[Dynamic changes of locus coeruleus damage in Parkinson's disease-like mice induced by paraquat].

Objective: To observe the dynamic changes of brainstem locus coeruleus (LC) damage in Parkinson' s disease (PD) -like mice by paraquat (PQ) . Methods: In October 2019, 36 male C57BL/6 mice were randomly divided into the exposure group and the control group, with 18 mice in each group. The mice in the exposure group were given intraperitoneal injection of 15 mg/kg PQ, and the mice in the control group were given intraperitoneal injection of 0.9% saline, twice a week for 8 weeks. Neurobehavioral changes (pole climbing test, swimming test, open field test, tail hanging test, high plus maze test and water maze test) were observed at 4 weeks, 6 weeks and 8 weeks, respectively, and the changes of motor ability, emotion and cognitive function were evaluated. The brain tissue of mice were taken and stained with Hematoxylin-Eosin (HE) to observe the pathological changes of LC. Nissl staining was used to detect the changes of neuronal Nissl bodies in LC. Immunohistochemistry (IHC) staining was used to detect the expression of neuron nuclear antigen (NeuN) , dopamine (DA) neurons and norepinephrine (NE) neuron markers tyrosine hydroxylase (TH) , α-synuclein (α-syn) in substantia nigra (SN) and LC. The expression levels of NeuN, TH and α-syn in the midbrain and brainstem were detected by Western blotting. TUNEL staining was used to detect neuronal apoptosis in LC. Results: Compared with the 4th week of PQ exposure group, the time of pole climbing and swimming immobility were gradually increased, the ratio of open arm residence time of high plus maze test and the number of times of the platform and the residence time of platform quadrant in water maze test were gradually decreased (P<0.05) in the exposure group with the progress of exposure time. The results of HE and Nissl staining showed that the neurons in LC gradually arranged loosely, the nucleus were deeply stained, the cytoplasm was pyknosis, and the number of Nissl bodies gradually decreased (P<0.05) in the exposure group with the progress of exposure time. IHC results showed that the number of NeuN and TH positive cells in SN and LC of mice were gradually decreased, and the positive expression of α-syn was gradually increased (P<0.05) in the exposure group with the progress of exposure time. Western blotting results showed that the expression levels of NeuN and TH in the midbrain and brainstem were gradually decreased, and the expression level of α-syn was gradually increased (P<0.05) in the exposure group with the progress of exposure time. TUNEL staining showed that the apoptosis rates of neurons in LC were gradually increased (P<0.05) in the exposure group with the progress of exposure time. Conclusion: PQ induces progressive damage in the LC area of PD-like mice, which may be caused by the abnormal accumulation of pathological α-syn in the LC area.目的： 观察百草枯（PQ）诱导的帕金森病（PD）样小鼠脑干蓝斑核（LC）损伤的动态变化。 方法： 于2019年10月，将36只雄性C57BL/6小鼠按随机数字表法分为染毒组和对照组，各18只。染毒组小鼠给予腹腔注射15 mg/kg PQ，对照组小鼠腹腔注射等容量0.9%生理盐水，每周2次，连续8周。分别于4周、6周、8周末次给药后，观察各组小鼠的神经行为学（爬杆、游泳、旷场、悬尾、高架十字迷宫及水迷宫实验）变化，评估小鼠的运动能力、情绪和认知功能变化。取小鼠脑组织，苏木素-伊红（HE）染色观察LC区病理变化；尼氏染色检测LC区神经元尼氏体数量变化；免疫组织化学（IHC）染色检测黑质（SN）区与LC区神经元标志物神经元核抗原（NeuN），多巴胺（DA）能神经元和去甲肾上腺素（NE）能神经元标志物酪氨酸羟化酶（TH），以及α突触核蛋白（α-syn）表达情况；蛋白免疫印迹（Western blotting）法检测中脑及脑干NeuN、TH、α-syn蛋白表达水平；TUNEL染色检测LC区神经元凋亡情况。 结果： 与PQ染毒第4周组比较，随着染毒时间的进展，PQ染毒组小鼠爬杆时间、游泳不动时间逐渐增加，高架十字迷宫实验开放臂停留时间比和水迷宫实验小鼠穿越平台次数、平台象限停留时间逐渐减少（P<0.05）。HE与尼氏染色结果显示，随着染毒时间的进展，染毒组小鼠LC区神经元逐渐排列疏松，细胞核深染，胞质发生固缩，尼氏体数目逐渐减少（P<0.05）。IHC结果显示，随着染毒时间的进展，染毒组小鼠SN区与LC区神经元NeuN、TH阳性细胞数目逐渐减少，α-syn阳性表达量逐渐增多（P<0.05）。Western blotting结果显示，随着染毒时间的进展，染毒组小鼠中脑和脑干NeuN、TH表达水平逐渐降低，α-syn寡聚体表达水平逐渐升高（P<0.05）。TUNEL染色显示，随着染毒时间的进展，染毒组小鼠LC区神经元凋亡率逐渐升高（P<0.05）。 结论： PQ诱导PD样小鼠LC区出现渐进性损伤，可能是LC区出现病理性α-syn的异常聚集所引起的。.