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。     

脑内灌流6-羟基多巴胺对大鼠纹状体细胞外液氨基酸类神经递质的影响及美多芭的作用

目的探讨脑内灌流6-羟基多巴胺(6-OHDA)对大鼠纹状体细胞外液氨基酸类神经递质的影响以及美多芭的作用。方法SD大鼠,随机分为对照组、模型组及美多芭高、低剂量组。采用微透析技术脑内灌流6-OHDA造模,邻苯二甲醛柱前衍生,高效液相-荧光检测技术,动态监测清醒自由活动大鼠纹状体细胞外液氨基酸神经递质水平。结果6-OHDA脑内灌流40 min后,模型组大鼠纹状体细胞外液谷氨酸(Glu)浓度有4个时间点、γ-氨基丁酸(GABA)浓度有1个时间点、牛磺酸(Tau)浓度有8个时间点较对照组升高(P<0.05或P<0.01)。美多芭高、低剂量组与模型组比较分别有5个和4个时间点Glu浓度降低、有7个和2个时间点GABA水平升高(P<0.05或P<0.01)。结论6-OHDA脑内灌流引起大鼠纹状体细胞外液氨基酸类神经递质变化,美多芭可以剂量依赖性地抑制其引起的Glu升高,并进一步增加GABA的浓度。     

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。     

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。     

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。     

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。     

应用脑微透析技术建立L-DOPA引起的PD大鼠脑氧化损伤的模型

目的应用脑微透析技术建立左旋多巴(L-DOPA)引起的PD大鼠脑氧化损伤的模型。方法6-羟基多巴胺(6-OHDA)脑内注射造成PD大鼠模型,采用微透析技术,脑内灌流L-DOPA、水杨酸捕获羟自由基和高效液相-电化学检测,动态观察L-DOPA处理前后大鼠纹状体细胞外液多巴胺(DA)及其代谢产物,羟自由基被捕获所形成的2,3-二羟基苯甲酸(2,3-DHBA)、2,5-二羟基苯甲酸(2,5-DHBA)浓度的变化。结果L-DOPA处理后,模型组大鼠2,3-DHBA和2,5-DHBA浓度分别有6个和7个时间点较对照组明显升高(P<0.05,P<0.01);还原型谷胱甘肽在多个时间点抑制了这种升高(P<0.05,P<0.01)。结论应用脑微透析技术建立L-DOPA引起的PD大鼠脑氧化应激损伤的急性模型,可以进行在体的和动态的监测,为筛选协同L-DOPA治疗PD的抗氧化损伤药物提供有用的方法。     

用在体脑微透析技术研究大鼠纹状体中单胺递质的释放和代谢

采用脑微透析技术与高效液相色谱-电化学检测器联用测定了清醒自由活动大鼠纹状体细胞外液中多巴胺(DA)及其酸性代谢物3,4-二羟苯乙酸(DOPAC)和高香草酸(HVA)以及5-羟色胺代谢物5-羟吲哚乙酸(5-HIAA)的含量.透析液中DA为0.44 pmol/40μl,DOPAC和HVA含量较DA高约80倍.右旋苯丙胺2 mg/kg,ip使纹状体DA释放显著增加,DOPAC和HVA含量明显下降.     

微透析技术在中药脑保护机理研究中的应用

应用微透析和高效液相色谱技术，分别对脑内灌流叠氮钠（NaN3）所致的脑线粒体损伤大鼠以及双侧颈总动脉结扎再灌引起的不完全脑缺血大鼠的脑细胞外液乙酰胆碱（ACh）、儿茶酚胺和氨基酸类神经递质的水平进行动态监测，探讨天麻方（TMF）脑保护作用的机理。[第一段]     

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。     

Anaesthesia effects on in vivo acetylcholine transmission; comparisons of radioenzymatic and HPLC assays

The effect of general anaesthesia on extracellular levels of acetylcholine (ACh) in the caudate-putamen of freely moving rats was studied by microdialysis. ACh concentrations were determined in the same perfusate samples by radioenzymatic and HPLC/electrochemical procedures in order to compare the assays. The concentration of ACh in perfusate samples was estimated to be 0.30 microM in conscious unrestrained rats. However, when these rats were administered chloral hydrate (400 mg/kg i.p.), the level of ACh was decreased immediately by 50%, attaining a value of 0.06 microM within 20-40 min following the injection. Upon recovery of the righting reflex, ACh levels were once again re-elevated. The levels of choline (Ch), the precursor of ACh, were unaffected by anaesthesia. It was apparent that the level of consciousness (i.e. awake vs. anaesthetized) is an important factor determining ACh overflow. Radioenzymatic and HPLC assays proved to give identical results for the analysis of ACh and Ch.     

