TNF-α介导丙泊酚致发育期大脑的神经毒性

发布时间：2018-06-03 22:46

本文选题：丙泊酚 + 暴露次数　；参考：《广西医科大学》2017年硕士论文

【摘要】：第一部分丙泊酚单次与多次暴露对新生大鼠远期学习记忆功能的影响目的:探讨丙泊酚不同暴露次数对新生大鼠远期学习记忆功能的影响方法:SPF级7日龄(P7)雄性SD大鼠,体重13~18g,按随机数字表法分为4组:空白对照组,除无腹腔注射外其它条件同其它组;溶剂对照组,分别在P7、P8、P9每日腹腔注射0.1%DMSO 5m L/kg;丙泊酚单次暴露组,在P7、P8时每日腹腔注射0.1%DMSO 5m L/kg,P9时腹腔注射丙泊酚50mg/kg;丙泊酚多次暴露组,分别在P7、P8和P9每日腹腔注射丙泊酚50mg/kg。麻醉苏醒后,各组取部分大鼠经左心室采血行血气分析。余大鼠于P36行Morris水迷宫实验检测远期空间学习记忆功能。结果:麻醉期间,各组大鼠血气分析各指标差异无统计学意义(P0.05)。与空白对照组比较,丙泊酚单次暴露组大鼠的游泳速度,逃避潜伏期及平台穿越次数差异无统计学意义(P0.05);而丙泊酚多次暴露组大鼠的逃避潜伏期明显延长,平台穿越次数明显减少,差异有统计学意义(P0.05),游泳速度无显著差异;溶剂对照组的各项行为学指标与空白对照组差异无统计学意义(P0.05)。结论:丙泊酚单次暴露不影响大鼠远期学习记忆功能;丙泊酚多次暴露可损害大鼠远期学习记忆功能。第二部分丙泊酚多次而非单次暴露损害新生大鼠远期学习记忆功能的机制目的:探讨丙泊酚多次而非单次暴露损害新生大鼠远期学习记忆功能的机制方法:SPF级7日龄(P7)雄性SD大鼠,按随机数字表法分为4组:空白对照组,除无腹腔注射外其它条件同其它组;溶剂对照组,分别在P7、P8、P9每日腹腔注射0.1%DMSO 5m L/kg;丙泊酚单次暴露组,在P7、P8时每日腹腔注射0.1%DMSO 5m L/kg,P9时腹腔注射丙泊酚50mg/kg;丙泊酚多次暴露组,分别在P7、P8和P9每日腹腔注射丙泊酚50mg/kg。麻醉苏醒后5h(P9)、P14、P21和P35将大鼠处死取左侧大脑行尼氏染色观察海马CA1区和前额叶皮层Pr L区神经元密度,行免疫组化染色观察activated caspase-3阳性细胞密度;取右侧大脑行免疫荧光观察synaptophysin阳性颗粒密度。结果:与空白对照组比较,丙泊酚单次暴露组海马CA1区和前额叶皮层Pr L区于P9 activated caspase-3阳性细胞密度显著增多(P0.05),神经元密度减少(P0.05),而于P14、P21和P35各指标差异无统计学意义(P0.05),synaptophysin阳性颗粒密度于P9、P14、P21和P35差异均无统计学意义(P0.05);丙泊酚多次暴露组海马CA1区和前额叶皮层Pr L区于P9、P14、P21和P35 activated caspase-3阳性细胞密度显著增多(P0.05),神经元密度减少(P0.05),synaptophysin阳性颗粒密度减少(P0.05)。溶剂对照组各时间点的各项指标与空白对照组比较差异无统计学意义(P0.05)。结论:丙泊酚单次暴露仅诱发短暂的海马及前额叶皮层神经元凋亡和神经元缺失;而丙泊酚多次暴露可诱发持续的海马及前额叶皮层神经元凋亡,突触发育抑制,神经元缺失,这可能是丙泊酚多次而非单次暴露导致新生大鼠远期学习记忆功能损害的原因。第三部分TNF-α介导丙泊酚多次暴露致发育期大脑神经毒性的分子机制目的:探讨TNF-α介导丙泊酚多次暴露致发育期大脑神经毒性的分子机制方法:SPF级7日龄(P7)雄性SD大鼠,按随机数字表法分为3组:空白对照组,除无腹腔注射外其它条件同余两组;溶剂对照组,分别在P7、P8、P9每日腹腔注射0.1%DMSO 5m L/kg;丙泊酚组,分别在P7、P8和P9每日腹腔注射丙泊酚50mg/kg。分别于P7、P8和P9麻醉后6h、P10和P11各时间点经侧脑室采集脑脊液行ELISA观察释放的TNF-α水平,取海马和前额叶皮层组织行Western blot观察释放的TNF-α蛋白水平。另取大鼠按随机数字表法分为4组:空白对照组;假手术对照组,与ETN组同时间点侧脑室注射等体积a CSF,丙泊酚组;ETN组,于P7丙泊酚腹腔注射前30min侧脑室注射ETN(5μg/2μL)。麻醉苏醒后5h(P9)和P35经左心室灌注多聚甲醛固定后取左侧大脑行尼氏染色观察海马CA1区和前额叶皮层Pr L区神经元密度,行免疫组化染色观察activated caspase-3阳性细胞密度;取右侧大脑行免疫荧光观察synaptophysin阳性颗粒密度,P36行Morris水迷宫实验检测远期空间学习记忆功能。另取大鼠按随机数字表法分为3组:空白对照组;溶剂对照组和丙泊酚组。于P9麻醉苏醒后取脑行免疫荧光双标染色观察海马及前额叶皮层释放增多的TNF-α来源。结果:与空白对照组比较,丙泊酚组脑脊液、海马及前额叶皮层TNF-α水平均于P7、P8、P9和P10呈显著增高(P0.05),P11回到空白对照水平(P0.05);丙泊酚组中,海马CA1区和前额叶皮层Pr L区于P9和P35与空白对照组比较activated caspase-3阳性细胞密度显著增多(P0.05),神经元密度减少(P0.05),synaptophysin阳性颗粒密度减少(P0.05),水迷宫实验逃避潜伏期明显延长(P0.05),平台穿越次数明显减少(P0.05);ETN组海马CA1区和前额叶皮层Pr L区于P9和P35与空白对照组比较activated caspase-3阳性细胞密度,神经元密度和synaptophysin阳性颗粒密差异均无统计学意义(P0.05),水迷宫实验逃避潜伏期及平台穿越次数差异无统计学意义(P0.05);假手术对照组与空白对照组比较各指标差异亦无统计学意义(P0.05);丙泊酚麻醉后,海马区内仅有小胶质细胞合成TNF-α增多,在前额叶皮层区内小胶质细胞和神经元均合成TNF-α增多。结论:丙泊酚可导致发育期大脑TNF-α释放增多,TNF-α可能参与或介导了丙泊酚多次暴露致发育期大脑的神经毒性,小胶质细胞是丙泊酚麻醉后脑内增多的TNF-α的主要来源,而神经元合成TNF-α增多呈脑区依赖性。
[Abstract]:The first part of the effect of the single and multiple exposure of propofol on the long-term learning and memory function of neonatal rats: To explore the effect of different exposure times of propofol on the long-term learning and memory function of neonatal rats: SPF grade 7 days old (P7) male SD rats, weight 13~18g, and divided into 4 groups according to random numbers: blank control group, except for no intraperitoneal injection The other conditions were the same as those in the other groups; 0.1%DMSO 5m L/kg was intraperitoneally injected daily in P7, P8, P9, and the single exposure group of propofol was intraperitoneally injected with 0.1%DMSO 5m L/kg every day at P7 and P8, and the peritoneal injection of propofol was intraperitoneally injected into the P9. The blood gas analysis of the left ventricular blood sampling was taken in some rats. The long-term spatial learning and memory function was detected by the Morris water maze test