苄丝肼对大鼠纹状体细胞外液左旋多巴药动学的影响

目的探讨苄丝肼对左旋多巴大鼠纹状体细胞外液药动学影响。方法大鼠随机分3组（每组7只）：左旋多巴合并苄丝肼组,大鼠灌胃给予左旋多巴48mg·kg^-1和苄丝肼12mg·kg^-1;单用左旋多巴组,大鼠灌胃给予左旋多巴48mg·kg^-1;生理盐水对照组,大鼠灌胃给予等体积生理盐水。采用脑微透析活体取样和高效液相色谱技术,测定给药后6h内不同时间点大鼠血浆和纹状体细胞外液左旋多巴浓度,应用3P87药动学程序拟合药动学参数。结果左旋多巴在血浆及纹状体细胞外液药-时曲线符合一室模型。单用左旋多巴在纹状体细胞外液t1/2、tmax、ρmax和AUC0→∞分别为（21．55±13．74）min、（46．82±27．49）min、（9．283±2．130）μg·L^-1、（2762±1257）μg·L^-1;左旋多巴合用苄丝肼组纹状体细胞外液t1/2、tmax、ρmax和AUC0→∞分别为（83．50±10．24）min、（49．97±11．72）min、（119．62±81．12）μg·L^-1、（18431±9115）μg·L^-1。其中,除tmax外,单用左旋多巴组t1/2、ρmax和AUC0→∞均显著小于左旋多巴合用苄丝肼组（P〈0．01）。结论苄丝肼可显著提高左旋多巴进入纹状体药量。     

建立脑内灌流叠氮钠所致的大鼠纹状体细胞外液乙酰胆碱水平下降的模型

目的　建立可用于筛选防治老年退行性病变和其他脑细胞损伤所致智能减退药物的模型。方法应用脑内微透析技术 ,在清醒自由活动大鼠纹状体内灌流含叠氮钠 (NaN3,30mmol·L- 1)和新斯的明(10 μmol·L- 1)的林格液 ,连续收集透析液。用高效液相色谱 柱后固定化酶反应器 电化学检测器检测乙酰胆碱 (ACh)和胆碱水平。结果　脑内灌流NaN390min后该脑区细胞外液ACh水平较正常组持续下降 (P <0 .0 5～ 0 .0 1)。最低值在停止NaN3灌流后6 0min时出现 ,为相同时间点对照组水平的 10 .0 % ;此后有所恢复 ,但在恢复期 180min内未能达到对照组水平。阳性药甲磺酸阿米三嗪 (10mg·kg- 1·d- 1,ig ,灌流前连续给药 14d)可在停止NaN3灌流后 30和 6 0min时明显减缓ACh水平的下降 (均P <0 .0 5 )。结论　大鼠脑内灌流NaN3使局部脑区胆碱能神经功能损伤 ,可造成ACh水平持续下降的急性模型。     

高效液相色谱-电化学法测定大鼠纹状体细胞外液中单胺类神经递质含量

目的:建立应用常规高效液相色谱-电化学法(HPLC-ECD)测定大鼠脑微透析液中单胺类神经递质及其代谢产物。方法:将探针插入大鼠右侧纹状体,在清醒自由活动状态下,用Ringer氏液以1.5μL.min-1的速度灌流,每20 min收集l管透析液,将其注入HPLC-ECD仪,考察本方法的专属性、线性范围、精密度和准确度等,并对其中所含的NE、DA、DOPAC、HIAA、HVA和5-HT进行含量测定。结果:各递质在0.025～0.5 ng.μL-1范围内线性关系良好,方法加样回收完全,日间日内测定均RSD<3.8%。结论:本实验选用了高效液相色谱与电化学检测器连用的方法测定神经递质的含量,该方法简单准确、灵敏、特异性好。     

