【作者】 曹娟；

【导师】 贾天明；

【作者基本信息】 郑州大学 ， 儿科学， 2010， 硕士

【摘要】 新生儿缺氧缺血性脑损伤(hypoxic-ischemic brain damage, HIBD)是指在围产期窒息缺氧等因素导致脑的缺氧缺血性损害,临床上出现一系列脑病的表现。重度患儿常遗留脑瘫、癫痫、智力障碍、共济失调等后遗症,给家庭和社会造成极大的负担。因此,寻找有效的防治HIBD方法对于提高新生儿的存活率,降低致残率,搞好优生优育,提高我国人口素质,具有重要的意义。缺氧缺血后脑组织血运供应的改善与神经元功能的恢复密切相关。血管内皮细胞生长因子(vascular endothelial growth factor, VEGF)是一组近年来新发现的血管新生因子,是一种特异性作用于血管内皮细胞的多功能细胞因子,也是血管发生和神经营养因子,不仅能促进血管的新生,还能直接作用于神经细胞发挥神经营养和神经保护的作用。目的近年来,物理治疗方法如电刺激、功能训练、针灸、按摩等在脑缺血疾病的治疗过程中越来越受到重视,而电刺激目前治疗成年脑缺血性疾病的疗效已得到肯定,但对HIE恢复期的疗效在国内外却少有报道,更缺少实验证据。本实验通过制作新生大鼠缺氧缺血性脑损伤模型,给予电刺激其小脑顶核,利用免疫组化方法来观察脑组织中VEGF及其受体的表达,并通过记忆能力测试,探讨电刺激对脑保护作用的机制。材料与方法1分组新生7日龄健康SD大鼠105只,雌雄不限,体重12-16g,随机分成A、B两组。每组又分为对照组、模型组、电刺激组,A组各25只,B组各10只。A组在造模术后又按1天、3天、7天、14天、21天随机分成5组,每组各5只。2 Rice法模型制作新生7日龄健康SD大鼠,乙醚吸入麻醉,取颈左侧切口,游离左颈总动脉,用丝线结扎,缝合切口,置于缺氧箱中,持续充以气流量为2L/min的8：92氧氮混合气体2h。对照组仅分离出左侧颈总动脉,不结扎也不缺氧。术后夹尾左旋是制模成功的标志,据此纳入实验。3电刺激治疗方法电刺激组在术后12h开始刺激治疗,每次电刺激30分钟,刺激部位为耳后相当于人乳突处,每日1次,电刺激强度为3.5V、频率50HZ,使大鼠肢体稍有震颤为宜。对照组与模型组不予电刺激治疗,仅予相应时段捕捉固定。4标本采集与制备A组三组大鼠每组分别于术后1天、3天、7天、14天、21天随机各取5只大鼠,应用乙醚麻醉后,剪开胸腔,暴露心脏,剪开右心耳,以20ml注射器连接5号头皮针经左心室心尖部先灌注9g/L盐水50ml,然后灌注40g/L多聚甲醛溶液。沿颈部断头,眼科镊剥离颅骨,离断视神经和延髓完整取出脑组织,取以海马为中心的脑组织,40g/L多聚甲醛固定24h,将脑组织沿视交叉和正中隆起处做冠状切开,分别做冠状切片,进行VEGF及VEGFR1、VEGFR2的免疫组化检测,并对切片进行病理学指标的检测。5指标测定5.1脑组织HE染色光学显微镜下观察结果。5.2脑组织VEGF及VEGFR1、VEGFR2的测定高倍光镜下观察大鼠海马区VEGF及VEGFR1、VEGFR2阳性细胞数的表达,胞核染成棕黄色者判为阳性表达产物。利用Image-pro plus 6.0软件系统对切片进行平均光密度测定,每张切片随机选取5个视野,取其平均值。5.3记忆能力测试采用Y迷宫实验。于手术后第28天对B组各组大鼠进行迷宫实验测试,以连续10次测试中有9次正确的反应为达到学会的标准,记录每一动物迷路分辨学习达到标准前所需要的测试数和正确反应率。24 h后重复以上实验的过程。6统计学分析：采用SPSS for windows 13.0进行统计分析,方法为单因素方差分析,组间两两比较采用LSD-t检验,数据均以x±s表示,以α=0.05为显著性检验水准。结果1 HE染色结果：对照组神经细胞形态正常,胞质丰富,细胞排列整齐紧密,细胞核清楚,核仁明显,胞浆染色均匀,胞膜完整。模型组海马区锥体细胞变性,细胞间隙增大,排列紊乱,正常神经元减少,可见大量明显的坏死灶,坏死中心区呈空泡样变,神经细胞数量减少,胞质皱缩,核浓缩深染,核仁消失,胞浆疏松,间质水肿。电刺激组神经细胞变性坏死较少,损伤程度明显减轻,海马区锥体细胞及神经元存活数量多,多数细胞形态相对正常。2 VEGF、VEGFR1、VEGFR2免疫组化染色：电刺激组各个时间点VEGF及VEGFR1、VEGFR2表达均高于模型组和对照组(P<0.05)；模型组各个时间点VEGF及VEGFR1、VEGFR2表达均高于对照组(P<0.05)；电刺激组和模型组VEGF表达于第3天达高峰,持续14天开始下降至21天仍有表达,VEGFR1、VEGFR2表达于第7天达到高峰,持续14天开始下降至21天仍有表达；对照组各个时间点的VEGF及VEGFR1、VEGFR2表达无明显差异(P>0.05)。3记忆能力测试：模型组大鼠第1天达标所需反应次数显著多于对照组,正确反应率显著低于对照组,第2天复测仍有相似结果,而电刺激组第1天达标所需反应次数显著低于模型组,正确反应率也显著高于模型组,各组相比均有显著意义(P<0.05)；在模型组,第2天正确率未见明显提高,与第1天相比P>0.05,余均见明显提高(P<0.05)结论1电刺激可促进HIBD新生大鼠脑组织VEGF及其受体VEGFR1、VEGFR2的表达。2电刺激能改善HIBD新生大鼠的学习和记忆功能。更多还原

【Abstract】 Neonatal hypoxic-ischemic brain damage (HIBD) refers to factors such as perinatal asphyxia with hypoxic-ischemic brain damage, the clinical manifestations appeared on a series of encephalopathy. Children often left with severe cerebral palsy, epilepsy, mental retardation, ataxia and other after-effects, to families and society is a great burden. Therefore, the search for an effective method for improving the prevention and treatment of HIBD newborn survival and reduce disability, improve prenatal and postnatal care, improve the quality of China’s population has important significance.Hypoxic-ischemic brain tissue supplied by blood circulation improvement and recovery of neuronal function is closely related to. Vascular endothelial growth factor (VEGF) is a group in recent years, the newly discovered angiogenesis factor, is a specific role in vascular endothelial cells of the multifunctional cytokine, but also angiogenesis and neurotrophic factor, not