中枢胆碱能系统退化在术后认知功能障碍中的作用及其机制研究

发布时间：2018-04-16 16:07

本文选题：术后认知功能障碍 + 中枢胆碱能神经　；参考：《上海交通大学》2014年博士论文

【摘要】：背景术后认知功能障碍（POCD）是患者麻醉手术后出现的记忆力下降、注意力不能集中等认知功能改变，严重影响患者生活质量，增加术后死亡率。然而POCD的发病机制仍然不明。虽然老龄是目前POCD唯一明确的诱发因素，但增龄导致POCD发病增加的原因还不清楚。中枢胆碱能神经系统在学习、记忆等认知功能方面起着重要调节作用，而且中枢胆碱能系统随着年龄增长的退化可以造成其对抗中枢伤害性反应的能力下降。因此，本课题拟证明中枢胆碱能系统的退化是造成老年动物术后认知功能损伤的主要原因，并探讨其具体机制与神经生长因子（NGF）的关系，为POCD的防治提供新的思路。快速老化模型小鼠（SAM）是一种近交系衰老模型鼠，其中SAMP-8品系是目前用于研究阿尔茨海默病（AD）早期神经退行性病变较理想的模型。本课题拟评估SAMP-8小鼠作为非转基因AD动物模型对吸入麻醉药的敏感性变化，为临床AD患者优化麻醉用药提供参考。方法与结果 1、中枢胆碱能神经退化在手术造成老年小鼠POCD中的作用方法：实验一：选取2月龄、18月龄C57BL/6小鼠行阑尾切除术，术后第3天应用Morris水迷宫试验测试小鼠空间学习和记忆能力的改变，水迷宫结束后采用Western Blot和生物化学发光技术检测小鼠海马胆碱能神经主要标志物的表达变化，包括乙酰胆碱转移酶（ChAT）、乙酰胆碱酯酶（AChE）、囊泡乙酰胆碱转运体（VAChT）、胆碱转运体（CHT）以及海马乙酰胆碱（ACh）的含量；实验二：为验证中枢胆碱能系统在手术引起老年小鼠学习记忆减退中的作用，给予老年小鼠5mg/kg/day多奈哌齐连续灌胃4周，提高中枢胆碱能神经的活性，灌胃结束后第1天行阑尾切除术，术后第3天水迷宫试验测试小鼠空间学习和记忆能力的改变，水迷宫结束后采用Western Blot和生物化学发光技术检测海马胆碱能神经主要标志物的蛋白表达和ACh含量的变化；实验三：为进一步证明中枢胆碱能系统的退化是手术引起小鼠学习记忆减退的重要原因，给予成年小鼠侧脑室内注射0.8μg免疫神经毒mu-p75-saporin适度损伤基底前脑胆碱能神经，20天后行阑尾切除术，术后第3天水迷宫试验测试其认知功能的变化。结果：阑尾切除术对成年小鼠的空间学习和记忆能力没有影响，但能引起老年小鼠的空间学习和记忆力下降。相应地，手术并不改变成年小鼠海马胆碱能神经主要标志物的表达，但明显下调了老年小鼠海马ChAT、VAChT的水平及ACh含量。应用多奈哌齐能预防老年小鼠术后空间学习和记忆力的下降，并上调海马内ACh含量。而应用mu-p75-saporin适度地损伤成年小鼠基底前脑胆碱能神经后再行手术则会引起其学习记忆力的下降。 2、手术造成老年小鼠中枢胆碱能神经损伤的机制研究方法：实验一：为探究手术对老年小鼠中枢NGF及调节NGF生成和降解的相关蛋白的影响，老年小鼠行阑尾切除术后采用Western Blot检测其海马内NGF、NGF前体蛋白（proNGF）和受体TrkA、p75NTR蛋白量的变化，，同时检测小鼠海马内纤维蛋白溶酶原、组织纤溶酶原激活物（tPA）和基质金属蛋白酶9（MMP9）的表达变化；实验二：为验证NGF在手术引起老年小鼠中枢胆碱能系统损伤中的作用，给予老年小鼠双侧海马内注射10μg重组小鼠β-NGF，注射后30min立即行阑尾切除术，术后第3天水迷宫试验测试其认知功能的变化，水迷宫结束后采用Western Blot检测海马胆碱能神经主要标志物的表达变化，包括ChAT、AChE、VAChT和CHT。结果：手术下调了老年小鼠海马内NGF及其活化受体pTrkA的表达水平，但对proNGF、TrkA和p75NTR受体的表达没有影响。同时，手术上调了MMP9的表达水平，不影响纤维蛋白溶酶原和tPA的表达。给予外源性NGF能预防手术导致的老年小鼠学习和记忆能力的下降，同时逆转手术对老年小鼠中枢胆碱能神经的损伤。 3、快速老化小鼠SAMP-8对吸入麻醉药敏感性的研究方法：为观察小鼠对挥发性吸入麻醉药的敏感性变化，采用夹尾法检测4、6、8、10月龄快速老化小鼠SAMP-8和正常老化小鼠SAMR-1异氟醚、七氟醚及地氟醚的最低肺泡有效浓度（MAC）值。结果：统计学分析表明年龄和品系因素对MAC值均有影响，SAMP-8小鼠三种吸入麻醉药的MAC明显低于SAMR-1小鼠，并且随着年龄增长SAMP-8小鼠的MAC明显降低。结论 1、中枢胆碱能系统退化是造成老年动物术后认知功能损伤的重要原因。 2、手术造成老年小鼠中枢胆碱能神经损伤的机制可能与NGF的降解增加相关。外源性NGF能预防手术造成的老年小鼠学习和记忆能力的减退。 3、与正常老化小鼠SAMR-1相比，快速老化小鼠SAMP-8对三种常用吸入麻醉药（异氟醚、七氟醚和地氟醚）的敏感性更强。
[Abstract]:Background

