左旋多巴和多巴胺对中脑原代细胞的毒性作用

目的:观察左旋多巴和DA对中脑原代培养细胞的毒性作用.方法:采用大鼠胚胎中脑原代细胞培养法,运用TH免疫荧光染色和[3H]DA摄取率检测DA能神经元的存活数和功能;GFAP免疫荧光染色检测星形胶质细胞的存活数;以及MTT检测非DA能神经元的存活数.结果:左旋多巴或DA处理后的TH阳性和GFAP阳性细胞数以及细胞存活率均显著低于加药前基数,且呈剂量依赖性;同时残存细胞体积变小,突起减少、变短或断裂.TH阳性细胞和GFAP阳性细胞比非DA能神经元更易受损.结论:左旋多巴和DA对中脑原代细胞培养中的DA能神经元和非DA能神经元均有毒性作用.     

利噜唑拮抗左旋多巴和多巴胺对中脑原代细胞的毒性作用

目的:研究左旋多巴(L-DOPA)和多巴胺(DA)对大鼠胚胎中脑原代细胞的毒性损害以及利噜唑拮抗L-DOPA和DA的毒性作用。方法:通过体外大鼠胚胎中脑原代细胞培养,采用MTT细胞活性和[~3H]DA摄取率检测中脑原代细胞的存活数和DA能神经元的[~3H]DA摄取功能。结果:当L-DOPA和DA浓度增至500 μmol·L~(-1)时,细胞存活率明显下降(P<0.05),均呈剂量依赖性。当L-DOPA和DA浓度分别增至1mmol·L~(-1)和200μmol·L~(-1)时,[~3H]DA摄取率明显下降(P<0.05),均呈剂量依赖性。利噜唑2～10μmol·L~(-1)能抑制L-DOPA和DA对细胞存活率和[~3H]DA摄取率的影响(P<0.05)。结论:大剂量L-DOPA和DA(≥500 μmol·L~(-1))对中脑原代细胞产生毒性损害,利噜唑能拮抗L-DOPA和DA对中脑原代细胞的毒性效应,具有神经保护作用。     

鱼藤酮对多巴胺能神经元的神经毒性作用

目的　探讨鱼藤酮 (rotenone)对多巴胺能神经元的神经毒性作用的机制。方法　用神经生长因子 (NGF)将PC12细胞诱导分化成多巴胺能神经元的细胞模型 ,经不同浓度的鱼藤酮处理 ,观察细胞形态改变 ,四甲基偶氮唑盐 (MTT)法检测细胞活性及代谢状态 ,磷脂酰丝氨酸外翻法(Annexin V)检测细胞凋亡 ,流式细胞术检测细胞凋亡率 ,α synuclein、硫磺素T(thioflavinT)染色研究细胞内蛋白聚集情况。结果　经鱼藤酮处理 2 4h后PC12细胞突起样结构消失 ,细胞体积变小、形态变圆 ;随着鱼藤酮浓度或作用时间增加 ,细胞活性进一步下降 ,呈量效和时效依赖性。与对照组比较 ,细胞活力在浓度为 10nmol/L鱼藤酮作用 2 4h时即出现明显下降 ,吸光度A570 值为 0 4 15± 0 0 13(P <0 0 5 ) ;可见Annexin V呈阳性的早期凋亡细胞 ;凋亡率在 5nmol/L为 7 35 %± 0 5 2 % (P <0 0 5 ) ,在 10nmol/L为 13 30 %± 1 80 % (P <0 0 1) ;细胞内出现α synuclein和硫磺素T双标染色呈阳性的蛋白聚集。结论　鱼藤酮在体外对多巴胺能神经元有毒性作用 ,可诱导出现细胞凋亡并出现类包涵体 ,表明鱼藤酮可能通过影响α synuclein的代谢而在帕金森病发病机制中起作用。     

