神经干细胞联合胶质细胞源性神经营养因子脑内移植对Parkinson病大鼠纹状体内多巴胺含量的影响

将原代培养的胚鼠(E14.5)腹侧中脑神经干细胞(NSCs)单独或联合胶质细胞源性神经营养因子(GDNF)移植入Parkinson病(PD)大鼠纹状体内,术后各组动物存活一段时间,观察旋转行为后处死,取纹状体组织做高效液相色谱检测多巴胺(DA)含量。探讨NSCs单独或联合GDNF脑内移植对PD大鼠纹状体内DA含量的影响。结果显示:与PD模型相比,NSCs单独或联合GDNF脑内移植均能提高PD大鼠纹状体内的DA含量(P<0.05);在术后30d和60d,DA含量在GDNF+NSCs组的增加幅度比NSCs组更加明显,动物旋转行为亦得到明显改善(P<0.05)。上述结果表明,NSCs单独或联合GDNF移植均对PD大鼠有一定的治疗作用,而NSCs联合GDNF移植的治疗效果更好。     

大鼠黑质损毁后多巴胺能神经元形态及纹状体c-fos表达的变化

目的:观察毁损黑质后,多巴胺(DA)能神经元形态学变化和纹状体内相关神经元c-fos表达情况,探讨c-fos表达与毁损程度的关系。方法:利用6羟多巴胺(6OHDA)特异毁损大鼠黑质DA能神经元,采用阿朴吗啡(APO)诱导旋转实验观察术后1、7、14和21d行为学变化;利用HE染色、Nissl染色、免疫组织化学方法和电镜,观察各时间点黑质DA能神经元形态学变化和纹状体c-fos表达。结果:毁损侧DA能神经元逐渐减少,超微结构损伤逐渐加重;DA神经元丢失比例≥80%时,纹状体毁损侧c-fos表达上调,APO诱导的旋转实验>7r/min。结论:黑质DA能神经元丢失是毁损大鼠行为改变的病理学基础;cfos的表达与DA能神经元的毁损程度、行为改变有一定的关系。     

大鼠胚胎中脑腹侧细胞和大脑皮质神经干细胞移植治疗帕金森病大鼠的比较

体外培养的大鼠胚胎中脑腹侧(VM)细胞和大脑皮质神经干细胞(NSCs)分别移植入帕金森病(PD)模型大鼠毁损侧纹状体。移植后2,4,8周采用脑微透析术结合高效液相色谱(HPLC)动态监测毁损侧纹状体内多巴胺水平,同期观察大鼠的旋转行为。移植后8周采用免疫组化法检测植入细胞的存活及分化情况。结果显示:移植后4,8周VM移植组多巴胺水平明显高于NSCs移植组或对照组,同期VM移植组较其余两组旋转行为有显著改善,而NSCs移植组与对照组多巴胺水平及旋转行为无显著差异。VM移植组较NSCs移植组植入的细胞易存活和分化。以上结果提示,大鼠胚胎VM细胞移植治疗PD模型的疗效明显优于胚胎大脑皮质NSCs。     

组胺合成酶抑制剂α-FMH对帕金森病大鼠旋转行为和脑内多巴胺及组胺能神经元的影响

目的探讨内源性组胺在PD发病中的作用。方法采用6-羟基多巴（6-OHDA）制备偏侧毁损的PD大鼠模型,连续7d侧脑室给予组胺合成酶抑制剂α-FMH（12．5μg,25μg）以降低脑内组胺的含量。术后第7d观察如下指标：阿朴吗啡诱导的旋转行为;免疫组织化学法检测黑质内多巴胺能神经元和下丘脑后部结节乳头核（TMN）内组胺能神经元的免疫反应活性;高效液相色谱法检测纹状体内多巴胺的含量。结果与模型组相比,α-FMH（25pg）明显降低了阿朴吗啡诱导的PD大鼠的旋转行为（4．09与6．18r／min相比,P〈0．05）;延缓了毁损侧黑质内多巴胺神经元的缺失;轻微地增加了纹状体内多巴胺的含量。此外,与假手术组相比,模型组和α-FMH给药组大鼠TMN内组胺能神经元均无明显改变。结论内源性组胺介入了PD的发病机制,但并不伴有组胺能神经元的病理改变。     

