1-甲基-4-苯基-1,2,3,6-四氢吡啶制备的帕金森病模型小鼠对帕金森病自主神经功能障碍的适用性研究

Applicability research of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-prepared mouse model for autonomic nervous dysfunction in Parkinson’s disease

目的探讨1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)诱导的帕金森病(PD)小鼠模型对PD自主神经功能障碍研究的适用性。方法将20只2月龄小鼠随机分为MPTP组和对照组,每组10只。MPTP组皮下注射MPTP(20 mg/kg)联合腹腔注射丙磺舒(250 mg/kg),每周2次,连续5周;对照组注射相同剂量、频次的生理盐水及丙磺舒溶液。于第2、3、4、5、6周评估小鼠自主神经功能(胃肠功能、泌尿功能、心血管功能),同时采用转棒、爬竿测试评价小鼠的运动功能。采用免疫组化法测量中脑黑质阳性多巴胺能神经元的数量,Western blotting法检测酪氨酸羟化酶(TH)的表达。结果与对照组相比,MPTP组第4~6周排便颗粒数显著减少,第5~6周粪便含水量显著降低(P<0.05~0.01)。与对照组相比,MPTP组第5~6周1 h排尿次数显著增多,平均单次尿量显著下降(均P<0.01)。两组小鼠各时间点心率及连续RR间隔差的均方根(RMSSD)差异无统计学意义(均P>0.05)。两组小鼠第2~5周运动功能差异无统计学意义,第6周MPTP组小鼠转头时间及爬杆时间显著延长,而转棒时间显著减少(均P<0.05)。MPTP组小鼠黑质区域多巴胺能神经元数量及中脑TH表达均显著降低(t=19.00,P<0.001;t=3.67,P<0.01)。MPTP组小鼠中脑多巴胺能神经元数量与排便颗粒数、粪便含水量以及平均单次尿量呈正相关(r=0.6930,P<0.05;r=0.6718,P<0.05;r=0.8149,P<0.05),与1 h排尿次数呈负相关(r=-0.7155,P<0.05)。结论慢性MPTP诱导的小鼠模型可用于PD早期胃肠道功能及泌尿系统功能的研究,但不适用于心血管功能的研究。

Objective To investigate the applicability of mouse model of Parkinson’s disease(PD)induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)on study of autonomic nervous dysfunction in PD.Methods A total of twenty 2-month-old mice were randomly divided into control and model groups with 10 in each group.For MPTP group,MPTP(20 mg/kg)was administrated subcutaneously combining with an intraperitoneal injection of probenecid(250 mg/kg)twice a week for 5 weeks,while the control group was injected with physiological saline and probenecid at the same dose and frequency.The autonomic nerve function(gastrointestinal,urinary and cardiovascular function)of mice at week 2,3,4,5,6 were assessed,and the rotating rod and climbing pole test were used to evaluate the motor function of mice at the same time.The immunohistochemical method was employed to measure the number of tyrosine hydroxylase(TH)-positive neurons in the substantia nigra of the midbrain,while Western blotting was used to examine the expression of TH.Results Compared with those in control group,the number of defecation particles in the MPTP group were reduced significantly at weeks 4-6,and the fecal water content was decreased remarkably at weeks 5-6(P<0.05-0.01).Compared with those in control group,MPTP group exhibited a significant increase in number of 1 hour urination and a remarked decrease in average single urination volume at weeks 5-6(all P<0.01).There were no significant differences in heart rate and root mean square of successive RR interval difference(RMSSD)between the two groups at each time point(all P>0.05).The motor function of the two groups had no statistical difference at weeks 2-5.The head turning time and pole-climbing time in MPTP group elevated significantly at week 6,while the time in the rotarod was declined significantly(all P<0.05).Number of dopaminergic neurons in substantia nigra and the expression of TH in midbrain were both significantly decreased in MPTP group(t=19.00,P<0.001;t=3.67,P<0.01).The number of dopaminergic neurons in the substantia nigra of MPTP group was positively correlated with the number of defecation particles,fecal water content and average single urination volume(r=0.6930,P<0.05;r=0.6718,P<0.05;r=0.8149,P<0.05),while negatively correlated with the number of 1 hour urination(r=-0.7155,P<0.05).Conclusion Chronic MPTP-induced mouse model can be used to study gastrointestinal and urinary system functions in the early stages of PD,but not cardiovascular function.