左旋多巴对帕金森病鼠行为及多巴胺神经纤维的影响

目的 了解左旋多巴对帕金森鼠行为及多巴胺神经纤维的影响。方法 采用左旋多巴和生理盐水分别治疗不同程度损伤的帕金森鼠，观察TH阳性神经纤维及神经元的表达和鼠旋转行为。结果 左旋多巴治疗的重度帕金森病鼠出现旋转行为，而中、轻度帕金森病鼠和生理盐水治疗组未出现鼠旋转行为；左旋多巴治疗的中、轻度帕金森病鼠TH阳性神经纤维表达增加。结论 左旋多巴对中、轻度帕金森病鼠有益。     

左旋多巴和多巴胺对多巴胺转运体数量和功能的影响

目的:了解左旋多巴和多巴胺对多巴胺转运体的影响.方法:通过体外永久表达大鼠多巴胺转运体(DAT)的CHO细胞(CHO/DAT,即D8细胞)的培养,采用MTT细胞活性检测和流式细胞仪观察不同剂量左旋多巴和多巴胺对D8细胞的毒性作用;利用[3H]WIN35,428结合率和[3H]DA摄取率检测DAT的数量和功能.结果:左旋多巴和多巴胺不仅对D8细胞有毒性损害,呈剂量、时间依赖(P＜0.01),还能使DAT数量明显减少,与剂量相关(P＜0.01),一定剂量的左旋多巴(200μmol/L～1 mmol/L)可增加细胞对[3H]DA的摄取,而大剂量左旋多巴(5 mmol/L)则抑制DAT的功能(P＜0.01).左旋多巴和多巴胺呈剂量依赖性地促进细胞凋亡(P＜0.01).结论:左旋多巴和多巴胺不仅对D8细胞有毒性损害,还能使DAT数量明显减少,DAT结合位点的减少比细胞活性的下降更明显.一定剂量左旋多巴可增强DAT的功能.     

胶质细胞源性神经营养因子对中脑多巴胺能神经元生物活性观察

胶质细胞源性神经营养因子 (ＧＤＮＦ)是一种新型强效多巴胺 (ＤＡ)能神经元营养因子 ,本实验用大鼠中脑原代培养细胞观察ＧＤＮＦ对ＤＡ能神经元的营养、保护和损伤修复作用 ,为其应用于帕金森病 (ＰＤ)治疗提供依据。材料和方法 :取Ｅ14ｄ大鼠胚胎中脑组织 ,制成细胞悬液 ,按每孔 1× 10 5接种于涂有Ｐｏｌｙ Ｌｙｓｉｎｅ的 48孔培养板。在细胞接种后不同时间添加重组人ＧＤＮＦ或甲基 苯基 吡啶离子 (ＭＰＰ )、6 羟基多巴胺 (6 ＯＨＤＡ)进行处理 ,通过酪氨酸羟化酶 (ＴＨ)免疫荧光染色观察ＴＨ阳性 (ＴＨ )细胞形态并计数每孔所…     

左旋多巴对帕金森病大鼠黑质多巴胺能神经元及纹状体多巴胺递质的影响

目的　研究长期应用左旋多巴对帕金森病 (PD)大鼠黑质多巴胺 (DA)能神经元和DA递质的影响。方法　采用 6 羟基多巴胺 (6 OHDA)制备部分损毁和严重损毁的PD大鼠模型 ,给两种模型口服不同剂量左旋多巴 /苄丝肼 3个月 ,通过观察大鼠旋转行为、酪氨酸羟化酶 (TH)免疫组化染色和高效液相色谱 电化学检测仪 (HPLC ECD)检测纹状体单胺类递质 ,研究左旋多巴对PD大鼠残存的黑质DA能神经元的影响。结果　 (1)左旋多巴对PD大鼠的旋转行为无明显影响 ;(2 )TH阳性细胞数损毁侧 /非损毁侧比值在左旋多巴喂药组和不喂药对照组的差异无显著意义 (P >0 0 5 ) ;(3)在严重损毁组 ,大剂量左旋多巴使PD大鼠损毁侧DA和 3,4二羟基苯乙酸 (DOPAC)水平明显升高(P <0 0 1)。结论　长期使用左旋多巴对 6 OHDA单侧损毁的PD大鼠残存的黑质DA能神经元无毒性作用。     