PD大鼠模型的行为学评价和黑质-纹状体通路中单胺类神经递质的含量变化

目的建立帕金森病(Parkinson’s disease,PD)大鼠模型,观测和评价其行为学改变,检测和分析PD大鼠模型黑质-纹状体通路中单胺类神经递质含量变化。方法采用6-羟基多巴胺立体定向注射至成年SD大鼠单侧前脑内侧束,制作偏侧PD大鼠模型,观测和评价其行为学改变(悬尾测试、旋转行为的旋转速度、启动时间和维持时间),通过高效液相色谱法测定和分析单胺类神经递质的含量在PD大鼠模型黑质-纹状体通路中的变化。结果 (1)PD大鼠出现了尾僵直、少动、运动迟缓、震颤、竖毛、咬尾或足、姿势和步态不稳等异常行为;(2)悬尾测试对于评价PD模型没有统计学意义,旋转行为的旋转速度、启动时间和维持时间的结果具有统计学意义且呈时间依赖性;(3)PD大鼠模型毁损侧的黑质与纹状体部位的多巴胺(Dopamine,DA)和五羟色胺(Serotonin,5-HT)含量均降低,DA的含量具有逐渐下降的趋势,术后第2、3、4及5周黑质部位的DA含量相对于正常对照组分别下降62.96%、82.88%、92.71%、91.50%,纹状体部位的DA含量分别下降77.47%、91.39%、96.25%、96.18%;术后第3、4及5周黑质部位的DA含量平均下降(89.03±5.36)%(第3、4及5周的DA含量无统计学差异),纹状体部位的DA含量平均下降(93.61±2.79)%(第3、4及5周的DA含量无统计学差异)。结论 (1)PD大鼠模型具有类似人类PD的运动症状,除旋转速度作为旋转行为经典的行为学指标外,启动时间和维持时间可作为PD模型旋转行为的辅助指标;(2)PD大鼠脑内毁损侧的黑质和纹状体部位的DA和5-HT含量均降低。     

帕金森病大鼠模型纹状体中谷氨酸、γ-氨基丁酸与多巴胺之间的关系

目的:了解帕金森病大鼠模型纹状体内谷氨酸(glutamate,Glu)、γ-氨基丁酸(gama-aminobutyric acid,GABA)和多巴胺(dopamine,DA)之间的关系,从而进一步探讨帕金森病的发病机制。方法:动物分为溶剂对照组、假手术组和帕金森模型组。大脑右侧黑质致密部和前脑内侧束两点注射6-羟基多巴胺(6-hydroxydopamine,6-OHDA)建立帕金森病大鼠模型,溶剂对照组注入生理盐水,假手术组不注射任何药物,采用脑微透析术于建模后第3,4,5,6周连续动态透析大鼠毁损侧纹状体,结合高效液相色谱(HPLC)动态监测各组谷氨酸、GABA和多巴胺的变化。结果:(1)PD组纹状体内多巴胺含量到第5周仅为溶剂对照组和假手术组的1/5;(2)谷氨酸含量随建模时间逐渐升高,到第6周PD组是溶剂对照组和假手术组的1倍以上;(3)GABA含量呈下降趋势,到第6周约降至溶剂对照组、假手术组的1/2。结论:帕金森病大鼠模型纹状体内谷氨酸的变化与多巴胺分泌可能存在某种联系;GABA含量随建模时间的增加而下降。     

