促红细胞生成素对实验性脑出血大鼠神经突触重塑的影响

发布时间：2018-05-05 02:46

本文选题：脑出血 + 促红细胞生成素(EPO)　；参考：《西南医科大学》2017年硕士论文

【摘要】：目的:脑出血(Intracerebral hemorrhage ICH)是急性脑血管疾病的一种,为神经内科多发疾病,因其死亡率及致残率较高,严重危害着患者的生命健康及生活质量。脑出血后血肿的占位效应、血肿刺激周围组织释放的血管活性物质和血肿的分解产物、凝血酶级联放大反应及补体系统的激活,这些因素相互作用直接或间接的引起脑组织损害,诱发炎症反应及细胞凋亡,导致神经功能缺损;针对发病率及死亡率高的现状,我国目前针对脑出血的治疗方案尚无突破性进展,目前的治疗主要集中于降颅压、清除氧自由基、营养神经、调控血压、血糖、维持水盐电解质平衡等对症支持治疗或外科手术治疗,缺乏特异有效的治疗方案,且就后期神经功能的恢复方面的治疗更是缺乏。神经突触重塑与ICH后神经功能恢复密切相关,较长时间内都可受到调节,有利于受损神经功能的恢复。促红细胞生成素(erythropoietin,EPO)是一种由肾脏分泌的相对分子量为34000的糖蛋白,以往认为促红细胞生成素(EPO)在体内的主要作用是与红系祖细胞表面的特异性促红细胞生成素受体(erythropoietin receptor,EPOR)结合,从而刺激其增值分化,其在临床上的应用也局限于纠正慢性贫血。近年来大量研究表明脑组织中有促红细胞生成素(EPO)和促红细胞生成素受体(EPOR)的表达;同时发现促红细胞生成素(EPO)的生成量与脑组织的供血供氧情况密切相关,当脑组织缺血缺氧时促红细胞生成素(EPO)的生成量成倍增加,对神经元起保护作用,并且通过降低神经元对缺血缺氧的敏感性,增强神经元的存活能力,促进神经祖细胞的增值和抑制凋亡。但就促红细胞生成素(EPO)是否能够有助于神经突触重塑方面的研究尚不多,因此关于促红细胞生成素(EPO)对脑出血后神经突触的影响方面的研究很有必要。本实验通过予以重组人促红细胞生成素(rh EPO)干预后观察ICH后血肿周围突触素(Synaptophysin,SYP)和突触后致密物-95(Post synaptic density,PSD-95)蛋白的变化情况,探讨促红细胞生成素(EPO)对实验性脑出血大鼠神经突触重塑的影响。方法:1.在造模前根据Morris水迷宫视频跟踪分析系统训练预备大鼠,连续训练4天后测试,选取逃避潜伏期在10-40秒之间的大鼠进入造模;2.根据《大鼠脑立体定向仪图谱》以右侧基底节区尾状核(前囟前0.2mm,中线右侧3.0mm)为造模注射点,采用断尾取自体血法制作ICH模型。按照Zea-longa评分标准,将评分在1-3分之间的SD大鼠入选为实验大鼠;3.动物的分组及处理:将经Morris水迷宫筛选出的105只健康雄性SD大鼠随机分为三组:假手术组(Sham组)35只,脑出血组(ICH组)35只,脑出血+促红细胞生成素治疗组(EPO组)35只;将Sham组、ICH组、EPO组按时间点分为术后6h、24h、48h、72h、7d、14d、21d共七组,每组5只大鼠。实验大鼠的具体处理如下:(1)Sham组用微量注射器在右侧基底节区尾状核处定位并缓慢刺入,不注入自体血,其余步骤与ICH组一致;(2)ICH组用微量注射器在右侧基底节区尾状核处注入不抗凝自体血50μl,同时腹腔注射与EPO组所用EPO剂量相等的生理盐水;(3)EPO组用微量注射器在右侧基底节区尾状核处注入不抗凝自体血50μl,同时按3000U/kg的剂量向腹腔注射EPO。4.标本制备及检测:将造模成功的大鼠于术后6h、24h、48h、72h、7d、14d、21d各对应时间点先用Garcia神经功能评分法评价神经功能缺损情况后进行Morris水迷宫测试,之后予以水合氯醛麻醉,麻醉后立即断头取脑,制作脑组织标本,用干湿重法测定各组大鼠血肿周围脑组织含水量变化,用HE染色观察血肿周围脑组织基本形态和结构,用免疫组化法检测各时间点血肿周围SYP、PSD-95蛋白表达。结果:1.脑组织含水量:6h、24h、48h、72h、7d各时间点ICH组和EPO组血肿周围脑组织含水量均明显高于Sham组(P0.05);6h时间点EPO组血肿周围脑组织含水量与ICH组无差异(P0.05),但24h、48h、72h、7d各时间点EPO组血肿周围脑组织含水量较ICH组明显减少(P0.05)。2.HE染色观察各组脑组织病理改变:(1)Sham组无血肿形成,各时间点脑组织结构完整;(2)ICH组72h时间点血肿周围可见大量红细胞,脑组织结构紊乱,炎症细胞浸润明显,神经细胞水肿严重甚至变性坏死,7d、14d时病灶周围的血细胞逐渐减少,炎性细胞逐渐减少,神经细胞水肿程度减轻,到21d时病灶周围血细胞更少甚至消失、炎症细胞几乎消失,纤维组织、胶质细胞增生,受损的组织结构较前有所恢复。(3)与ICH相比,EPO组各时间点的水肿程度和神经细胞变性坏死都有所减轻,受损组织的修复都较ICH组好。3.神经功能评分:各实验组大鼠于术后6h、24h、48h、72h、7d、14d、21d各时间点予以神经功能评分,其中(1)Sham组未见明显神经功能缺损情况;ICH组、EPO组各时间点神经功能评分均低于Sham组,有统计学差异(P0.05);(2)各时间点EPO组神经功能评分均高于ICH组,具有统计学差异(P0.05)。4.水迷宫测试结果:大鼠造模前进行Morris水迷宫连续训练4天,每天4次,第5天测试逃避潜伏期、穿越平台次数、平台象限时间百分比和平台象限路程百分比,各组无显著差异(P0.05);造模后再次行水迷宫测试上述指标,Sham组各指标较前均无明显变化(P0.05),ICH组、EPO组的逃避潜伏期、穿越平台次数、平台象限时间百分比和平台象限路程百分比等多种反应学习记忆能力的指标,较前均有所下降,并随着ICH后时间的延长各指标逐渐上升,且EPO组的上升程度及速度要优于ICH组(P0.05)。5.血肿周围脑组织SYP蛋白含量测定:(1)Sham组仅有少量SYP蛋白表达,各个时间点无明显差异(P0.05);(2)在6h、24h、48h、72h时间点ICH组、EPO组仅有少量SYP蛋白表达,与Sham组比较无明显差异(P0.05);7d、14d、21d时间点,ICH组、EPO组SYP蛋白表达明显多于Sham组(P0.05);(3)7d、14d、21d时间点,EPO组SYP蛋白表达明显高于ICH组,具有统计学差异(P0.05);(4)ICH组、EPO组从72h后SYP蛋白开始增多,14d达峰值(P0.05),之后表达下降,但21d时血肿周围仍有较多表达。6.血肿周围脑组织PSD-95蛋白含量测定:(1)Sham组仅有少量PSD-95蛋白表达,各个时间点无明显差异(P0.05);(2)在6h、24h、48h、72h时ICH组、EPO组仅有少量PSD-95蛋白表达,与Sham组比较无明显差异(P0.05),在7d、14d、21d时ICH组、EPO组PSD-95蛋白表达明显多于Sham组(P0.05);(3)在7d、14d、21d时间点,EPO组PSD-95蛋白表达明显高于ICH组,具有统计学差异(P0.05);(4)ICH组、EPO组从72h后PSD-95蛋白开始增多,14d达峰值(P0.05),之后表达下降,但21d时血肿周围仍有较多表达。7.各组大鼠神经功能评分与SYP、PSD-95蛋白含量行相关性分析:(1)在14d时间点,ICH组、EPO组的SYP蛋白含量与相应实验大鼠神经功能评分呈正相关(r=0.940,P0.001);(2)在14d时间点,ICH组、EPO组的PSD-95蛋白含量与相应实验大鼠神经功能评分呈正相关(r=0.848,P0.001)。结论:1、采取不抗凝自体血注入法制作大鼠ICH模型方法较简单,易于操作,且与临床上人体自发性ICH的病理变化过程相似,是比较理想的研究ICH的动物模型;2、ICH后血肿周围SYP、PSD-95蛋白表达增多,利于神经功能恢复;3、促红细胞生成素(EPO)能上调血肿周围SYP、PSD-95蛋白表达,促进ICH后神经突触重塑;4、促红细胞生成素(EPO)能促进ICH大鼠肢体功能及学习记忆能力的恢复。
