老年小鼠术后认知功能障碍与NMDA受体-NF-Kappa B-nNOS信号通路关系的探讨

发布时间：2018-04-13 13:42

本文选题：术后认知功能障碍 + NMDA受体　；参考：《中国人民解放军医学院》2016年博士论文

【摘要】：第一部分手术创伤对老年小鼠POCD行为学影响目的：通过左肝外叶切除术构建C57BL/6老年小鼠术后认知功能障碍(postoperative cognitive dysfunction or decline,POCD)模型,探索老年小鼠POCD模型的构建,以及手术创伤对老年小鼠术后认知功能的影响。方法：15月龄健康C57BL/6老年小鼠共80只,按随机数组表法分为空白对照组、麻醉组、假手术组和手术组。小鼠在术前5天接受训练,每天3次,寻找固定隐藏平台。第6天接受左肝外叶切除术。术后第1,3,7天用“空间探索实验”评价其空间记忆能力,记录穿台次数和目标象限停留时间百分比。结果：在空间获得性实验中,各组C57BL/6老年小鼠学习记忆能力随着时间的延长显著提高(P0.05)并在第5天趋于稳定,且组间没有差异。Morris水迷宫测试老年小鼠穿台次数和目标象限停留百分比结果显示：与空白对照组比较,麻醉组无差异,假手术组(除术后7d)和手术组明显下降(P0.05)：与假手术组相比,手术组在各时间点明显下降(P0.05)。组内比较,与术后1d比较,空白组和麻醉组术后3d、7d无差异;假手术组术后3d、7d逐渐恢复,至术后7d时间点基本恢复正常(P0.05);手术组术后3d、7d虽有所恢复,但仍较正常值明显下降(P0.05)。结论：左肝外叶切除术能成功建立C57BL/6老年小鼠POCD模型,且空间记忆能力的损伤在肝切除术老年小鼠中较假手术老年小鼠重。第二部分POCD与NMDA受体-NF-Kappa B-nNOS信号通路的关系实验一目的：对C57BL/6老年小鼠进行手术并抑制围术期应激因素(镇痛,镇静),研究中枢神经POCD过程中NMDA受体-NFKB-nNOS信号通路的激活。方法：15月龄健康C57BL/6老年小鼠共80只,按随机数字表法分为空白对照组、麻醉组、假手术组和手术组。观察内容如下：分别于术后1,3,7d三个时间点,各组分别有8只动物断头取脑,ELISA测定左侧脑组织NMDA受体NR2B,nNOS,NF-κB,NOS以及NO含量测定;右侧脑组织RT-PCR测定突触素mNRA, PSD-95蛋白mNRA.结果：检测结果显示,与空白对照组比较,各检测指标(NMDA受体NR2B，NF-κB, nNOS, NOS)蛋白水平以及NO含量,麻醉组在术后各时间点无差异;假手术组(除外术后7d时间点)和手术组在术后各时间点明显下降(P0.05)：与假手术组比较,各时间点手术组明显下降(P0.05)。组内比较：与术后1d比较,假手术组至7d时基本恢复正常(P0.05);手术组则逐渐下降,至7d时降至最低(P0.05)。RT-PCR测定老年小鼠脑内突触素mRNA和PSD-95mRNA表达水平发现,与空白对照组比较,麻醉组在术后各时间点无差异;假手术组(除外术后7d时间点)和手术组在术后各时间点明显下降(P0.05);与假手术组比较，各时间点手术组明显下降(P0.05)。组内比较：与术后1d比较,假手术组老年小鼠至7d时基本恢复正常(P0.05);手术组则逐渐下降,至7d时降至最低(P0.05)。结论：C57BL/6老年小鼠在构建POCD模型后,脑内NMDA受体、NF-κB、 nNOS蛋白水平的变化趋势均呈渐进性下降,且7d为最低值。这与脑内与突触相关的突触素mRNA和PSD-95蛋白mRNA表达趋势相同,因此提示脑内NMDA受体、NF-κB、nNOS蛋白水平的变化是引起老年小鼠POCD的可能机制。实验二目的：对C57BL/6老年小鼠进行手术,抑制围术期应激因素(镇痛,镇静),通过应用NMDA受体激动剂(15mg/kg NMDA)验证老年小鼠POCD过程中存在NMDA受体-NFκB-nNOS通路的激活。方法：15月龄健康C57BL/6老年小鼠共56只，按随机数字表法分为空白对照组、假手术组和手术组。腹腔内注射NMDA受体激动剂(15mg/kgNMDA) 1小时后行手术。观察内容如下：分别于术后1,3,7d三个时间点,各组分别有8只动物断头取脑,ELISA测定左侧脑组织NMDA受体NR2B,nNOS,NF-KB,NOS以及NO含量测定;右侧脑组织RT-PCR测定突触素mRNA,PSD-95蛋白mRNA.结果：检测结果显示,与空白对照组比较,各检测指标(NMDA受体NR2B、 NF-κB、nNOS、NOS)蛋白水平以及NO含量，，假手术组在术后各时间点无差异：手术组在术后各时间点明显下降(P0.05)。手术组内：与术后1d比较,其余时间点明显下降(P0.05),但术后7d与3d比较则无差异(P0.05)。RT-PCR测定老年小鼠脑内突触素mRNA和PSD-95mRNA表达水平发现，与空白对照组比较，假手术组在术后各时间点(除术后1d)无差异;手术组在术后各时间点明显下降(P0.05)。手术组内：与术后1d比较,其余时间点明显下降(P0.05),但术后7d与3d比较则无差异(P0.05)。结论：腹腔内注射NMDA受体激动剂后，发现老年POCD小鼠脑内海马组织NMDA受体NR2B、NF-κB、nNOS蛋白水平以及NO含量的降低趋势减缓,且7d与3d比较无明显差异，这与神经元突触素mRNA以及PSD-95mRNA表达趋势相同。提示脑内NMDA受体,NF-κB,nNOS蛋白水平的变化是引起老年小鼠POCD的可能机制，并为我们治疗POCD提供了新的思路。
[Abstract]:Part one the effect of surgical trauma on the behavior of POCD in aged mice objective by left hepatic lobectomy to construct C57BL/6 aged mice of postoperative cognitive dysfunction (postoperative cognitive dysfunction or decline, POCD) model was constructed to explore the old mice POCD model, as well as the influence of surgical trauma on postoperative cognitive function in elderly mice. Methods: 15 month old a total of 80 healthy aged C57BL/6 mice, according to the random array table were divided into blank control group, anesthesia group, sham operation group and operation group. The mice in the 5 days before training, 3 times a day, looking for hidden fixed platform. Sixth days underwent left hepatic lobectomy. 