【作者】 张润峰；

【导师】 陈运贞；

【作者基本信息】 重庆医科大学 ， 内科学， 2004， 博士

【摘要】 目的当前临床上用来防治心肌梗死（MI）后心脏性猝死的有效措施尚不完善 ，若干抗严重心律失常的药物 ，还可能促发严重的心律失常 ，使死亡率增高 ；寻找改善MI预后的有效方法已成为亟待解决的研究课题 。近年来发现 ，电刺激小脑顶核（FNS）具有中枢神经源性神经保护作用 ，可抑制神经细胞凋亡 ，促进神经组织的结构重建 。预先FNS 1 h ，可使随后发生的脑梗死体积平均缩小50% 。应用FNS治疗大鼠局灶性脑梗死的实验中 ，使用同心圆电极从小脑顶核引导的电信号显示 ，体外电刺激可到达小脑顶核 ，提示体外刺激也可能在实验或临床中产生效果 。目前 ，采用仿生物电 ，模拟实验性小脑顶核电刺激而研制的小脑电刺激仪已用于许多疾病的临床治疗 ；尤其在脑梗死患者中 ，证实FNS可显著改善心率变异性（HRV） ，增强迷走神经活性 。大量研究结果证实 ，定量评价自主神经功能状况的HRV降低与<WP=9>MI的总死亡率和猝死率密切相关 。受FNS已有研究的启发 ，本课题拟探索FNS对心肌梗死后大鼠HRV及心脏神经再生的作用 ，并探讨FNS对心脏神经生长因子（NGF） 、炎性细胞因子基因表达以及氧自由基 、神经递质水平的影响 ，为FNS预防MI后的心脏性猝死提供实验依据 。方法分别采用小脑顶核电刺激（FNS）或小脑顶核毁损（FNL）技术及左冠状动脉前降支（LAD）结扎法建立大鼠实验模型 。实验分组 ：①MI组 ，单纯结扎LAD ；②FNS+MI组 ，即先FNS ，后结扎LAD组 ；③FNL+FNS+MI组 ，即毁损小脑顶核后电刺激小脑顶核部位 ，再行LAD结扎组 ；④假手术组（Sham组） ，仅在LAD下穿线不结扎 。各组按结扎LAD后的时程又随机分为1 、7 、 21d三个时间点进行研究 。通过微机大鼠心率变异性记录分析系统测定大鼠心率功率谱密度 ，统计MI后大鼠存活率 ；应用免疫组织化学方法分别检测心肌梗死区（IZ）与非梗死区（NIZ）交感神经与迷走神经纤维密度分布 ； 应用RT-PCR法检测IZ与NIZ的NGF 、 TNF-α 、IL-1β 、IL-6 mRNA的表达 ； 应用四唑氮蓝(NBT)法测定心肌梗死面积 ；应用高效液相色谱法测定心脏去甲肾上腺素含量 ； 应用生化方法检测心脏乙酰胆碱酯酶（ACHE）活性 、 超氧化物歧化酶（SOD）活性 、总抗氧化能力（TAOC） 、丙二醛含量 、肌酸激酶（CK）和乳酸脱氢酶（LDH）活性 。 <WP=10>结果 1. FNS对HRV的作用MI后1 、7 、21天 ， MI组与Sham组比较 ， LF 、HF均显著降低 ，FD 减小 ，HFnorm也明显降低 ; LFnorm增高 ，LF/HF比值升高 。如果预先电刺激小脑顶核 ，MI后各时间点（1 ，7 ，21d）出现HRV参数HF 、LF 、HFnorm 、FD等明显升高 ， LFnorm 、LF/HF降低 。使用鹅膏氨酸提前毁损双侧小脑顶核 ，则FNS对HRV的改善作用消失 。2. FNS对大鼠存活率的影响FNS+MI组与MI组的21天存活率分别为86.6% 、40.0% ，二者行卡方检验比较 ，χ2 =7.03 ， p<0.01 ，统计学差异十分显著 。3. FNS对心脏自主神经纤维密度变化的影响FNS+MI组大鼠 ， MI后第1天 、7天 、21天IZ与NIZ的TH 、CHAT阳性神经纤维较MI组显著增多 。 毁损大鼠双侧小脑顶核后 ，与FNS+MI组比较 ，电刺激顶核部位对心脏神经无显著影响 。4. FNS对心肌梗死面积的影响FNS+MI组心肌梗死面积明显小于MI组 ， 血清LDH 、CK活性降低 ； 而毁损小脑顶核后再预电刺激小脑顶核部位 ， 上述血清酶学及心肌梗死面积与单纯心肌梗死大鼠相比均无统计学差异 。5. FNS对心脏氧化应激的影响与MI组相比 ， FNS+MI组心脏组织SOD活性 、TAOC显著升高（P<0.01） ， MDA含量显著降低（P<0.01）; 而FNL后再电刺激小<WP=11>脑顶核部位 ，与单纯心肌梗死大鼠相比 ，上述指标间无统计学显著性差异（P＞0.05） 。6. FNS对心脏神经生长因子表达的影响结扎大鼠LAD后 ， IZ和NIZ心肌组织NGF基因表达均显著下调 。 与单纯MI组比较 ， 预先电刺激小脑顶核 ，冠脉结扎后第1和第7天IZ和NIZ NGFmRNA表达明显增加 ，第21天则表达增加不明显 。预先毁损小脑顶核后 , FNS对心脏NGF mRNA在梗死后不同时间点的表达无显著影响 。7. FNS对心脏细胞因子表达的影响MI后TNF-α、IL-β和IL-6 mRNA表达不论在IZ还是NIZ均明显增加 ，其中以冠状动脉结扎后第7天最明显 。经FNS预处理后 ，上述细胞因子在MI后各时间点的表达明显下降 ； 提前毁损小脑顶核后再给予电刺激则无明显变化 。8. FNS对心脏神经递质的影响与假手术组比较 ， MI第1天IZ与NIZ心肌组织中NE水平升高 ，反映ACH释放量的ACHE活性减少 ; 而第7 、21天无明显改变 。FNS可显著降低MI后1天IZ与NIZ心肌组织NE含量 ， 增加ACH释放 ，表现为ACHE活性升高 。FNS对MI后7天和21天IZ与NIZ心肌组织NE与ACHE无明显影响 。结论1．FNS可缩小心肌梗死面积 ，增加HRV ，提高存活率 ，对MI后心脏有保护作用 。 <WP=12>2．FNS使MI后IZ和NIZ心肌组织TH和CHAT染色阳性神经纤维密度增加 ，促进心肌梗死后心脏自主神经纤维再生 。3．FNS可上调心脏NGF mRNA表达 ，下调炎性细胞因子TNF-α 、IL-1β 、IL-6 mRNA的表达。4．FNS增强组织抗氧化防御能力 ，减少氧自由基产生 。5．FNS调节心脏神经递质NE和ACH的释放更多还原