only to promote new blood vessels, but also to play a direct role in the nerve cells neurotrophic and neuroprotective role.ObjectiveIn recent years, physical therapy methods such as electrical stimulation, functional training, acupuncture, massage in the treatment of cerebral ischemia in the course of the disease more and more attention, while the electrical stimulation of the current treatment of adult diseases, the efficacy of cerebral ischemia has been affirmed, but for HIE the efficacy of recovery at home and abroad rarely reported, but the lack of empirical evidence. In this study, through the production of hypoxic-ischemic brain injury model, given electrical stimulation of the cerebellar fastigial nucleus, using immunohistochemical method to observe the brain in the expression of VEGF and its receptors to explore the protective effect of electrical stimulation on brain mechanisms.Materials and methods1 Groupingnew 7-day-old SD rats were 105 healthy male and female open, weight 12-16g, were randomly divided into A, B groups. Each group was divided into control group, model group, electrical stimulation group, A group of 25, B group of all 10. A group made after they die by 1 day,3 days,7 days,14 days,21 days were randomly divided into five groups, each group 5.2 Rice Law modeling7-day-old healthy newborn SD rats, ether inhalation anesthesia, take the left neck incision, free left common carotid artery ligation with silk suture incision, placed in oxygen tank, continuous filled with gas The eight ninety-two flow 2L/min oxygen-nitrogen gas mixture 2h. The control group, only the left common carotid artery was isolated, non-ligation nor hypoxia. Folder after the end of L is a sign of the success of the system model accordingly included in the experiment.3 Electrical stimulation treatmentElectrical stimulation group 12 h after stimulation treatment began, each 30 minutes of electrical stimulation to stimulate the parts of the ear at the equivalent of human mastoid day 1, electrical stimulation intensity 3.5V, frequency 50HZ, a slight tremor of rat limb is appropriate. The control group and model group, no electrical stimulation therapy, only with the corresponding capture time.4 Specimen collection and preparationA group of three rats in each group,respectively, after Id,3d,7d,14d,21d randomly from each of five rats, application of ether anesthesia, cut the chest, exposing the heart, cut right atrial appendage in order to connect 5 Scalp 20ml syringe needle before left ventricular apical perfusion 9g/L saline 50ml, and then perfusion 40g/L paraformaldehyde solution. Along the neck, decapitation, ophthalmic forceps stripping the skull, breaking away from the integrity of the optic nerve and bulbar brain tissue removed,40g/L paraformaldehyde 24h, the brain along the optic chiasm and median eminence at doing coronal incision, separately coronal slices, for VEGF, VEGFR1, VEGFR2 immunohistochemistry testing, and for taking the part of the pathological slices target detection.5 Index Determination5.1 Brain tissue HE stainingOptical microscope observations.5.2 Brain tissue determination of VEGF, VEGFR1, VEGFR2High-powered light microscope, the hippocampus of rat VEGF and