1,3,7 days after operation with "space to explore the spatial memory ability evaluation experiment, record the times across the platform and the percentage of target quadrant time. Results: experiments in space, each C57BL/6 in aged mice learning and memory ability with The time significantly improve (P0.05) and tend to be stable in fifth days, and there were no group differences in the.Morris water maze test in aged mice Taiwan number and target quadrant percentage results show: compared with the control group, no difference in anesthesia group, sham operation group (except 7D) and the operation group decreased significantly (P0.05): compared with sham operation group, operation group was significantly decreased at each time point (P0.05). Compared with the comparison group, postoperative 1D, blank group and anesthesia group after 3D, 7d no difference; sham operation group after 3D, 7d gradually recovered to the recovery time of postoperative normal base 7d (P0.05); surgery group 3D 7d, despite the recovery, but still lower than the normal value decreased significantly (P0.05). Conclusion: left hepatic lobectomy can be successfully established C57BL/6 POCD in the senile mice model, and the spatial memory ability of injury during liver resection in aged mice compared with sham operated mice. Second elderly part POCD The relationship between NMDA and -NF-Kappa receptor B-nNOS signaling pathway Objective: a surgical C57BL/6 in old mice and inhibit the perioperative stress factors (analgesia, sedation), activation of central nerves in the process of POCD NMDA receptor -NFKB-nNOS signaling pathway. Methods: 15 month old healthy C57BL/6 mice aged 80, anesthesia group randomly divided into blank control group, sham operation group and operation group. Observe the contents are as follows: respectively after 1,3,7d three time points, each group has 8 animal brains were removed for determination of NMDA, ELISA receptor NR2B, left brain tissue nNOS, NF- kappa B, NOS and NO determination of content; the right side of the brain tissue was determined by RT-PCR the expression of mNRA, PSD-95 protein mNRA. results: test results show that, compared with the control group, all indexes (NMDA receptor NR2B, NF- kappa B, nNOS, NOS) protein level and the content of NO, anesthesia group at each time point after surgery no difference Different; sham operation group (except after 7d time point) and the operation group at each time point after operation was significantly decreased (P0.05) compared with the sham operation group, operation group at each time point was significantly decreased (P0.05). Group comparison: compared with postoperative 1D, sham operation group and 7d returned to normal (P0.05); operation group decreased to the lowest at 7d (P0.05).RT-PCR expression level was found the expression of mRNA and PSD-95mRNA in elderly mice, compared with the blank control group, anesthesia group at each time point after operation there is no difference; sham operation group (except after 7d time point) and the operation group in at each time point after operation was significantly decreased (P0.05); compared with the sham operation group, operation group at each time point was