药物干预对6-羟基多巴胺处理大鼠的纹状体细胞外液多巴胺的影响

目的 研究古拉定、川芎嗪、丹参对6—羟基多巴胺(6—OHDA)处理后大鼠的纹状体细胞外液多巴胺(DA)及其代谢产物的影响。方法 运用6—OHDA损毁大鼠黑质，设立正常对照组、6—OHDA损毁组、损毁后古拉定治疗组、川芎嗪治疗组和丹参治疗组。损毁后8周用微透析法采集各组大鼠纹状体细胞外液，用高效液相色谱-电化学法(HPLC—ECD)检测DA、DOPAC、HVA。结果 发现四组损伤组的DA及其代谢产物含量明显低于正常对照组(P＜0．001)，而三组治疗组与非治疗组组间比较无统计学差异(P＞0．05)。结论三种药物不能改变6—OHDA处理后大鼠纹状体细胞外液DA的含量。     

都可喜对脑能量代谢障碍大鼠纹状体细胞外液乙酰胆碱、葡萄糖和乳酸水平的影响

目的探讨阳性药都可喜对脑内灌流叠氮钠(NaN3)所致脑能量代谢障碍大鼠纹状体细胞外液乙酰胆碱(ACh)、葡萄糖(Glucose)和乳酸(LD)水平的作用。方法大鼠分3组:对照组、模型组和都可喜组。都可喜组以10mg.kg-1.d-1灌胃14d,其余2组以普通水灌胃14d。2wk后,应用脑内微透析技术,纹状体内灌流NaN3,并动态监测清醒自由活动大鼠纹状体细胞外液ACh、Glucose和LD水平的变化。结果在NaN3灌流(90min)和停止灌流(180,240,360min)期间,模型组大鼠纹状体细胞外液ACh水平分别明显低于对照组(P<0.01、0.05、0.01、0.01);模型组细胞外液Glucose水平在90、180、240min时则明显低于对照组(P<0.01、0.01、0.05);模型组的LD水平在各时间点则明显高于对照组(P均<0.01);都可喜组在360min时间点的ACh水平明显高于模型组(P<0.05),在240min时Glucose水平明显高于模型组(P<0.05),而LD水平在各时间点与模型组无差异(P均>0.05)。结论叠氮钠脑内灌流所致ACh水平下降的模型大鼠纹状体细胞外液葡萄糖和乳酸水平的变化反映了脑能量代谢的障碍,都可喜有一定的干预作用。为改善脑能量代谢的脑保护作用的药物研究提供了可用的方法。     

帕金森病6-OHDA模型大鼠左旋多巴血药浓度与纹状体细胞外液氨基酸类递质结合研究的方法

目的建立对清醒自由活动的帕金森病模型大鼠进行左旋多巴(L-DOPA)血药浓度与纹状体细胞外液氨基酸类神经递质水平同步动态监测、结合研究的方法。方法 SD大鼠,脑内注射6-羟基多巴胺(6-OHDA)造模,血、脑双位点微透析采样、高效液相-荧光方法(HPLC-FLD)检测相关物质的浓度。结果大鼠腹腔给药吸收迅速,L-DOPA药时曲线符合一室模型;美多巴高剂量组的AUC、Cmax明显高于美多巴低剂量组(P<0.05或P<0.01)。与对照组相比,模型组大鼠纹状体细胞外液谷氨酸(Glu)水平明显升高,γ-氨基丁酸(GABA)水平明显降低;美多巴投药改善了6-OHDA导致的脑内氨基酸递质紊乱(P<0.05或P<0.01)。相同时间点血中L-DOPA浓度与脑内Glu/GABA值进行回归分析,r2值为0.7950。结论采用血、脑双位点微透析采样、HPLC-FLD检测技术,实现了对清醒自由活动大鼠L-DOPA血药浓度与纹状体细胞外液药效指标同步、动态的观察,提供了反映L-DOPA血药浓度-纹状体效应-时间三维关系的直接依据。     

大鼠左旋多巴给药后纹状体高香草酸水平变化

目的:探讨左旋多巴给药后大鼠纹状体细胞外液高香草酸浓度随时间变化规律。方法:大鼠单次灌胃给予左旋多巴48mg.kg-1和苄丝肼12mg.kg-1后,采用脑微透析活体取样和高效液相色谱技术,测定给药后6h内不同时间点大鼠纹状体细胞外液高香草酸和左旋多巴浓度。结果:左旋多巴+苄丝肼给药后大鼠纹状体细胞外液高香草酸水平升高明显迟于左旋多巴,显示左旋多巴代谢为高香草酸需一定时间;左旋多巴水平在给药4h后基本恢复到基础水平,而高香草酸水平给药6h后仍远高于给药前水平。结论:大鼠如以左旋多巴48mg.kg-1和苄丝肼12mg.kg-1每6h一次给药,高香草酸水平可能存在蓄积现象,其临床意义有待进一步研究。     