神经干细胞移植对帕金森病样模型大鼠的治疗作用

目的 观察大鼠神经干细胞(NSC)植入帕金森病(PD)模型大鼠的脑纹状体后的存活、分化及功能状态.方法 体外分离培养NSC,单克隆培养,免疫细胞化学检测多向分化潜能和特异性标记nestin.建立PD大鼠模型,纹状体内植入DAPI标记的NSC,检测6-羟基多巴胺(6-HHDA)诱发的旋转行为,荧光检测标记细胞的分布情况,免疫细胞化学和高效液相色谱检测细胞分化和神经递质含量.结果 培养的NSC表达nestin,具有自我更新和多向分化能力;NSC植入PD模型大鼠纹状体后大鼠诱导旋转行为显著改善(P＜0.05);NSC在脑内迁移,并在纹状体内形成少量酪胺酸羟化酶(TH)阳性细胞、纤维;植入后纹状体内多巴胺(DA)及其代谢产物含量上升,2个月时分别增加3.6倍和2.8倍,4个月时增加3.4倍和2.4倍(P＜0.01).结论 神经干细胞在体外大量扩增后植入PD脑内能长期存活并分化、分泌神经递质,从而部分改善PD症状.     

鼠胚神经干细胞移植对帕金森模型大鼠的治疗作用

背景：干细胞移植是治疗帕金森的有潜力的方法之一。 目的：观察神经干细胞纹状体移植对帕金森模型大鼠旋转行为及脑内多巴胺含量的影响。 方法：采用6-羟基多巴胺定点注射毁损黑质纹状体的方法构建帕金森大鼠模型;向造模成功的大鼠纹状体内分别移植1×106(共计20 μL)的第3代胚鼠神经干细胞或等量生理盐水。 结果与结论：神经干细胞移植后,帕金森大鼠的旋转行为明显改善。干细胞移植后3周,免疫组化检测发现移植干细胞的帕金森大鼠脑黑质部位酪氨酸羟化酶阳性细胞数增多,纹状体内可见酪氨酸羟化酶阳性细胞;荧光显微镜下观察发现Hoechst 33324d标记神经干细胞在移植针道附近最为密集,并向远隔部位迁徙。干细胞移植后8周,高效液相色谱检测显示移植干细胞的帕金森大鼠纹状体内多巴胺含量明显增高(P < 0.01)。说明神经干细胞脑内移植能够减轻6-羟基多巴胺引起的大鼠中脑黑质多巴胺能神经元的损伤,改善大鼠的旋转行为。     

龟板对帕金森病大鼠行为和脑内多巴胺水平的影响

目的　探讨益肾中药龟板对帕金森病大鼠的治疗作用。方法　 6 羟基多巴胺脑内定位注射制备帕金森病模型 ,随机将 4 0只SD大鼠分为实验组和对照组 ,观察动物单侧旋转行为以及多巴胺及其代谢产物 3、4 二羟苯乙酸和高草酸含量的变化。结果　造模 8周后实验组帕金森病大鼠旋转圈数 6 97± 1 6 7比对照组 9 4 5± 1 75明显减少 (P <0 0 5 ) ;其纹状体内多巴胺 (DA)及其代谢产物 3、4二羟苯乙酸 (DOPAC)、高香草酸 (HVA)含量显著提高 ,分别为 3 12± 0 4 8,0 2 7± 0 0 6和 0 35±0 0 6 ,而对照组仅为 0 4 9± 0 0 4 ,0 0 7± 0 0 3和 0 2 7± 0 0 3(P <0 0 5 )。结论　益肾中药龟板对大鼠帕金森病具有潜在的临床应用价值。     

