本文選題：丙泊酚 + 暴露次數(shù)��； 參考：《廣西醫(yī)科大學》2017年碩士論文

【摘要】：第一部分丙泊酚單次與多次暴露對新生大鼠遠期學習記憶功能的影響目的:探討丙泊酚不同暴露次數(shù)對新生大鼠遠期學習記憶功能的影響方法:SPF級7日齡(P7)雄性SD大鼠,體重13~18g,按隨機數(shù)字表法分為4組:空白對照組,除無腹腔注射外其它條件同其它組;溶劑對照組,分別在P7、P8、P9每日腹腔注射0.1%DMSO 5m L/kg;丙泊酚單次暴露組,在P7、P8時每日腹腔注射0.1%DMSO 5m L/kg,P9時腹腔注射丙泊酚50mg/kg;丙泊酚多次暴露組,分別在P7、P8和P9每日腹腔注射丙泊酚50mg/kg。麻醉蘇醒后,各組取部分大鼠經(jīng)左心室采血行血氣分析。余大鼠于P36行Morris水迷宮實驗檢測遠期空間學習記憶功能。結(jié)果:麻醉期間,各組大鼠血氣分析各指標差異無統(tǒng)計學意義(P0.05)。與空白對照組比較,丙泊酚單次暴露組大鼠的游泳速度,逃避潛伏期及平臺穿越次數(shù)差異無統(tǒng)計學意義(P0.05);而丙泊酚多次暴露組大鼠的逃避潛伏期明顯延長,平臺穿越次數(shù)明顯減少,差異有統(tǒng)計學意義(P0.05),游泳速度無顯著差異;溶劑對照組的各項行為學指標與空白對照組差異無統(tǒng)計學意義(P0.05)。結(jié)論:丙泊酚單次暴露不影響大鼠遠期學習記憶功能;丙泊酚多次暴露可損害大鼠遠期學習記憶功能。第二部分丙泊酚多次而非單次暴露損害新生大鼠遠期學習記憶功能的機制目的:探討丙泊酚多次而非單次暴露損害新生大鼠遠期學習記憶功能的機制方法:SPF級7日齡(P7)雄性SD大鼠,按隨機數(shù)字表法分為4組:空白對照組,除無腹腔注射外其它條件同其它組;溶劑對照組,分別在P7、P8、P9每日腹腔注射0.1%DMSO 5m L/kg;丙泊酚單次暴露組,在P7、P8時每日腹腔注射0.1%DMSO 5m L/kg,P9時腹腔注射丙泊酚50mg/kg;丙泊酚多次暴露組,分別在P7、P8和P9每日腹腔注射丙泊酚50mg/kg。麻醉蘇醒后5h(P9)、P14、P21和P35將大鼠處死取左側(cè)大腦行尼氏染色觀察海馬CA1區(qū)和前額葉皮層Pr L區(qū)神經(jīng)元密度,行免疫組化染色觀察activated caspase-3陽性細胞密度;取右側(cè)大腦行免疫熒光觀察synaptophysin陽性顆粒密度。結(jié)果:與空白對照組比較,丙泊酚單次暴露組海馬CA1區(qū)和前額葉皮層Pr L區(qū)于P9 activated caspase-3陽性細胞密度顯著增多(P0.05),神經(jīng)元密度減少(P0.05),而于P14、P21和P35各指標差異無統(tǒng)計學意義(P0.05),synaptophysin陽性顆粒密度于P9、P14、P21和P35差異均無統(tǒng)計學意義(P0.05);丙泊酚多次暴露組海馬CA1區(qū)和前額葉皮層Pr L區(qū)于P9、P14、P21和P35 activated caspase-3陽性細胞密度顯著增多(P0.05),神經(jīng)元密度減少(P0.05),synaptophysin陽性顆粒密度減少(P0.05)。溶劑對照組各時間點的各項指標與空白對照組比較差異無統(tǒng)計學意義(P0.05)。結(jié)論:丙泊酚單次暴露僅誘發(fā)短暫的海馬及前額葉皮層神經(jīng)元凋亡和神經(jīng)元缺失;而丙泊酚多次暴露可誘發(fā)持續(xù)的海馬及前額葉皮層神經(jīng)元凋亡,突觸發(fā)育抑制,神經(jīng)元缺失,這可能是丙泊酚多次而非單次暴露導致新生大鼠遠期學習記憶功能損害的原因。