建立脑双位点微透析结合高效液相色谱-柱后固定化酶反应器-电化学方法同步监测大鼠腹内侧前额叶皮质和海马中乙酰胆碱和胆碱含量

To establish method of determination of acetylcholine and choline in medial prefrontal cortex and hippocampus of rats by doubleprobe brain synchronous micro-dialysis combined with high performance liquid chromatography with post column immobilized enzyme r

目的建立同步动态监测清醒自由活动状态下大鼠腹内侧前额叶皮质（mPFC）和海马细胞外液中乙酰胆碱（ACh）和胆碱（Ch）方法,用于药物干预下不同脑区胆碱类神经递质变化研究。方法建立mPFC和海马双位点双通道同步微透析采样技术和高效液相色谱-柱后固定化酶反应器-电化学（HPLCIMER-ECD）方法。通过对清醒自由活动大鼠mPFC和海马同步灌流透析取样,HPLC-IMER-ECD分析药物作用下大鼠mPFC和海马细胞外液中ACh和Ch的动态变化,验证该技术方法的可行性和适用性。结果大鼠mPFC和海马双套管植入术及透析采样后状态表现良好,表明脑双位点双通道同步微透析采样技术可行。HPLC-IMER-ECD法测定ACh和Ch,两者浓度在0.02-2.00μmo·lL-1范围内线性关系良好,ACh最低检测限为10 nmo·lL-1,Ch最低检测限为5 nmol·L-1（信噪比为3∶1）。ACh 0.1,0.4和2.0μmol·L-1日内精密度相对标准偏差为0.87%-1.83%,日间精密度为5.38%-6.80%;Ch日内精密度相对标准偏差为1.60%-2.91%,日间精密度相对标准偏差为5.56%-7.38%;ACh相对回收率为96.0%-113.0%,Ch为102.3%-109.2%。叠氮钠能明显降低mPFC和海马细胞外液中ACh（P〈0.05）,但能显著升高两脑区内Ch含量（P〈0.05）。人参皂苷Rg1可逆转叠氮钠导致的海马区ACh降低（P〈0.05）及mPFC和海马区Ch含量的升高（P〈0.05）。mPFC细胞外液中ACh和Ch较高,受到叠氮钠影响较海马区更敏感。结论脑双位点微透析结合HPLC-IMER-ECD方法能同步监测大鼠mPFC和海马中ACh和Ch含量变化。人参皂苷Rg1可通过调节脑内ACh和Ch动态平衡,增强中枢胆碱能系统的功能。

OBJECTIVE To establish a method of synchronous dynamic monitoring of extracellular acetylcholine（ ACh） and choline（ Ch） in the medial prefrontal cortex（ mPFC） and hippocampus of freely moving rats,which is used to analyze the changes of ACh and Ch in different brain regions through medicine intervention. METHODS The double-probe synchronous brain microdialysis and high performance liquid chromatography with post column immobilized enzyme reactor electrochemical detection（ HPLC-IMER-ECD） were established and used to monitor dynamically ACh and Ch of extracellular fluid in the mPFC and hippocampus of freely moving rats. RESULTS After the double-cannula implantation and dialysis sampling in the mPFC and hippocampus,the rat performance was normal. The dual-probe brain microdialysis was feasible and practical. Determination of ACh and Ch by HPLC-IMER-ECD had good linear relationship within the range of 0. 02 to 2. 00 μmol·L- 1. The detection limit was 10 nmol·L- 1for ACh and 5 nmol·L- 1for Ch. Intra-day precision of ACh at three concntrations（ 0. 1,0. 4 and2. 0 μmo·lL- 1） was 0.87%-1.83%,inter-day precision of ACh was 5.38%-6.80%; Intra-day precision of Ch was 1. 60%- 2. 91%,inter-day precision of Ch was 5. 56%- 7. 38%. Relative recovery of ACh was 96. 0%- 113. 0%,and that of Ch was 102. 3%- 110. 2%. NaN3could result in the decrease of ACh（ P 0. 05） and the increase of Ch（ P 0. 05,P 0. 01） in both the mPFC and hippocampus.Ginsenoside Rg1 reversed the effect induced by NaN3,increased significantly ACh（ P 0.05） in the hippocampus,and decreased Ch of the mPFC and hippocampus（ P 0. 05）. ACh and Ch in the mPFC were higher than in the hippocampus,and the impact of NaN3was more sensitive to changes in mPFC.CONCLUSION Double-probe and dual-channel brain microdialysis technique combined with HPLC-IMERECD can monitor synchronously the dynamic changes of ACh and Ch in the mPFC and hippocampus of conscious rats. Ginsenoside Rg1 can up-regulate the dynamic balance of ACh and Ch in the mPFC and hippocampus while enhancing central cholinergic system functions.