Postoperatively , cognitive impairment ( POCD ) is the only one in the pathogenesis of POCD , but the pathogenesis of POCD is still unknown . However , the pathogenesis of POCD is still unknown . Although old age is the only definite inducing factor of POCD , it is not clear that the degeneration of central cholinergic system can lead to the injury of cognitive function in old animals . Therefore , the relationship between the specific mechanism and nerve growth factor ( NGF ) is discussed , which provides a new idea for the prevention and treatment of POCD .

Rapid aging model mouse ( SAM ) is a kind of inbred mouse with aging model , in which SAMP - 8 strain is the ideal model for studying early neurodegenerative diseases of Alzheimer ' s disease ( AD ) .

Methods and Results

1 . Role of central cholinergic nerve degeneration in POCD of senile mice caused by operation

Methods : The changes of spatial learning and memory ability of mouse hippocampus were measured by Morris water maze test on the 3rd day after the end of the water maze . Western Blot and biochemiluminescence techniques were used to detect the changes of the main markers of cholinergic nerve in the hippocampus of mice .
Experiment 2 : In order to verify the effect of central cholinergic system on learning and memory loss of senile mice , 5 mg / kg / day old mice were given orally for 4 weeks to improve the activity of central cholinergic nerves . After the end of the stomach , appendix resection was performed on the first day . After the end of the water maze test , the changes of spatial learning and memory ability were measured in mice . Western Blot and biochemiluminescence techniques were used to detect the protein expression and the changes of the protein expression of the main marker of cholinergic nerve in hippocampus after the end of the water maze .
Experiment 3 : To further prove that the degeneration of the central cholinergic system is an important cause of memory loss in mice induced by surgery , the rats were given a moderate damage to the basal anterior cerebral cholinergic nerve in the lateral ventricle of the adult mouse . After 20 days of appendix resection , the changes of cognitive function were measured in the third Tianshui maze test .

Results : The spatial learning and memory ability of adult mice were not affected by appendix resection , but the spatial learning and memory ability of aged mice were decreased . Accordingly , the levels of ChAT and VAChT in hippocampus of aged mice were not changed .

2 . Study on the mechanism of nerve injury in the central cholinergic nerve of old mice caused by surgery

Methods : The expression of NGF , proNGF and TrkA and p75NTR protein in the hippocampus of aged mice were examined by Western Blot to investigate the changes of NGF , proNGF and TrkA and p75NTR protein in aged mice .
Experiment 2 : To verify the role of NGF in the injury of old mouse central cholinergic system caused by operation , 10 渭g recombinant mouse 尾 - NGF was injected into bilateral hippocampus of old mice . After injection , appendix resection was performed 30 min after injection . The changes of cognitive function were measured in the third day after injection . Western Blot was used to detect the changes of the main markers of cholinergic nerve in hippocampus .

Results : The expression level of NGF and its activated receptor pTrkA in the hippocampus of aged mice was regulated by surgery , but the expression of proNGF , TrkA and p75NTR receptors was not affected . At the same time , the expression level of MMP - 9 was raised , and the expression of plasminogen activator was not affected .

3 . Study on the sensitivity of SAMP - 8 to inhalation anesthetics in mice with rapid aging

Methods : To observe the sensitivity of mice to volatile inhalation anesthetics , the minimum alveolar effective concentration ( MAC ) values of SAMP - 8 and normal aging mice ( SAMP - 8 ) and normal aging mice ( SAMP - 1 isoflurane , heptafluoroether and desmethyl ether ) were determined by means of clip - tail method .

Results : The statistical analysis showed that age and strain factors had a significant influence on MAC values . The MAC of three inhaled anesthetics in SAMP - 8 mice was significantly lower than that of SAMR - 1 mice , and the MAC of SAMP - 8 mice decreased with age .

Conclusion

1 . The degeneration of central cholinergic system is an important cause of impairment of cognitive function in old animals .

2 . The mechanism of nerve injury in the central cholinergic nerve of old mice caused by the operation may be related to the increase of NGF . Exogenous NGF can prevent the aging mice from learning and memory loss caused by operation .

3 . Compared with normal aged mouse SAMR - 1 , the sensitivity of SAMP - 8 to three commonly used inhalational anesthetics ( isoflurane , heptafluoroether and desmethyl ether ) was stronger than that of normal aging mice .

【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R614

【参考文献】