第三部分TNF-α介導丙泊酚多次暴露致發(fā)育期大腦神經(jīng)毒性的分子機制目的:探討TNF-α介導丙泊酚多次暴露致發(fā)育期大腦神經(jīng)毒性的分子機制方法:SPF級7日齡(P7)雄性SD大鼠,按隨機數(shù)字表法分為3組:空白對照組,除無腹腔注射外其它條件同余兩組;溶劑對照組,分別在P7、P8、P9每日腹腔注射0.1%DMSO 5m L/kg;丙泊酚組,分別在P7、P8和P9每日腹腔注射丙泊酚50mg/kg。分別于P7、P8和P9麻醉后6h、P10和P11各時間點經(jīng)側(cè)腦室采集腦脊液行ELISA觀察釋放的TNF-α水平,取海馬和前額葉皮層組織行Western blot觀察釋放的TNF-α蛋白水平。另取大鼠按隨機數(shù)字表法分為4組:空白對照組;假手術(shù)對照組,與ETN組同時間點側(cè)腦室注射等體積a CSF,丙泊酚組;ETN組,于P7丙泊酚腹腔注射前30min側(cè)腦室注射ETN(5μg/2μL)。麻醉蘇醒后5h(P9)和P35經(jīng)左心室灌注多聚甲醛固定后取左側(cè)大腦行尼氏染色觀察海馬CA1區(qū)和前額葉皮層Pr L區(qū)神經(jīng)元密度,行免疫組化染色觀察activated caspase-3陽性細胞密度;取右側(cè)大腦行免疫熒光觀察synaptophysin陽性顆粒密度,P36行Morris水迷宮實驗檢測遠期空間學習記憶功能。另取大鼠按隨機數(shù)字表法分為3組:空白對照組;溶劑對照組和丙泊酚組。于P9麻醉蘇醒后取腦行免疫熒光雙標染色觀察海馬及前額葉皮層釋放增多的TNF-α來源。結(jié)果:與空白對照組比較,丙泊酚組腦脊液、海馬及前額葉皮層TNF-α水平均于P7、P8、P9和P10呈顯著增高(P0.05),P11回到空白對照水平(P0.05);丙泊酚組中,海馬CA1區(qū)和前額葉皮層Pr L區(qū)于P9和P35與空白對照組比較activated caspase-3陽性細胞密度顯著增多(P0.05),神經(jīng)元密度減少(P0.05),synaptophysin陽性顆粒密度減少(P0.05),水迷宮實驗逃避潛伏期明顯延長(P0.05),平臺穿越次數(shù)明顯減少(P0.05);ETN組海馬CA1區(qū)和前額葉皮層Pr L區(qū)于P9和P35與空白對照組比較activated caspase-3陽性細胞密度,神經(jīng)元密度和synaptophysin陽性顆粒密差異均無統(tǒng)計學意義(P0.05),水迷宮實驗逃避潛伏期及平臺穿越次數(shù)差異無統(tǒng)計學意義(P0.05);假手術(shù)對照組與空白對照組比較各指標差異亦無統(tǒng)計學意義(P0.05);丙泊酚麻醉后,海馬區(qū)內(nèi)僅有小膠質(zhì)細胞合成TNF-α增多,在前額葉皮層區(qū)內(nèi)小膠質(zhì)細胞和神經(jīng)元均合成TNF-α增多。結(jié)論:丙泊酚可導致發(fā)育期大腦TNF-α釋放增多,TNF-α可能參與或介導了丙泊酚多次暴露致發(fā)育期大腦的神經(jīng)毒性,小膠質(zhì)細胞是丙泊酚麻醉后腦內(nèi)增多的TNF-α的主要來源,而神經(jīng)元合成TNF-α增多呈腦區(qū)依賴性。
[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.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R965

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本文編號：1974546