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。     

首乌方对左旋多巴诱导异动症大鼠的干预作用及其机制

目的探讨首乌方对左旋多巴诱导的异动症(levodopa induced dyskinesia,LID)大鼠的干预作用及其机制。方法 32只雄性SPF级SD大鼠,随机分为4组:假手术组(sham operation,SO组)、异动症模型组(levodopa induced dyskinesia,LID组)、首乌方高剂量组(shouwufang-high dose,SWF-H组)、首乌方低剂量组(shouwufang-low dose,SWF-L组),每组8只。单侧双位点注射6-羟基多巴胺造成PD模型后,灌胃给予多巴丝肼(30 mg/kg)22 d诱发LID,首乌方剂量组同时灌胃(SWF-H含生药27 g/kg,SWF-L含生药18g/kg)。观察各组LID发病率并进行动物AIM评分;清醒自由活动状态下脑内纹状体微透析、水杨酸捕获羟自由基方法采样,高效液相-电化学检测动态观察病变靶器官细胞外液中羟自由基指标(2,3-DHBA、2,5-DHBA)的浓度变化;检测大鼠体内超氧化物歧化酶(SOD)及丙二醛(MDA)水平的变化。结果首乌方可明显降低大鼠LID的发病率及AIM评分,降低纹状体细胞外液2,3-DHBA及2,5-DHBA的浓度、并显著降低血清内MDA的水平。结论首乌方可以改善大鼠LID的行为学指标,有预防及干预LID的作用,其机制可能与减轻整体特别是病变靶器官的氧化应激损伤有关。     

大鼠脑微透析样品中左旋多巴、多巴胺及其代谢产物、羟自由基的测定

目的:建立快速、准确、同时测定大鼠脑微透析样品中左旋多巴(L-DOPA)、多巴胺(DA)及其代谢产物、羟自由基指标的高效液相色谱-电化学检测方法。方法:应用玻璃碳工作电极、银/氯化银(Ag/AgCl)参比电极、Antec Leyden BV C18分析柱,工作电压为0.52 V,单一流动相,检测大鼠脑微透析样品。结果:清醒自由活动大鼠纹状体微透析液中L-DOPA、DA及其代谢产物、羟自由基指标等10种物质在15 min内分离。标准曲线在一定浓度内线性良好。结论:所建方法可用于大鼠脑微透析样品中L-DOPA、DA及其代谢产物等的同时测定,为脑内L-DOPA药物浓度-药物效应-药物副作用同步研究提供可用的方法。     

HPLC检测在体大鼠脑缺血再灌期羟自由基的变化及维生素E的影响

选用水杨酸为捕捉剂,用HPLC—UV动态检测大鼠全脑缺血再灌期纹状体细胞外液羟自由基的变化。主要测定羟自由基与水杨酸生成稳定化合物2,5-二羟基苯甲酸(DHBA)的浓度。结果表明,2,5-DHBA在重灌15～25 min时达最高值,而在缺血期无明显变化。缺血前20 min分别ip Vit E30,60或120 mg·kg^-1均能抑制重灌期2,5-DHBA的升高。提示,本法可用于大鼠脑缺血再灌期羟自由基的测定,并可观察药物对在体脑缺血动物羟自由基反应的影响。     

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。     

依达拉奉对6-OHDA诱导的帕金森病大鼠黑质纹状体神经元的保护作用

目的探讨依达拉奉对6-羟基多巴胺(6-OHDA)诱导的帕金森病(PD)大鼠模型的神经保护作用及其作用机制。方法用6-OHDA制备SD大鼠PD模型,观察依达拉奉对PD模型大鼠行为学改变、酪氨酸羟化酶(TH)阳性细胞数和超氧化物歧化酶(SOD)、羟自由基(.OH)的影响。结果依达拉奉呈剂量依赖性减少阿朴吗啡(APO)诱发的大鼠PD模型旋转次数(P<0.01);依达拉奉(3mg/kg×14d,×28d)可阻止模型大鼠TH阳性细胞的减少,增加大鼠黑质纹状体SOD含量(P<0.05),降低.OH含量。结论依达拉奉增加大鼠黑质纹状体SOD含量,降低.OH含量,对6-OHDA诱导的PD动物模型具有神经保护作用。     