Effects of the centrally acting cholinesterase inhibitors tetrahydroaminoacridine and E2020 on the basal concentration of extracellular acetylcholine in the hippocampus of freely moving rats

The effects of the centrally acting cholinesterase (ChE) inhibitors, tetrahydroaminoacridine (THA) and E2020 (1-benzyl-4-[(5,6-dimethoxy-l-indanon)-2-yl] methylpiperidine hydrochloride), potential drugs for the treatment of senile dementia, on the basal extracellular acetylcholine (ACh) concentration in the hippocampus of freely moving rats, were determined using a microdialysis technique without the use of a ChE inhibitor in the perfusion fluid and a sensitive RIA. The mean (±SEM) basal ACh content in the perfusate was 103.1 ± 3.6 fmol/sample collected over 30 min when microdialysis probes with a length of 3 mm dialysis membrane were used. The content of ACh decreased to an almost undetectable level upon perfusion of magnesium, suggesting that, in the present study, most of the ACh detected in the perfusates was due to cholinergic neuronal activity. THA (1.65 mg/kg, i.p.) produced an insignificant increase in the extracellular ACh concentration, but a dose of 5 mg/kg, i.p. caused a prolonged and significant 5.5-fold increase from the control value. E2020 (0.65 and 2 mg/kg, i.p.) produced significant, prolonged and dose-dependent increases (4 and 12 times the control value, respectively), the peak effect occurring within 1 h. Perfusion with 10 mol/l physostigmine produced an about 30-fold increase of ACh output, suggesting that the basal extracellular ACh concentration is highly dependent on ChE activity. When ChE was inhibited locally by perfusion with physostigmine, THA (5 mg/kg) produced a transient and, at its maximum, a 1.42-fold increase in extracellular ACh concentration. These results demonstrate that the basal, physiological, extracellular ACh concentration in the hippocampus of freely moving rats can be determined using a microdialysis technique and a sensitive RIA, and suggest that THA and E 2020 increase ACh concentration in the synaptic cleft of the hippocampus in a dose-dependent manner mostly through ChE inhibition. Correspondence to: K. Kawashima at the above address     

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.     

Systemic morphine simultaneously decreases extracellular acetylcholine and increases dopamine in the nucleus accumbens of freely moving rats.

Microdialysis was used to measure extracellular levels of acetylcholine (ACh) and dopamine (DA) simultaneously in the nucleus accumbens (NAC) of freely moving rats. Systemic injection of morphine (20 mg/kg) significantly decreased ACh (30%, p less than .01) while it increased DA (55%, p less than .01). The effects of morphine were eliminated by naloxone. The results confirm that morphine increases DA and in addition, demonstrate an inhibitory influence of this opiate on extracellular levels of ACh in the NAC.     

Pharmacological characterization of nicotine-induced acetylcholine release in the rat hippocampus in vivo: evidence for a permissive dopamine synapse.

In this study, the mechanism of nicotine-induced hippocampal acetylcholine (ACh) release in awake, freely moving rats was examined using in vivo microdialysis. Systemic administration of nicotine (0.4 mg kg(-1), s.c.) increased the levels of ACh in hippocampal dialysates. The nicotine-induced hippocampal ACh release was sensitive to the pretreatment of neuronal nicotinic acetylcholine receptor (nAChR) antagonists mecamylamine (3.0 mg kg(-1), s.c.) and dihydro-beta-erythrodine (DHbetaE; 4.0 mg kg(-1), s.c.) as well as systemic administration of the dopamine (DA) D1 receptor antagonist SCH-23390 (R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-benzaz epine; 0.3 mg kg(-1), s.c.). Local perfusion of mecamylamine (100 microM), DHbetaE (100 microM) or SCH-23390 (10 microM) through microdialysis probe did not increase basal hippocampal ACh release. Hippocampal ACh release elicited by systemic administration of nicotine (0.4 mg kg(-1), s.c.) was antagonized by local perfusion of SCH-23390 (10 microM), but not by MEC (100 microM) or DHbetaE (100 microM). Direct perfusion of nicotine (1 mM, but not 0.1 mM) increased hippocampal ACh levels; however, this effect was relatively insensitive to blockade by co-perfusion of either mecamylamine (100 microM) or SCH-23390 (10 microM). These results suggest that nicotine-induced hippocampal ACh release occurs by two distinct mechanisms: (1) activation of nAChRs outside the hippocampus leading to DA release and subsequent ACh release involving a permissive DA synapse, and (2) direct action of nicotine within the hippocampus leading to ACh release via non-DA-ergic mechanism.     