on P36. Results: during the anesthesia, there was no significant difference in the blood gas analysis of each group (P0.05). The swimming speed of the rats in the single exposure group of propofol was compared with the blank control group, and the escape latency and the latency period were compared. There was no significant difference in the number of platform crossing times (P0.05), but the escape latency of the multiple exposure groups of propofol was significantly prolonged, the number of platform crossing times decreased significantly, the difference was statistically significant (P0.05), and there was no significant difference in swimming speed, and there was no significant difference between the behavioral indexes of the solvent control group and the blank control group (P0.05). The single exposure of propofol does not affect the long-term learning and memory function of rats; the multiple exposure of propofol can damage the long-term learning and memory function of rats. Second the mechanism of the long-term learning and memory function of the neonatal rats with multiple propofol, not single exposure, is to explore the long-term learning of the neonatal rats with multiple propofol instead of single exposure. The mechanism and method of memory function: SPF grade 7 days old (P7) male SD rats were divided into 4 groups according to random number table method: blank control group, except for no abdominal injection, other conditions and other groups, and solvent control group, P7, P8, P9 were intraperitoneally injected with 0.1%DMSO 5m L/kg daily, and the single exposure group of propofol was intraperitoneally injected into P7 and P8. Intraperitoneal injection of propofol 50mg/kg, propofol multiple exposure group, 5h (P9), P14, P21 and P35 respectively after 50mg/kg. anesthesia in P7, P8 and P9, respectively, 5h (P9), P14, P21 and P35, were executed with Nissl's staining to observe the neuron density in the CA1 region of the hippocampus and the prefrontal cortex. The density of synaptophysin positive particles in the right brain was observed by immunofluorescence. Results: compared with the blank control group, the density of the P9 activated caspase-3 positive cells in the hippocampal CA1 and prefrontal cortex of the propofol group was significantly increased (P0.05) and the neuron density decreased (P0.05) in the single exposure group of the propofol (Pr L), but in P14, P21 and P35. There was no statistical significance (P0.05). There was no significant difference in the density of synaptophysin positive particles in P9, P14, P21 and P35 (P0.05), and Pr L region in the hippocampal CA1 and prefrontal cortex of the propofol group. There was no significant difference between the various time points of the solvent control group and the blank control group (P0.05). Conclusion: the single exposure of propofol only induces the transient hippocampal and prefrontal cortical neurons apoptosis and neuron loss, while the repeated exposure of propofol can induce the continuous hippocampal and prefrontal cortex. Neuronal apoptosis, synaptic development inhibition, and neuron loss, this may be the cause of long-term learning and memory impairment in neonatal rats. The third part of the molecular mechanism of TNF- alpha mediated the multiple exposure of propofol to the developmental stage of brain neurotoxicity: TNF- alpha mediates the development of propofol multiple exposures. The molecular mechanism of cerebral neurotoxicity: SPF grade 7 days old (P7) male SD rats, divided into 3 groups according to random number table method: blank control group, except two groups