微透析—高效液相色谱—化学发光法动态监测6—羟基多巴帕金森病模型大鼠纹状体内多巴胺水平

目的:建立动态监测自由清醒大鼠纹状体内多巴胺水平的微透析-高效液相色谱-化学发光分析法，研究天然冰片联合左旋多巴对6-羟基多巴诱导的帕金森病(PD)模型大鼠纹状体内多巴胺水平的影响。方法:雄性SD大鼠随机分为正常组、6-羟基多巴PD模型组、左旋多巴组和天然冰片联合左旋多巴组。模型组大鼠麻醉后于右侧前脑内侧束缓慢注入4μg·μL^-1 6-羟基多巴，术后4周腹腔注射0.5 mg·kg^-1阿扑吗啡，筛选旋转圈数≥7 r·min^-1(或每30 min 210 r)的大鼠作为6-羟基多巴诱导的PD模型动物。采用脑微透析活体采样联用高效液相色谱-化学发光法(HPLC-CL)监测自由清醒大鼠纹状体内多巴胺水平的动态变化。HPLC分离系统包括Shim pack ODS色谱柱(250 mm×4.6 mm, 5μm),三水合乙酸钠缓冲液-乙腈(80∶20)作为流动相，流速为1.0 mL·min^-1,柱温为37℃。CL检测系统包括3μmol·L^-1鲁米诺溶液、稀释1 000倍的纳米金溶液和50μmo...     

白藜芦醇抗帕金森病黑质细胞凋亡的实验性研究

目的　研究帕金森病(PD)动物模型黑质细胞的凋亡情况及白藜芦醇对神经的保护作用。方法　成年健康SD大鼠100只,分为5组,每组20只。PD模型组应用脑立体定位仪注入6-羟基多巴胺和0.2 %抗坏血酸生理盐水混合液至中脑黑质,诱发PD模型;白藜芦醇实验组应用脑立体定位仪注入6-羟基多巴胺和白藜芦醇混合液;正常对照组应用脑立体定位仪注入0.2 %抗坏血酸生理盐水混合液,术后观察SD大鼠旋转行为,及应用TH免疫组化和Tunel法观察白藜芦醇对黑质神经元的保护作用。结果　TH染色可见白藜芦醇实验组黑质部位损毁较轻、有较多的残存TH阳性细胞。6-羟基多巴胺模型组TH染色黑质大部毁损,几乎无残存的TH阳性细胞。旋转试验结果,白藜芦醇实验组大鼠旋转次数平均206圈/ 30分;6-羟基多巴胺模型组模型鼠旋转次数平均314圈/ 30分;两者存在统计学差异(P<0.05 )。术后第2天检测黑质细胞凋亡情况,白藜芦醇实验组黑质细胞凋亡较少,细胞棕黑色,可见凋亡小体;6-羟基多巴胺模型组黑质细胞凋亡数量较多。结论　白藜芦醇具有神经保护作用。     

左旋多巴在治疗6-OHDA损毁制作的大鼠帕金森病模型中首次诱发明显运动并发症

目的：观察左旋多巴首次治疗6-羟基多巴胺（6-hydroxydopamine,6-OHDA）损毁致大鼠帕金森病模型诱发运动并发症的表现。方法：将成年雄性SD大鼠随机分为正常对照组和帕金森病组,帕金森病组用6-OHDA损毁进行大鼠帕金森病（Parkin-son’s disease,PD）造模。将PD成功模型随机分为左旋多巴胺（Levodopa,L-DOPA）给药组和生理盐水（NS）给药组,其中L-DOPA给药组又分别分为阿朴吗啡检测后L-DOPA给药组,阿朴吗啡检测前L-DOPA给药组,并各分为不同剂量标准。各组大鼠首次给药后进行不自主运动（abnormal involuntary movement ,AIM）评分。结果：L-DOPA给药的各组大鼠均出现了不同程度的不自主运动,而正常对照组、NS、PD组均没有出现。结论：左旋多巴胺可以在6-OHDA损毁致大鼠帕金森病模型首次治疗中诱发明显不自主运动。     

Production of reactive oxygen species following acute ethanol or acetaldehyde and its reduction by acamprosate in chronically alcoholized rats

The salicylate trap method, combined with microdialysis, has been used to validate whether reactive oxygen species, particularly hydroxyl radicals, (^•OH), are generated in the hippocampus of male Wistar rats after acute intraperitoneal administration of either ethanol, 2 and 3 g/kg, or acetaldehyde, 200 mg, or during the initial stages of ethanol withdrawal after chronic ethanol intoxication. Salicylate (5 mM) was infused into the hippocampus via the microdialysis probe and the products of its metabolism by hydroxyl radical, particularly 2,3-dihydroxybenzoic acid (2,3-DHBA) as well as 2,5-dihydroxybenzoic acid (2,5-DHBA) assayed by HPLC (High Pressure Liquid Chromatography).