Effect of Toluene Inhalation on Extracellular Striatal Acetylcholine Release Studied with Microdialysis

Abstract: Intracerebral microdialysis was performed on awake, freely moving rats in order to record effect of toluene exposure on acetylcholine release in striatum. Acetylcholine release decreased during (about 20%) and after (about 60%) toluene exposure (2 hr, 2000 p.p.m.) Striatal acetylcholine release is thought to be mediated by dopamine. In a previous work we found that extracellular dopamine levels increase during toluene exposure. A dopamine uptake inhibitor (LU 19-005, 2 mg/kg) was therefore injected subcutaneously and the effect of increased extracellular dopamine on acetylcholine release within the striatum was monitored in the abscence of toluene exposure. LU 19-005 increased striatal dopamine levels six times and the acetylcholine levels increased to about 14% of basal value. The present study shows that toluene exposure decrease acetylcholine release while an injection of a dopamine uptake inhibitor fails to decrease acetylcholine release. Indicating that acute exposure of toluene decreases striatal acetylcholine release by a mechanism that is not mediated by increased extracellular dopamine levels. Our data suggest that toluene decrease acetylcholine release within the striatum and that this effect not is mediated by increased extracellular dopamine levels.     

Development of a liquid chromatography/tandem mass spectrometry method for the quantitation of acetylcholine and related neurotransmitters in brain microdialysis samples

Monitoring concentrations of acetylcholine (ACh) in specific brain regions is important in understanding disease pathology, as well as in designing and evaluating novel disease-modifying treatments where cholinergic dysfunction is a hallmark feature. We have developed a sensitive and quantitative liquid chromatography/tandem mass spectrometry method to analyze the extracellular concentrations of ACh, choline (Ch) and (3-carboxylpropyl)-trimethylammonium (iso-ACh) in brain microdialysis samples of freely moving animals. One immediate advantage of this new method is the ability to monitor ACh in its free form without having to use a cholinesterase inhibitor in the perfusate. The separation of ACh, Ch, iso-ACh and related endogenous compounds was carried out based on cation exchange chromatography with a volatile elution buffer consisting of ammonium formate, ammonium acetate and acetonitrile. An unknown interference of ACh, which was observed in brain microdialysates from many studies, was well separated from ACh to ensure the accuracy of the measurement. Optimization of electrospray ionization conditions for these quaternary ammonium compounds achieved the limits of detection (S/N=3) of 0.2 fmol for ACh, 2 fmol for Ch and 0.6 fmol for iso-ACh using a benchtop tandem quadrupole mass spectrometer with moderate sensitivity. The limit of quantitation (S/N=10) was 1 fmol for ACh, 3 fmol for iso-ACh and 10 fmol for Ch. This method was selective, precise (<10% R.S.D.), and sensitive over a range of 0.05-10nM for ACh, 0.25-50 nM for iso-ACh and 15-3000 nM for Ch. To demonstrate that the developed method can be applied to monitoring changes in ACh concentrations in vivo, reference agents that have previously been shown to influence ACh levels were studied in rat dorsal hippocampus. This includes the 5-HT6 receptor antagonist, SB-271046, and the cholinesterase inhibitor, donepezil. Moreover, levels of ACh were demonstrated to be sensitive to infusion of tetrodotoxin (TTX) suggesting that the ACh being measured in vivo was of neuronal origin. Collectively, these biological data provided in vivo validation of this analytical method.     

Evaluation of acetylcholine,seizure activity and neuropathology following high-dose nerve agent exposure and delayed neuroprotective treatment drugs in freely moving rats

Organophosphorus nerve agents such as soman (GD) inhibit acetylcholinesterase, producing an excess of acetylcholine (ACh), which results in respiratory distress, convulsions and status epilepticus that leads to neuropathology. Several drugs (topiramate, clobazam, pregnanolone, allopregnanolone, UBP 302, cyclopentyladenosine [CPA], ketamine, midazolam and scopolamine) have been identified as potential neuroprotectants that may terminate seizures and reduce brain damage. To systematically evaluate their efficacy, this study employed in vivo striatal microdialysis and liquid chromatography to respectively collect and analyze extracellular ACh in freely moving rats treated with these drugs 20?min after seizure onset induced by a high dose of GD. Along with microdialysis, EEG activity was recorded and neuropathology assessed at 24?h. GD induced a marked increase of ACh, which peaked at 30?min post-exposure to 800% of control levels and then steadily decreased toward baseline levels. Approximately 40?min after treatment, only midazolam (10?mg/kg) and CPA (60?mg/kg) caused a significant reduction of ACh levels, with CPA reducing ACh levels more rapidly than midazolam. Both drugs facilitated a return to baseline levels at least 55?min after treatment. At 24?h, only animals treated with CPA (67%), midazolam (18%) and scopolamine (27%) exhibited seizure termination. While all treatments except for topiramate reduced neuropathology, CPA, midazolam and scopolamine showed the greatest reduction in pathology. Our results suggest that delayed treatment with CPA, midazolam, or scopolamine is effective at reducing GD-induced seizure activity and neuropathology, with CPA and midazolam capable of facilitating a reduction in GD-induced ACh elevation.