[Abstract]:Objective: Intracerebral hemorrhage ICH is a kind of acute cerebrovascular disease, which is a multiple disease of Neurology. The mortality and disability rate are high, which seriously harm the life health and quality of life of the patients. The occupying effect of hematoma after intracerebral hemorrhage and the decomposition of vasoactive substances and hematoma released in the surrounding tissues by hematoma. The product, the cascade amplification reaction of thrombin and the activation of the complement system, these factors interact directly or indirectly to the damage of brain tissue, induce the inflammatory reaction and cell apoptosis, and lead to the nerve function defect. In view of the current situation of high morbidity and mortality, there is no breakthrough progress in the treatment of cerebral hemorrhage at present in China. The treatment is mainly focused on craniofacial pressure, oxygen free radical scavenging, nourishment nerve, regulation of blood pressure, blood sugar, maintenance of water and salt electrolyte balance and other symptomatic support treatment or surgical treatment, lack of specific and effective treatment, and lack of treatment for the recovery of neural function later. Neural synaptic remodeling is closely related to the recovery of nerve function after ICH. Erythropoietin (EPO) is a glycoprotein of the relative molecular weight of 34000, which was secreted by the kidney. It was previously thought that the main use of erythropoietin (EPO) in the body is specific erythropoiesis with the surface of the erythroid progenitor cells. The combination of erythropoietin receptor (EPOR) stimulates its value-added differentiation, and its clinical application is limited to the correction of chronic anemia. In recent years, a large number of studies have shown that the expression of erythropoietin (EPO) and erythropoietin receptor (EPOR) in the brain tissue; and the formation of erythropoietin (EPO) and the brain in the brain The blood supply and supply of oxygen in the tissue is closely related. When cerebral ischemia and hypoxia, the production of erythropoietin (EPO) increases exponentially, protects the neurons, and enhances the viability of neurons by reducing the sensitivity of neurons to ischemia and anoxia, and promotes the increment and inhibition of apoptosis of the progenitor cells of the deity. It is necessary to study the effect of erythropoietin (EPO) on neural synapses after intracerebral hemorrhage, and the study of the effect of erythropoietin (RH EPO) on the posthematoma peripheral synaptophysin (Synaptophysin, SYP) after ICH is necessary for the study of whether EPO can contribute to neural synapse remodeling. The changes of -95 (Post synaptic density, PSD-95) protein and the effect of erythropoietin (EPO) on neural synapse remodeling in experimental intracerebral hemorrhage rats were investigated. Methods: 1. before modeling, the rats were trained on the basis of the Morris water maze video tracking analysis system for 4 days after continuous training, and the escape latency was 1. The rats between 0-40 seconds entered the model; 2. according to the atlas of the rat brain stereotactic apparatus, the injection point was made by the caudate nucleus of the right basal ganglia region (0.2mm of the anterior fontanelle, the right 3.0mm of the middle line), and