Acetaldehyde, 200 mg/kg, and the higher acute dose of ethanol, 3 g/kg, induced transitory increases in 2,3-DHBA and 2,5-DHBA microdialysate content. At the cessation of four weeks of chronic ethanol intoxication, (by the vapour inhalation method), the mean blood alcohol level was 1.90 g/l. Significant increases of microdialysate 2,3-DHBA and 2,5-DHBA levels were assayed 3 h after alcohol withdrawal which were sustained for a further 5 and 1 h 40 min respectively. Oral administration of Acamprosate, 400 mg/kg/day, during the chronic ethanol intoxication procedure prevented the increased formation of 2,3- and 2,5-DHBA by comparison to rats chronically ethanol intoxicated alone.     

Effect of desferrioxamine, a strong iron (III) chelator, on 1-methyl-4-phenylpyridinium ion (MPP+)-induced hydroxyl radical generation in the rat striatum

The present study was examined that the desferrioxamine, a strong iron (III) chelator, enhanced 1 methyl-4-phenylpyridinium ion (MPP+)-induced hydroxyl radical (*OH) generation in the extracellular fluid of caudate nucleus anesthetized rats. Rats were anesthetized, and sodium salicylate in Ringer's solution (0.5 nmol/microl/min) was infused through a microdialysis probe to detect the generation of *OH as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the striatum. Induction of desferrioxamine (50 microM) drastically increased the formation of *OH trapped as 2,3-DHBA by the action of MPP+, as compared with MPP+-only-treated animals. Although desferrioxamine did not change the levels of MPP+-induced dopamine, a marked elevation of *OH formation trapped as 2,3-DHBA was observed. When corresponding experiments were performed with reserpinized animals, the level of dopamine and 2,3-DHBA drastically decreased. However, the level of dopamine did not change, but desferrioxamine significantly increased the level of 2,3-DHBA in reserpinized animals. Iron (III) decreased MPP+-induced 2,3-DHBA formations in the presence of dopamine (10 microM). Moreover, when iron (II) was administered to desferrioxamine-treated animals, a marked elevation of 2,3-DHBA was observed, compared with MPP+-only-treated animals, that showed a positive linear correlation between iron (II) and *OH formation trapped as 2,3-DHBA (R2=0.981) in the dialysate. The present study indicates that the suppression of MPP+-induced *OH formation by iron (III) may play a key role in protective effect of iron (III) on the rat brain.     

In vivo evidence against clomethiazole being neuroprotective against MDMA ('ecstasy')-induced degeneration of rat brain 5-HT nerve terminals by a free radical scavenging mechanism

Clomethiazole is an effective neuroprotective agent against the degeneration of 5-HT neurones that follows administration of 3,4-methylenedioxymethamphetamine (MDMA or 'ecstasy'). Since there is good evidence that free radical formation resulting from auto-oxidation of MDMA metabolites is responsible for the degeneration we have examined whether clomethiazole is a free radical scavenger. MDMA (15 mg/kg i.p.) increased the formation of 2,3- and 2,5-dihydroxybenzoic acids (2,3-DHBA and 2,5-DHBA) from salicylic acid perfused through a microdialysis tube implanted in the hippocampus, indicating increased free radical formation. Clomethiazole (50 mg/kg i.p.) administered 5 min prior and 55 min post MDMA prevented both the acute MDMA-induced hyperthermia and the rise in 2,3- and 2,5-DHBA. However, when the temperature of the MDMA + clomethiazole treated rats was kept elevated to that of the MDMA treated rats with a homeothermic blanket there was no inhibition of the MDMA-induced increase in 2,3-DHBA or 2,5-DHBA. These data suggest firstly that free radical formation is inhibited when the acute MDMA-induced hyperthermia is prevented. Secondly the data further indicate that clomethiazole has no free radical scavenging activity since the drug produces substantial neuroprotection when MDMA + clomethiazole treated rats are kept hyperthermic. This conclusion was strengthened by our observation that clomethiazole is a weak inhibitor (IC50 > 1 mM) of lipid peroxidation in synaptosomes when it had been induced by addition of FeCl2 + ascorbic acid.     