significantly decreased (P0.05). Group comparison: compared with postoperative 1D, sham operation group aged mice to 7d normal (P0.05); operation group decreased to the lowest at 7d (P0.05). Conclusion: the old C57BL/6 In mice in the POCD model, NMDA receptors in the brain, NF- kappa B, the change trend of nNOS protein level showed a gradual decline, and 7d is the lowest. The brain and synaptic associated synaptophysin mRNA and PSD-95 protein expression of mRNA in the same trend, thus suggesting that NMDA receptors in the brain, the expression of NF- B. Changes in the level of nNOS protein is the possible mechanism of aged mice caused by POCD. Experiment two objective: surgical C57BL/6 in old mice, inhibition of perioperative stress factors (analgesia, sedation), through the application of NMDA receptor agonist (15mg/kg NMDA) of NMDA receptor activation -NF B-nNOS pathway of the validation process of aged mice. Methods POCD 15 month old: C57BL/6 total of 56 healthy aged mice, were randomly divided into control group, sham operation group and operation group. Intraperitoneal injection of NMDA receptor agonist (15mg/kgNMDA) 1 hours after surgery. Observe the contents are as follows: after operation. 1,3,7d three time points, each group has 8 animal brains were removed for determination of NMDA, ELISA receptor NR2B, left brain tissue nNOS, NF-KB, NOS and NO determination of content; the right side of the brain tissue RT-PCR determination of synapsin mRNA, PSD-95 protein mRNA. results: test results show that, compared with the control group, all indexes (NMDA receptor NR2B, NF-, nNOS, NOS) kappa B protein level and the content of NO, the sham operation group at each time point after operation, no difference in operation group at each time point after operation was significantly decreased (P0.05). The operation group: compared with 1D after operation, the rest of the time points were significantly decreased (P0.05), but after the operation 7d and 3D were no difference (P0.05) determination of.RT-PCR expression levels of synaptophysin mRNA and PSD-95mRNA found in the brain of aged mice, compared with the blank control group, sham operation group at each time point after operation (except 1D) no difference; surgical group at each time point after operation was significantly decreased (P0.05) operation group. In: Compared with the 1D after the operation, the rest of the time points were significantly decreased (P0.05), but the postoperative 7d compared with 3D had no difference (P0.05). Conclusion: intraperitoneal injection of NMDA receptor agonist, was found in the hippocampus of aged POCD mice NMDA receptor NR2B, NF- kappa B, slow decrease the protein level of nNOS and NO the content of 7D and 3D had no significant difference, this is the same as the neurons of synapsin mRNA and the expression of PSD-95mRNA receptors in the brain. The trend suggests that NMDA, NF- kappa B, nNOS protein level changes is the possible mechanism of aged mice caused by POCD, and provides a new way for us to treat POCD.

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R619

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