the autologous blood method was used to make the ICH model. In accordance with the Zea-longa score standard, the rats of 1-3 scores of SD were selected as experimental rats; 3. animals were selected. Group and treatment: 105 healthy male SD rats were randomly divided into three groups: 35 sham operation group (group Sham), 35 brain hemorrhage group (group ICH), cerebral hemorrhage + erythropoietin group (group EPO) 35, Sham group, ICH group and EPO group were divided into 6h, 24h, 48h, 72h, 72h, seven groups, 5 rats in each group. The specific treatment of rats was as follows: (1) group Sham was located at the caudate nucleus of the right basal ganglia with a micro injector and slowly pricking, and the other steps were consistent with the ICH group. (2) group ICH was injected with an anticoagulant autologous blood 50 mu l at the caudate nucleus of the right basal ganglia region, and the EPO agent used in the EPO group was injected into the abdominal cavity. Equal amount of physiological saline; (3) group EPO was injected with an anticoagulant autologous blood of 50 mu by injecting a micro injector to the right basal ganglia caudate nucleus and injected into the abdominal cavity at the dose of 3000U/kg to prepare and detect the EPO.4. specimens. The rats with the successful model were evaluated by the Garcia neural functional scoring method for 6h, 24h, 48h, 72h, 7d, 14d, 21d at the corresponding time points after the operation. After the Morris water maze test was carried out, chloral hydrate was followed by anaesthesia. After anesthesia, the brain tissue was cut off immediately after anesthesia and the brain tissue was made. The changes of water content around the hematoma around the hematoma were measured by dry wet weight method. The basic morphology and structure of the brain tissue around hematoma were observed by HE staining, and the time was detected by immunohistochemical method. SYP, PSD-95 protein expression around the hematoma. Results: 1. the water content of brain tissue: 6h, 24h, 48h, 72h, the water content around the hematoma in ICH group and EPO group of EPO group was significantly higher than that of Sham group (P0.05), and there was no difference in the brain tissue around the hematoma around the hematoma at the 6h time point. Compared with group ICH, the pathological changes of brain tissue were observed in group ICH (P0.05): (1) no hematoma was formed in group Sham, and the structure of brain tissue was complete at all time points. (2) a large number of red blood cells were seen around hematoma at 72h time point in group ICH, brain tissue structure disorder, inflammatory cells infiltrating obvious, nerve cell edema and even degeneration and necrosis, 7d, 14d disease The blood cells around the focal point decreased gradually, the inflammatory cells gradually decreased, the degree of edema of the nerve cells decreased, and the blood cells around the focus were less or even disappeared at 21d, the inflammatory cells almost disappeared, fibrous tissue, glial cells proliferated, and the damaged tissue was restored. (3) the degree of edema and nerve finer at each time point in group EPO compared with ICH The degeneration and necrosis of the cells were all relieved, and the repair of the damaged tissues was better than the ICH group.3. neural function score: the rats in the experimental group were given neurological function score at 6h, 24h, 48h, 