VEGFR1, VEGFR2-positive cells in the expression of the sentence of nuclei stained brown positive expression products. Using Image-pro plus 6.0 software system average optical density measurement slices, each slice randomly selected five horizons, whichever is the average.5.3 Memory testThe Y maze test. In the first 28 days after the operation of the B group of rats in each group maze experiment testing to 10 consecutive tests there are 9 times the right response to meet Institute standards, records of each animal discrimination learning labyrinth to reach the standards required before the test number and the correct response rates. Repeat the experiment after 24 h process.6 Statistical analysisUsing SPSS for windows 13.0 statistical analysis, methods for single-factor analysis of variance was used to compare between the two groups 22 LSD-t test, the data are s said toα=0.05 level for significance tests.Results1 HE stainingControl group, nerve cell morphologically normal, cytoplasm-rich cells arranged in neat rows close, clear nucleus, prominent nucleoli, cytoplasm stained uniform, membrane integrity. Pyramidal cells in hippocampus of model group, degeneration, cell gap increases, disordered, normal neuronal loss, we can see a large number of obvious necrosis and necrosis of the central area showed vacuolar change, reduce the number of nerve cells, cytoplasmic shrinkage, nuclear enrichment deeply stained, nucleolus disappeared, the cytoplasm loose, interstitial edema. Electrical stimulation of nerve cell degeneration and necrosis of less degree of injury significantly reduced hippocampal pyramidal cells and neuronal survival numbers. Most of cell morphology is relatively normal.2 VEGF, VEGFR1, VEGFR2 immunohistochemistryElectrical stimulation at each time point VEGF, VEGFR1, VEGFR2 expression was higher than model group and control group (P<0.05); model group at each time point VEGF, VEGFR1, VEGFR2 expression control group (P<0.05) electrical stimulation group and model group, VEGF expressed in the first 3 days and reached the peak last 14 days and begin to decline to 21 days still expressed, VEGFR1, VEGFR2 expression peaked on day 7, last 14 days and begin to decline to 21 days still expression; the control group at each time point of VEGF, VEGFR1, VEGFR2 expression was no significant difference (P> 0.05)3 Memory testsHIBD group (model group) rats 1st day compliance significantly more than the required response to the number of the control group, the correct response rate was significantly lower than the control group, the first two days retest still similar results, while the electrical stimulation Group 1 day compliance was significantly lower than the number required for response to the model group, the correct response rate was significantly higher than the model group, the groups were significant when compared (P<0.05); in the model group, the first two days was no obvious increase accuracy,1 day, compared with the first P> 0.05, all I see markedly improved (P<0.05)Conclusions1 Electrical stimulation can promote HIBD newborn rat brain tissue VEGF and its receptors VEGFR1, VEGFR2 expression.2 HIBD electrical stimulation can improve memory in neonatal rats and cognitive function.更多还原