清醒自由活动大鼠脑酪氨酸羟化酶活性的测定

The Measurement of Tyrosine Hydroxylase Activity in the Brain of Conscious Rats

目的 :建立清醒自由活动大鼠脑内酪氨酸羟化酶 (TH)活性的测定方法。方法 :应用脑微透析技术收集经 6 羟基多巴胺 (6 OHDA)预处理的大鼠 ,在L 芳香族氨基酸脱羧酶抑制剂 3 羟苄肼·盐酸 (NSD10 15 )灌流下 ,脑纹状体细胞外的透析液 ;用高效液相色谱 电化学检测方法测定透析液内多巴 (DOPA)水平 ,来反映脑内TH的活性。并观察TH抑制剂以及多巴胺受体的抑制剂和激动剂对其的影响。结果 :预先用 6 OHDA处理导致大鼠纹状体透析液内DOPA缺失 ,用NSD10 15阻断DOPA向多巴胺 (DA)转化 ,可测得DOPA的水平 ,且DA的代谢产物 3,4 二羟基苯乙酸水平逐步下降。但在TH抑制剂α 甲基 ρ 酪氨酸作用下DOPA又可消失 ,说明测定的DOPA是来自多巴胺神经末梢。另外 ,腹腔内给予多巴胺受体激动剂盐酸阿朴吗啡后 ,脑透析液中DOPA水平减少 ;而给予多巴胺受体抑制剂氟哌啶醇后 ,微透析液中DOPA水平增加。本研究测得在 0 .0 1mmol·L-1NSD10 15灌流下 ,经 6 OHDA预处理的 ,插入探针 2 4hSD大鼠纹状体中DOPA水平为 (0 .39± 0 .12 ) pmol·min-1(x±s,n =5 )。结论 :在脱羧酶抑制剂NSD 10 15灌流期间 ,测得预先用 6 OHDA处理大鼠纹状体透析液中DOPA水平可以认为是脑组织TH活性的指数 ,经工具药验证 。

Objective: To set the measuring method of tyrosine hydroxylase activity in the brain of conscious rats. Method: By using microdialysis and High Performance Liquid Chromatography Electrochemical Detector system, the 3,4 dihydrioxphenylalanine (DOPA) formation in the striatum of 6 hydroxdopamine pretreated rats during infusion of an L aromatic amino acid decarboxylase inhibitor (NSD1015) was monitored.Result: The absence of DOPA in dialysates of 6 hydroxdopamine pretreated rats, the measurable DOPA and the steady decreasing of 3,4 dihydroxyphenylacetic acid(DOPAC) during infusion of NSD1015 and the disappearance of DOPA after administration of α methyl ρ tyrosine indicated that the dialyzed DOPA was derived from dopaminergic nerve terminals.After intraperitoneal administration of dopamine receptor agonist apomorphine the DOPA output was deseased.After intraperitoneal administration of dopamine recepter antagonist haloperidol, the DOPA output was increased. The study showed that twenty four hours ofter implantation of the probe with infusion of 0.01 mmol·L -1 NSD1015, the DOPA level in the striatum of 6 hydroxdopamine pretreated rats was 0.39±0.12pmol/min(X±S,n=5).Conclusion:The DOPA concentration in striatal dialysates could be considered as an index of tyrosion hydroxlase activity during infusion of 0.01mM NSD1015.The method in vivo to monitor tyrosine hydroxlase activity in the brain is reliable.