建立脑内灌流6-OHDA所致大鼠纹状体细胞外液羟自由基升高的模型

目的建立脑内灌流6-羟基多巴胺(6-OHDA)所致大鼠纹状体细胞外液羟自由基升高的模型,为老年退行性病变和其它氧化应激所致脑细胞损伤的研究和药物筛选提供可利用的方法。方法微透析脑内灌流6-OHDA造模,采用水杨酸捕获羟自由基,高效液相-电化学检测技术,对活体脑内羟自由基所形成的2,3二羟基苯甲酸(2,3-DHBA)和2,5二羟基苯甲酸(2,5-DHBA)进行测定。结果6-OHDA脑内灌流后,模型组大鼠纹状体细胞外液2,3-DHBA和2,5-DH-BA在75 m in分别为对照组的6.6和3.4倍;2,3-DHBA在观察的全程中,一直高于对照组(P<0.01);2,5DHBA大部分时间点也高于对照组(P<0.05或P<0.01);维生素EC组的2,3-DHBA有4个时点低于模型组(P<0.05或P<0.01),2,5-DHBA各时点均低于模型组,但差异无显著性。结论6-OHDA脑内灌流可以造成大鼠纹状体细胞外液羟自由基升高的急性模型。     

大鼠脑透析液中羟自由基和单胺类递质及其代谢产物同步监测的方法

目的建立同时监测脑透析液中羟自由基和单胺类递质及其代谢产物水平的方法。方法应用脑内微透析技术、水杨酸捕获羟自由基和高效液相-电化学检测器(HPLC-ED)同步监测清醒自由活动大鼠纹状体细胞外液羟自由基和单胺类递质及其代谢产物的水平。结果从NE、EPI、DOPAC、DA、5-H IAA、2,5-DHBA、HVA、2,3-DHBA、3-MT和5-HT的保留时间、最小检测值、标准曲线在20~160μg.L-1浓度范围内与峰高的相关系数、同日4次测定混合标准的变异系数CV等表明本色谱分析的方法是可靠的;R inger液和水杨酸(SASS)-R inger液灌流时纹状体细胞外液单胺类递质及其代谢产物的水平无明显变化(P均>0.05)。SASS-R inger灌流时可测出羟自由基的水平,且不影响单胺类的测定。结论用SASS-R inger灌流收集的透析液可在本实验的条件下,一次进样后同时测定羟自由基和单胺类递质及其代谢产物。     

应用微透析技术和质谱法测定川芎嗪对大鼠脑内乙酰胆碱释放量的影响

本文结合脑内微透析技术与现代分析方法，考察川芎嗪对大鼠脑内乙酰胆碱释放的影响。采用脑内微透析技术进行取样，建立高效液相-串联四极杆质谱方法测定脑透析液中的乙酰胆碱含量。结果显示，川芎嗪皮下给药能够剂量相关地增加大鼠脑内不同脑区中乙酰胆碱的释放。该法能够准确反映药物对大鼠脑内乙酰胆碱释放量的影响，与传统方法相比，具有明显的优势。     

The effect of drugs on the choline and acetylcholine content of the rat striatum following two methods of sacrifice

Choline and acetylcholine concentrations were determined in the striatum of rats sacrificed either by decapitation or by microwave irradiation. In this structure, as shown by isotopic experiments using intravenously administered [³H]-choline, decapitation caused a 50% loss of acetylcholine which was quantitatively recovered as choline. After microwave irradiation, choline levels were identical in the striatum and in the frontal cortex. The levels of choline found in the two brain structures (23 dnmol/g wet weight) were almost identical to the blood choline concentration (19.4 nmol/mn plasma). Various cholinergic and dopaminergic drugs such as oxotremorine, atropine, haloperidol, reserpine, a-methyl-p-tyrosine and L-DOPA altered the striatal acetylcholine content measured after microwave irradiation.     