of other conditions without abdominal injection, P7, P8, P9, 0.1%DMSO 5m L/ kg respectively in P7, P8, and propofol group, respectively, in P7, P8, and daily peritoneal injection of propofol. The level of TNF- alpha released by ELISA observation in the lateral ventricle of the lateral ventricles of 6h, P10 and P11 after the anesthesia of P7, P8 and P9, respectively, was taken to observe the level of the TNF- alpha protein released by Western blot in the hippocampus and prefrontal cortex tissue, and the rats were divided into 4 groups according to the random number table method: the blank control group, the sham operation control group, and the same time. Intraventricular injection of a CSF, propofol group, group ETN, group ETN, ETN (5 u g/2 mu L) before intraperitoneal injection of P7 propofol. After the anesthesia was awakened, 5h (P9) and P35 were fixed to the left ventricle and fixed to the left ventricle to observe the neuron density of the hippocampal CA1 region and the prefrontal cortex, and the immunohistochemical staining was observed. The density of activated caspase-3 positive cells was observed and the density of synaptophysin positive particles was observed by immunofluorescence in the right brain. The long-term spatial learning and memory function was detected by the Morris water maze test of P36. The rats were divided into 3 groups according to the random number table method: the blank control group, the solvent control group and the propofol group. After the P9 anesthesia was awakened, the brain immunofluorescence was taken. Results: compared with the blank control group, the levels of TNF- alpha in the cerebrospinal fluid, hippocampus and prefrontal cortex were significantly higher in P7, P8, P9 and P10 in the propofol group than in the blank control group (P0.05), and P11 returned to the blank control level (P0.05). In the propofol group, the hippocampal CA1 region and the prefrontal cortex Pr L area were found in the propofol group. Compared with the blank control group, the density of activated caspase-3 positive cells increased significantly (P0.05), the density of neuron decreased (P0.05), the density of synaptophysin positive particles decreased (P0.05), the escape latency of the water maze experiment was significantly prolonged (P0.05), and the number of platform crossing times decreased significantly (P0.05), and the CA1 region and prefrontal cortex of ETN group were in the Pr region. Compared with P35 and blank control group, the density of activated caspase-3 positive cells, the density of neuron and the density of synaptophysin positive particles were not statistically significant (P0.05). There was no significant difference in the escape latency and the number of platform crossing times in the water maze test (P0.05), and there was no difference between the sham operation control group and the blank control group. Study significance (P0.05); after propofol anaesthesia, only microglia synthesized TNF- alpha in the hippocampus and increased TNF- alpha in the microglia and neurons in the prefrontal cortex. Conclusion: propofol may lead to the increase in the release of TNF- alpha in the developmental period of the brain, and TNF- alpha may participate in or mediate the multiple exposure of propofol to the deity of the developmental brain. Microtoxicity is the main source of increased TNF- alpha in the brain after anesthesia with propofol, while the increase in neuronal synthesis of TNF- alpha is brain region dependent.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R965

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