Studies on the role of dopamine in the degeneration of 5-HT nerve endings in the brain of Dark Agouti rats following 3,4-methylenedioxymethamphetamine (MDMA or 'ecstasy') administration

1. We investigated whether dopamine plays a role in the neurodegeneration of 5-hydroxytryptamine (5-HT) nerve endings occurring in Dark Agouti rat brain after 3,4-methylenedioxymethamphetamine (MDMA or 'ecstasy') administration. 2. Haloperidol (2 mg kg(-1) i.p.) injected 5 min prior and 55 min post MDMA (15 mg kg(-1) i.p.) abolished the acute MDMA-induced hyperthermia and attenuated the neurotoxic loss of 5-HT 7 days later. When the rectal temperature of MDMA + haloperidol treated rats was kept elevated, this protective effect was marginal. 3. MDMA (15 mg kg(-1)) increased the dopamine concentration in the dialysate from a striatal microdialysis probe by 800%. L-DOPA (25 mg kg(-1) i.p., plus benserazide, 6.25 mg kg(-1) i.p.) injected 2 h after MDMA (15 mg kg(-1)) enhanced the increase in dopamine in the dialysate, but subsequent neurodegeneration was unaltered. L-DOPA (25 mg kg(-1)) injected before a sub-toxic dose of MDMA (5 mg kg(-1)) failed to induce neurodegeneration. 4. The MDMA-induced increase in free radical formation in the hippocampus (indicated by increased 2,3- and 2,5-dihydroxybenzoic acid in a microdialysis probe perfused with salicylic acid) was unaltered by L-DOPA. 5. The neuroprotective drug clomethiazole (50 mg kg(-1) i.p.) did not influence the MDMA-induced increase in extracellular dopamine. 6. These data suggest that previous observations on the protective effect of haloperidol and potentiating effect of L-DOPA on MDMA-induced neurodegeneration may have resulted from effects on MDMA-induced hyperthermia. 7. The increased extracellular dopamine concentration following MDMA may result from effects of MDMA on dopamine re-uptake, monoamine oxidase and 5-HT release rather than an 'amphetamine-like' action on dopamine release, thus explaining why the drug does not induce degeneration of dopamine nerve endings.     

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。     

Evidence of Hydroxyl Free Radical Generation by Calcium Overload in Rat Myocardium

Although calcium (Ca²⁺) is important in cardiac dysfunction and has also been reported as a source of oxidative toxicity, the connection between Ca²⁺ overload and oxygen free radicals in the myocardium is not clear. We have investigated whether Ca²⁺ overload generates hydroxyl free radicals in rat ventricle. HPLC with electrochemical detection was used to measure the levels of 2,3- and 2,5-dihydroxybenzoic acid (DHBA) formed when the hydroxyl free radical reacts with salicylate. Ringer's solution containing salicylic acid (0.5 nmol μL^? min^?) was infused through a microdialysis probe in the region of the left anterior descending coronary artery of the rat ventricle. A positive linear correlation was obtained between Ca²⁺ and hydroxyl free radical formation trapped as 2,3-DHBA (r² = 0.976) and 2,5-DHBA (r² = 0–982) in the myocardial dialysate. The administration of ouabain (1 mg kg^?, i.v.), a Ca²⁺ elevator, into the femoral vein significantly increased the level of 2,3- and 2,5-DHBA. These results indicate that Ca²⁺ overload generates hydroxyl free radicals in rat heart.     

Preparation of Rotigotine-Loaded Microspheres and Their Combination Use with L-DOPA to Modify Dyskinesias in 6-OHDA-Lesioned Rats

Purpose

To prepare rotigotine loaded microspheres (RoMS) to achieve continuous dopaminergic stimulation (CDS) for the treatment of Parkinson??s disease (PD) and investigate both the therapeutic benefit and inducibility of AIMs of administration of RoMS combination with L-DOPA in 6-OHDA-leisioned rats.

Methods

Rotigotine was encapsulated into poly(lactic-co-glycolic acid) (PLGA) microspheres by an oil-in-water emulsion solvent evaporation technique. In vitro characteristics and in vivo pharmacokinetics of RoMS either in rat blood or brain (by microdialysis) were investigated. Contraversive rotations and AIMs were observed to investigate the therapeutic benefit and the propensity to induce dyskinesia of RoMS or RoMS combination with L-DOPA in 6-OHDA-lesioned rats.

Results

RoMS displayed continuous-release characteristics of rotigotine in animals and exhibited a steady efficacy lasted for 2?weeks in 6-OHDA-lesioned rats. No significant difference of the therapeutic benefit between the treatment of RoMS and pulsatile L-DOPA combination and mono L-DOPA was found. While the dyskinesia was significantly decreased with the treatment of RoMS and pulsatile L-DOPA combination compared to mono L-DOPA.

Conclusions

RoMS could supply an alternative of CDS for the treatment of PD and the study indicates a potential advantage of RoMS for the treatment of mild and advanced PD patient in combination with L-DOPA.