左旋多巴对帕金森病鼠行为及多巴胺神经纤维的影响

目的 了解左旋多巴对帕金森鼠行为及多巴胺神经纤维的影响。方法采用左旋多巴和生理盐水分别治疗不同程度损伤的帕金森鼠,观察TH阳性神经纤维及神经元的表达和鼠旋转行为。结果 左旋多巴治疗的重度帕金森病鼠出现旋转行为,而中、轻度帕金森病鼠和生理盐水治疗组未出现鼠旋转行为;左旋多巴治疗的中、轻度帕金森病鼠TH阳性神经纤维表达增加。结论 左旋多巴对中、轻度帕金森病鼠有益。     

MPP+对培养中脑多巴胺能神经元的影响

目的通过观察MPP+对体外培养的中脑多巴胺能神经元多巴胺及胆碱摄取能力、多巴胺含量变化及细胞形态的改变等,研究MPP+对中脑多巴胺能神经元功能的影响,探讨利用MPP+建立体外PD细胞模型的可能性及实际意义.方法用孕14天Wistar大鼠,氯胺酮麻醉后取胚胎,按本室常规方法分离培养中脑多巴胺能神经元,培养至第7天后将不同浓度的MPP+加入培养有神经元的培养基中,使其终浓度分别为0.1μM,1μM,10μM.分别测定不同时相点[3H]多巴胺和[3H]胆碱摄取能力及细胞内多巴胺含量变化,并进行TH免疫细胞化学染色.结果MPP+浓度为0.1μM、1μM和10μM时,其[3H]多巴胺摄取力分别是为6.2±0.7、5.9±0.5、5.4±04,较之对照组100±1.7的结果看,MPP+对中脑多巴胺能神经元多巴胺摄取力有明显抑制作用(P＜0.05);而[3H]胆碱摄取力其值为98±3.1,较之对照组100±2.4,差异不显著,说明MPP+对多巴胺能神经元胆碱摄取力无抑制作用(P＞0.05).另一个有趣的结果是胶质细胞的存在对MPP+的作用有明显影响,未抑制胶质细胞组其作用明显强于抑制胶质细胞组(P＜0.05);同时MPP+使中脑多巴胺能神经元细胞内多巴胺含量明显减少(P＜0.05);TH阳性细胞明显减少(P＜0.05).结论MPP+对体外培养的中脑多巴胺能神经元多巴胺摄取力有明显的抑制作用,不仅使多巴胺的摄取降低,而且细胞内的多巴胺含量也显著减少,TH免疫组化染色结果也支持这一结果.MPP+对多巴胺能神经元胆碱摄取能力无明显抑制作用.     

多巴胺对鱼藤酮细胞毒性介导作用研究

目的探讨多巴胺在鱼藤酮细胞毒性损伤中的作用。方法采用鱼藤酮(1μmol/L)处理PC12细胞,噻唑蓝比色法检测细胞活性,流式细胞术检测DHR123荧光强度;并采用多巴胺耗竭剂-利血平预处理3小时,观察上述指标。结果 1μmol/L鱼藤酮处理PC12细胞24小时,导致细胞活力较正常组显著下降(P<0.05),吸光度为0.730±0.01(正常组为1.112±0.025);利血平(1μmol/L和5μmol/L)预处理后再给予鱼藤酮,孵育24小时后,吸光度分别为0.945±0.02和1.06±0.03,细胞活力较鱼藤酮组显著升高(P<0.05)。鱼藤酮处理24小时,过氧化物水平升高至正常组的281%,而利血平(1μmol/L和5μmol/L)预处理后,分别降至正常组的248%和232%,与鱼藤酮组比较有显著差异(P<0.05)。结论多巴胺介导了鱼藤酮对多巴胺能细胞的毒性作用。     