72h, 7d, 14d, 21d at all time points after the operation, and (1) there was no obvious nerve function defect in the Sham group. Study difference (P0.05); (2) the score of EPO group in each time point was higher than that of group ICH, with statistical difference (P0.05).4. water maze test results: 4 days of continuous training for the rats before modeling, 4 times a day, fifth days to test the escape latency, the times of flat platform crossing, the percentage of platform quadrant time and the percentage of platform quadrant distance, There was no significant difference in each group (P0.05). The index of water maze was tested again after the model, and there was no significant change in each index in group Sham (P0.05). The escape latency, the number of crossing platform, the percentage of platform quadrant time, the percentage of platform quadrant and the percentage of platform quadrant were decreased in group ICH and EPO. With the prolongation of ICH, the index of each index increased gradually, and the level and speed of EPO group was better than that of SYP protein content in the brain tissue around.5. hematoma in group ICH (P0.05): (1) only a small amount of SYP protein was expressed in the Sham group, and there was no significant difference at every time point (P0.05); (2) there were only a small amount of protein expression in 6h, 24h, 48h, and time points. There was no significant difference in Sham group (P0.05); 7d, 14d, 21d time point, ICH group, SYP protein expression in group EPO was more than Sham group (P0.05). (3) 7d, 14d, and time points were significantly higher than those in the group. (4) There was still more expression of PSD-95 protein content around.6. hematoma around hematoma: (1) only a small amount of PSD-95 protein was expressed in group Sham, and there was no significant difference at each time point (P0.05). (2) there was only a small amount of PSD-95 protein expression in 6h, 24h, 48H, 72h, and there was no significant difference between the EPO group and the group. The expression of protein was significantly more than that of Sham group (P0.05); (3) the expression of PSD-95 protein in group EPO was significantly higher than that in ICH group at 7d, 14d and 21d, and there was a statistical difference (P0.05). (4) ICH group, EPO group began to increase from 72h. Correlation analysis with SYP and PSD-95 protein content: (1) at 14d time point, ICH group, EPO group SYP protein content was positively correlated with the corresponding experimental rat neural function score (r=0.940, P0.001). (2) in 14d time point, ICH group, EPO group PSD-95 protein content was positively correlated with the corresponding test of rat neural function score. Conclusion: 1, take The method of making ICH model of rats with anticoagulant autologous blood injection is simple and easy to operate, and it is similar to the pathological process of spontaneous ICH in clinic. It is an ideal animal model for studying ICH. 2, SYP, PSD-95 protein expression is increased around hematoma after ICH, and it is beneficial to the restoration of the function of the God, and 3, erythropoietin (EPO) can up the hematoma. The expression of SYP and PSD-95 protein promotes the synaptic remodeling after ICH. 4, erythropoietin (EPO) can promote the recovery of limb function and learning and memory ability of ICH rats.

【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.34

【参考文献】