Changes in extracellular striatal acetylcholine and brain seizure activity following acute exposure to nerve agents in freely moving guinea pigs

Organophosphorus nerve agents irreversibly inhibit acetylcholinesterase (AChE) in the peripheral and central nervous systems, causing an increase in the concentration of acetylcholine (ACh) in the synapse or neuromuscular junction and subsequent adverse effects. In this study, in vivo microdialysis was utilized to collect samples from the striatum for monitoring changes in extracellular ACh levels along with cortical electroencephalographic (EEG) recordings for identifying seizure activity after acute subcutaneous (s.c.) exposure to 1.0?×?LD₅₀ of the nerve agents sarin, soman, or one of two V-type agents (VX, or a Russian V-agent, designated VR) in unanesthetized freely moving guinea pigs. Based on EEG recordings, these animals were subsequently divided into groups that developed seizures (S) and those that did not develop seizures (NS). Maximum ACh levels in the striatum were observed at 60–70?min for sarin and soman S groups and 105?min for VX and VR S groups. In all NS groups the greatest increase in extracellular ACh occurred within 30?min after exposure, although in the sarin NS group a few sporadic increases of ACh from control occurred. Animals that developed seizures, regardless of the nerve agent, had significantly higher extracellular striatal ACh levels compared to the controls or those animals that did not develop seizures, yet both S and NS groups displayed similar levels of blood AChE inhibition. Regardless of the agent, all animals in the non-seizure groups survived 24?h, while lethality (25–42%) was observed only in animals that experienced seizure activity.     

Pharmacokinetics and dopamine/acetylcholine releasing effects of ginsenoside Re in hippocampus and mPFC of freely moving rats

Aim:

To investigate the pharmacokinetics and dopamine/acetylcholine-releasing effects of ginsenoside Re (Re) in brain regions related to learning and memory, and to clarify the neurochemical mechanisms underlying its anti-dementia activity.

Methods:

Microdialysis was conducted on awake, freely moving adult male SD rats with dialysis probes implanted into the hippocampus, medial prefrontal cortex (mPFC) or the third ventricle. The concentrations of Re, dopamine (DA) and acetylcholine (ACh) in dialysates were determined using LC-MS/MS.

Results:

Subcutaneous administration of a single dose of Re (12.5, 25 or 50 mg/kg) rapidly distributed to the cerebrospinal fluid and exhibited linear pharmacokinetics. The peak concentration (C_max) occurred at 60 min for all doses. Re was not detectable after 240 min in the dialysates for the low dose of 12.5 mg/kg. At the same time, Re dose-dependently increased extracellular levels of DA and ACh in the hippocampus and mPFC, and more prominent effects were observed in the hippocampus.

Conclusion:

The combined study of the pharmacokinetics and pharmacodynamics of Re demonstrate that increase of extracellular levels of DA and ACh, particularly in the hippocampus, may contribute, at least in part, to the anti-dementia activity of Re.     

Effects of scopolamine on extracellular acetylcholine and choline levels and on spontaneous motor activity in freely moving rats measured by brain dialysis

The present study demonstrates the feasibility of measuring acetylcholine (ACh) and choline in perfusate samples collected by in vivo brain dialysis in the frontal cortex and hippocampus of freely moving rats in which spontaneous motor activity could be measured simultaneously. Systemically administered scopolamine increased the output of ACh about 10-fold and 20-fold in the frontal cortex and hippocampus, respectively. By contrast, scopolamine decreased the choline level in the extracellular fluid about 2-fold in both brain regions, possibly owing to enhanced choline uptake into the presynaptic nerve terminals. Scopolamine also increased spontaneous motor activity over the same time course as the changes in ACh and choline. These results indicate that the in vivo brain dialysis technique applied to freely moving rats may be useful in investigating ACh turnover and in studying the relation between cholinergic transmission and behavioral functions.     