左旋多巴和多巴胺对多巴胺转运体数量和功能的影响

目的 :了解左旋多巴和多巴胺对多巴胺转运体的影响。 方法 :通过体外永久表达大鼠多巴胺转运体(DAT)的CHO细胞 (CHO/DAT ,即D8细胞 )的培养 ,采用MTT细胞活性检测和流式细胞仪观察不同剂量左旋多巴和多巴胺对D8细胞的毒性作用 ;利用 [3H]WIN35 ,4 2 8结合率和 [3H]DA摄取率检测DAT的数量和功能。 结果 :左旋多巴和多巴胺不仅对D8细胞有毒性损害 ,呈剂量、时间依赖 (P <0 .0 1) ,还能使DAT数量明显减少 ,与剂量相关 (P <0 .0 1) ,一定剂量的左旋多巴 (2 0 0 μmol/L～ 1mmol/L)可增加细胞对 [3H]DA的摄取 ,而大剂量左旋多巴 (5mmol/L)则抑制DAT的功能 (P <0 .0 1)。左旋多巴和多巴胺呈剂量依赖性地促进细胞凋亡 (P <0 .0 1)。结论 :左旋多巴和多巴胺不仅对D8细胞有毒性损害 ,还能使DAT数量明显减少 ,DAT结合位点的减少比细胞活性的下降更明显。一定剂量左旋多巴可增强DAT的功能。     

Different levodopa actions on the extracellular dopamine pools in the rat striatum

Levodopa has been the mainstay treatment for Parkinson's disease for several decades, but the precise mechanism for its therapeutic action is still not well understood. To date, little distinction has been made between the effects of levodopa on the different brain DA pools. We studied the levodopa action on two extracellular DA pools: one was analyzed by microdialysis (often considered as indicative of volume transmission) and the other by in vivo amperometry during nigrostriatal cell stimulation (more indicative of neurotransmission). Levodopa administration induced a moderate (increased 200%) and tardy (began at 60 min) increase in the DA-pool measured by microdialysis, an effect that increased (increased 500%) and accelerated (began at 10 min) after DA-cell degeneration. Levodopa action on the DA-pool measured by amperometry was very fast (10 min) and prominent (increased 600%) in normal rats. The DA-denervated striatum showed a fast exhaustion during cell stimulation, which prevented further study of the levodopa effect on the DA amperometry-pool under this condition. This study suggests a different kinetic for levodopa action on the volume transmitter and neurotransmitter DA-pool, showing marked changes in levodopa action in the denervated striatum.     

The mesencephalic locomotor region (MLR) in the rat

These studies demonstrate the presence of the MLR in the rat brain. Controlled locomotion on a treadmill could be induced by low level stimulation (< 50 μA) of an area in the posterior midbrain following a precollicular-prenigral brainstem transection. This area included the lateral part of the cuneiform nucleus and anterior as well as posterior portions of the pedunculopontine nucleus. In addition, the presence of a subthalamic locomotor region in the fields of Forel was determined in rats after prethalamic transections.     

Native human neocortex release-regulating dopamine D2 type autoreceptors are dopamine D2 subtype.

Dopamine (DA) autoreceptors expressed at DA nerve terminals regulate DA release. Considerable evidence has indicated that, in rodents, these autoreceptors belong to the D2 type of the DA receptor family, which, in turn, comprises the D2, D3 and D4 subtypes. We investigated here, for the first time, the subclassification of native human DA autoreceptors by studying the release of [3H]DA evoked by electrical stimulation in fresh human neocortical slices. The results have been compared with those obtained in three animal systems: rat neocortical and striatal slices and rat mesencephalic neuronal cultures. In human neocortical slices, the D2/D3 receptor agonist quinpirole (1 nM-10 microM) inhibited tritium release with a calculated EC50 of 17 nM and a maximal inhibition of approximately 75% reached at 1 microM. In the presence of the D2/D3 receptor antagonist (-)-sulpiride (0.1 and 1 microM), the concentration-response curve of quinpirole was shifted to the right, and the apparent pA2 mean value was 8.5 (8.14-8.77); on the other hand, the inhibitory effects of quinpirole were not affected by the D3 receptor-selective antagonist [7-N,N-dipropylamino-5,6,7, 8-tetrahydro-naphtho(2,3b) dihydro,2,3-furane] (S 14297) and the D4 receptor-selective antagonist 3-(4-[4-chlorophenyl]piperazin-1-yl)-methyl-1H-pyrrolo [2,3-b]pyridine (L-745,870) (0.01-1 microM in each case). Superimposable results have been obtained when the release was elicited from rat striatal slices or dopamine mesencephalic neurons in culture, whereas quantitative differences emerged in the case of rat cortical slices. It is concluded that in human brain, as well as in rat brain, the release of DA in the terminal region of midbrain dopaminergic neurons is regulated through autoreceptors of the D2 subtype.     