【Abstract】 Objective The methods used for preventing sudden cardiac death have been documented not to do well. A lot of drugs for severe arrhythmia may promote severe arrhythmia and increase mortality rates instead of what expected. Looking for the effective methods for improving prognosis of myocardial infarction (MI) has become the research subject that is urgent to be resolved in the fields of the <WP=14>cardivascular researches. The past several years have witnessed the recognition that fastigial nucleus stimulation (FNS) has neuroprotective effects against damage of ischemia and the others, including supressing neuronal apoptosis and promoting nerve tissue construction. One-hour electrical stimulation of fastigial nucleus (FN) can decrease infarction volume by 50% on average in cerebral infarction. It was reported that electrical stimulation of FN percutaneously in rats with local cerebral infarction had the similar effect as stimulation of FN directly, which was demonstrated by the electrical signal coming from the concentric circular electrode. The demonstration of similar effect of the direct or indirect FN stimulation implies that in vitro administration of percutaneous FNS could be of clinical relevance. Recently , cerebellum electric stimulation devices , which start from experimental FNS and minic biological electric current have used in clinical practise for treating a lot of diseases. It was confirmed that FNS significantly improved heart rate variability (HRV) or increased vagal activity on patients with cerebral infarction.Now it has been confirmed that reduced HRV as quantitative markers of autonomic nervous state is closely related to the overall postinfarction mortality and sudden cardiac death. Previous studies of FNS spurred efforts to explore the effect of FNS on HRV and cardiac nerve regeneration after MI in this study. Further, we investigate the effects of FNS on NGF，inflamatory cytokines genes expression and production of oxygen free radical as well as levels of nerve transmitters in <WP=15>post-MI hearts.Methods Animal models were created by stimulating FN with the flow of electricity, using ibotenic acid to produce lesions of FN neurons, ligating the left anterior artery (LAD), respectively. Wistar rats were randomly divided into four groups: group MI was rats with myocardial infarction alone; group FNS+MI was rats receiving FN stimulation for 1 hour before producing MI; group FNL+FNS+MI was rats undergoing the same procedure like group FNS+MI except that there were lesions of the FN five days before producing MI; group Sham was rats without ligating LAD and stimulating FN, just mimicing all the operations. Then every group was randomly divided into three subgroups according to the three time points, that is 1,7,21 days after MI for further study. Power spectral density (PSD) was measured by the HRV recording and analysing equipment; survial rate was calculated; sympathetic or vagal nerve fibers density in the infarcted zone(IZ) and non-infarcted zone(NIZ) were examined by immunohistochemistry ; the mRNA expression levels of NGF, TNF-α，IL-1β and IL-6 in IZ and NIZ were studied by RT-PCR; the infarction area was measured by nitroblue tetrazalium(NBT) staining method; cardiac norepinephrine(NE) content was measured with high performance liquid chromatography(HPLC); the activities of acetylcholinesterase (ACHE)，superoxide dismutese (SOD) ，content of malondialdehyde (MDA) and total anti-oxidative capability (TAOC) in cardiac tissues as well as serum creatine kinase (CK) and lactate dehydrogenase (LDH) activities were measured with biochemistry method.Results The main results were as follows:1. Effect of FNS on the HRV in ratsLF, HF, HFnorm and FD at day 1, 7, 21 after MI were significantly <WP=16>reduced compared with the Sham group; LFnorm and LF/HF increased significantly. Electrical stimulation of the FN could increase LF, HF, HFnorm, FD and decrease LFnorm, LF/HF at each time point. There showed no obvious effect on the HRV after lesion of FN in advance. 2. Influence of更多还原