons became pyknotic and deep-stained around the infarcs.
2.4 Expression of IGF-1
One-way ANOVE were used.There were a few IGF-1 positive cells stained lightly in the sham-operation group. The IGF-1 positive cells of model group were more than sham-operation group (P = 0.007) .The IGF-1 positive cells of pulsed magnetic field group and BYHWD group were more than model group (P = 0.000) . The IGF-1 positive cells in combination group were more than pulsed magnetic field group and BYHWD group (P = 0.000) .
Conclusion: The expression of IGF-1 was very low in normal brain tissues. The positive cells of IGF-1 higher expressions maintained from 2h to 7d after cerebral ischemia reperfusion, and reached the peak value at 3d, then decreased at 7d, but maintained a higher level than normal brain tissues. It suggests that cerebral ischemia reperfusion injury can enhanced expression of endogenous IGF-1, but the expression are limited and last for shorter durition. After cerebral ischemia reperfusion the neurological function is damaged, and it has a trend of self-recovery, but self-recovery are very weak. Pulsed magnetic field and BYHWD can promote recovery of neurological function deficit and decrease the infarct area. Pulsed magnetic field and BYHWD can protect the brain tissue after cerebral ischemia reperfusion, and promote IGF-1 expressing. The effect of pulsed magnetic field combined BYHWD on protecting the brain tissue after cerebral ischemia reperfusion, and promoting IGF-1 expressing were more than pulsed magnetic field and BYHWD.
引文
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