Attenuation of levodopa-induced toxicity in mesencephalic cultures by pramipexole

Summary The direct-acting dopamine (DA) agonist pramipexole (2 amino-4,5,6,7-tetrahydro-6-propyl-amino-benzthiazole-dihydrocfiloride) was evaluated for its ability to attenuate levodopa-induced loss of tyrosine hydroxylase immunoreactive (THir, a marker for dopamine neurons) cells in mesencephalic cultures. Pramipexole reduced levodopa-induced THir cell loss in a dosedependent and saturable fashion (ED₅₀=500 pM), its inactive stereoisomer was significantly less potent in this regard and pergolide and bromocriptine had negligible cytoprotective effects. Culture media from mesencephalic cultures incubated with pramipexole for 6 days increased THir cell counts in freshly harvested recipient cultures. The magnitude of this effect was directly proportional to the amount of pramipexole in the donor cultures and heatinactivation of the media abolished the growth promoting effect. The results from this exploratory set of experiments suggest that pramipexole may be cytoprotective to dopamine neurons in tissue culture. Pramipexole's affinity for DA receptors, its antioxidant action or its ability to enhance mesencephalic trophic activity could be responsible for this effect.     

On the morphology of the mesencephalic trigeminal cells. New data based on tracer studies

Injections of free horseradish peroxidase in the masticatory muscles of the cat resulted in retrograde labeling of not only large and small so-called pseudounipolar cells but also of multipolar neurons within the ipsilateral mesencephalic trigeminal nucleus. The latter cell type was present only in the pontine part of the nucleus, and usually more faintly labeled than the other cells. Several of the so-called pseudounipolar cells showed cell processes similar to dendrites of other cells, a finding indicating that the afferent connections of these cells are much more complex than hitherto assumed. The observations are discussed also with reference to the problem whether the multipolar cells should be considered as displaced locus coeruleus neurons.     

Toxicity of dopamine and dopaminochrome on cultured cells

Cultured rat fibroblasts, monkey kidney tumor cells (line Vero) and murine neuroblastoma cells were exposed to dopamine or dopaminochrome in the presence and absence of ascorbic acid. Ascorbic acid is able to potentiate the toxicity of both dopamine and dopaminochrome for all the tested cells. The toxicity of dopaminochrome was higher than that of dopamine. There is a correlation between toxicity and levels of bioreductive defenses of the cells, e.g. DT-diaphorase (NAD(P)H:quinone oxidoreductase EC 1.6.99.2) and glutathione. In general, tumor cells have lower defenses and seem to be more sensitive to the toxic action.     

复方地黄方干预帕金森病药物左旋多巴毒副反应大鼠纹状体多巴胺转运体含量的变化

Long-term application of levodopa (L-3,4-dihydroxyphenylalanine,L-DOPA) for Parkinson’s disease can lead to adverse effects and reduce the amount of dopamine transporter (DAT) in the corpus striatum.The present study attempted to verify whether increasing the amount of DAT can reduce the adverse effects of L-DOPA.The specific radioactive uptake value of DAT in the corpus striatum of the lesioned hemisphere was significantly decreased,but was significantly increased following administration of compound rehmannia formula [Radix rehmanniae preparata (prepared rehmannia root),Concha margaritifera usta (nacre),Radix paeoniae alba (white peony alba),Radix salviae miltiorrhizae (Danshen root),Scorpio (scorpion),green tea] for 4 weeks.The changes in DAT 125I-beta-carbomethoxy-3 beta-(4-iodophenyl) tropane autoradiography were consistent with those in radioactivity.The results revealed that the compound rehmannia formula can reduce the adverse effects of L-DOPA in treating Parkinson’s disease,possibly by increasing the amount of DAT.