Differential effects of the neuropeptide galanin on striatal acetylcholine release in anaesthetized and awake rats

1. In the present study the mechanisms were examined by which the neuropeptide galanin modulates the extracellular concentrations of striatal acetylcholine (ACh) in enflurane anaesthetized and in freely moving male rats by use of in vivo microdialysis and high performance liquid chromatography.
2. The perfusion of galanin through the microdialysis probe (0.3 nmol μl⁻¹, flow rate: 2 μl min⁻¹) caused a statistically significant increase in the basal striatal ACh levels in anaesthetized but a decrease in awake animals. No significant effect was revealed after a low dose (0.1 nmol μl⁻¹, flow rate: 2 μl min⁻¹) of galanin perfusion. Both the stimulating and inhibitory effects of galanin on basal ACh release were reversible.
3. The muscarinic antagonist scopolamine (0.1 mg kg⁻¹, subcutaneously (s.c.)) caused a significant increase in ACh release in both anaesthetized and awake animals.
4. The combination of galanin plus scopolamine attenuated the stimulant effect on ACh release caused by scopolamine alone in awake animals.
5. The putative galanin receptor antagonist M35 at 0.3 nmol μl⁻¹ but not at 0.1 nmol μl⁻¹ caused a significant reduction (20%) in ACh release, supporting the view that M35 at higher concentrations behaves as a partial agonist at the galanin receptor. When M35 (0.1 nmol μl⁻¹) was co-infused with galanin (0.3 nmol μl⁻¹) the galanin-evoked decrease in ACh release was completely blocked.
6. Taken together, these results indicate that galanin affects basal ACh release via stimulation of galanin receptors within the striatum. The mechanism involved is dependent on the anaesthesia procedure which may act via enhancement of γ-aminobutyric acid_A (GABA_A) mediated transmission within striatal and/or output neurones. In addition, anaesthesia may also decrease the activity of glutamatergic striatal afferents. The results with M35 indicate that the role of galanin perfused in striatum is permissive in the normal rat. Furthermore, galanin is a potent inhibitory modulator of basal ACh release also in the striatum, as recently was shown in the ventral hippocampus in awake animals.

     

Differential effects of pyrethroid insecticides on extracellular dopamine in the striatum of freely moving rats

In order to obtain a more complete understanding of pyrethroid neurotoxicity, effects of the pyrethroid insecticides, allethrin (type I), cyhalothrin (type II) and deltamethrin (type II) on extracellular levels of dopamine (DA) and its metabolites in the striatum of conscious rats were studied by in vivo microdialysis. Rats were treated i.p. with pyrethroids or vehicle. Allethrin had a dual effect on DA release. The increase in the extracellular level of striatal DA by 10 mg/kg allethrin reached a maximum of 178% of baseline but 20 and 60 mg/kg inhibited DA release to 63% and 52% of baseline with a peak effect at 60-80 min after injection. Cyhalothrin 10, 20 and 60 mg/kg inhibited DA release to 65%, 56% and 45% of basal release, respectively, with a peak time of inhibition 40-80 min past injection. Deltamethrin (10 and 20 mg/kg) increased DA release to maximum of 187% and 252% of basal release whereas 60 mg/kg first reduced the efflux for 40 min to 50% of basal release and then increased the efflux to a maximum of 344% of basal release with a peak time of 120 min. Local infusion of 1 microM tetrodotoxin, a Na(+) blocker through the dialysis probe completely prevented the effect of allethrin (10 and 60 mg/kg), cyhalothrin (60 mg/kg) and deltamethrin (20 mg/kg) on DA release but only partially blocked the effects of 60 mg/kg deltamethrin. The effect of deltamethrin (60 mg/kg) on DA release was completely prevented by local infusion of 10 microM nimodipine, an L-type Ca(++) channel blocker. All three pyrethroids did not alter the extracellular levels of DOPAC, 3-MT and HVA except that 20 and 60 mg/kg of allethrin and cyhalothrin increased 3-MT levels. Effect of the pyrethroids on synaptosomal DA uptake was also examined. The DA uptake was decreased in rats exposed to 60 mg/kg of allethrin and cyhalothrin but was increased in rats exposed to 60 mg/kg of deltamethrin. Our results demonstrate that striatal DA release and DA uptake are differentially affected by type I and the two type II pyrethroids indicating that dopaminergic circuitry, striatal DA in particular, may be a pyrethroid target and that pyrethroids may be acting on striatal DA by multiple mechanisms.