香椿子多酚通过抑制p38MAPK信号通路减轻帕金森病大鼠的神经炎症反应

目的探讨香椿子多酚(PTSS)对帕金森病(PD)大鼠神经炎症的抑制作用及机制。方法成年雄性SD大鼠单侧纹状体内注射6-羟多巴胺(6-OHDA)造模。造模成功大鼠随机分为模型组、PTSS处理组,另设一组正常大鼠为对照组,每组10只。30 d后,采用阿朴吗啡(APO)腹腔注射诱导大鼠的旋转行为,检测各组大鼠的行为改变。免疫组织化学染色法观测各组大鼠黑质酪氨酸羟化酶(TH)阳性的多巴胺(DA)能神经元、钙离子结合接头蛋白分子1(Iba1)阳性的小胶质细胞、胶质原纤维酸性蛋白(GFAP)阳性的星形胶质细胞形态和数量变化;免疫组织化学染色法检测黑质诱导型一氧化氮合酶(iNOS)、核因子κBp65(NF-κBp65)、p38丝裂原活化蛋白激酶(p38MAPK)和磷酸化的p38MAPK(p-p38MAPK)的表达; Western blot法检测大鼠黑质TH、GFAP、p38MAPK、p-p38MAPK蛋白水平。结果 PTSS处理组大鼠的旋转圈数较模型组明显减少。模型组大鼠损毁侧黑质TH阳性细胞较对照组显著减少, PTSS处理后, TH阳性细胞数量以及蛋白表达明显增加。与对照组相比,模型组大鼠损毁侧黑质Iba1、GFAP、iNOS、NF-κB、p38MAPK和p-p38MAPK蛋白含量显著增加。PTSS处理后,上述蛋白的表达均明显减弱,蛋白含量显著降低。结论 PTSS通过抑制p38MAPK信号通路,减少炎症因子的表达,对PD大鼠DA能神经元起保护作用。     

Increased utilization of dopamine in the nucleus accumbens but not in the cerebral cortex after dorsal raphe lesion in the rat

Dihydroxyphenylacetic acid (DOPAC) and dopamine (DA) levels were estimated in the frontal cortex, the nucleus accumbens and the striatum of the rat after electrolytical lesion of the dorsal raphe nucleus. The efficiency of this lesion was tested by measuring the decline in serotonin (5-HT) levels in the striatum. 5-HT levels were reduced by 90% when compared to those of sham-operated rats 11 days after the lesion. As revealed both by the increase in DOPAC levels and in the DOPAC/DA ratio, the rate of DA utilization was markedly increased in the nucleus accumbens, slightly enhanced in the striatum and in contrast remained unaffected in the frontal cerebral cortex 4 days after the lesion. Changes in DOPAC levels in the nucleus accumbens were also seen 11 and 30 days after the lesion but they were less pronounced than those observed at 4 days. These results suggest that neurons originating from the dorsal raphe and projecting to the ventro-tegmental area are regulating the activity of the meso-nucleus accumbens dopaminergic neurons but not that of the meso-cortical dopaminergic neurons.     

Pharmacological investigation on the role of dopamine in the rat locus coeruleus

The role of dopamine (DA) in the rat locus coeruleus (LC) was investigated by determining the levels of 3,4-dihydroxyphenylacetic acid (DOPAC), DA and noradrenaline (NA) in the LC after pharmacological treatments by pargyline, haloperidol, 6-hydroxydopamine (6-OHDA) and desmethylimipramine (DMI). The DA, DOPAC and NA contents of the LC were determined by high pressure liquid chromatography. Fifteen days after 6-OHDA, the DOPAC and NA levels were reduced by 60%, but they remained constant after 6-OHDA + DMI. Pargyline provoked highly significant increases in DA and NA but reduced DOPAC to non-measurable amounts. Haloperidol caused a 54% decrease in the DOPAC levels. Pargyline and haloperidol administered to rats having received 6-OHDA + DMI 15 days before, caused similar effects on DA, DOPAC and NA levels as those in non-treated rats. It is suggested that DOPAC is mainly located in noradrenergic neurons, thus eliminating the possibility of a significant DA cell body population in the rat LC.     

Cold restraint alters dopamine metabolism in frontal cortex, nucleus accumbens and neostriatum

The concentrations of dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) were assayed in the striatum, nucleus accumbens and frontal cortex of rats following 2 hours of cold restraint. The concentration of DA was significantly decreased in both the striatum (-16%) and nucleus accumbens (-41%) relative to unstressed controls. The content of DOPAC was significantly increased in both striatum (+56%) and frontal cortex (+76%), but not in nucleus accumbens. The DOPAC/DA ratio was increased in all three regions, that in frontal cortex approaching three-fold. These results extend earlier findings of an activation by acute stressors of frontal cortex DA metabolism, but suggest an involvement of other DA systems as well. The finding of the greatest response in frontal cortex, and the previous observations that this was the only region to show significant changes, may be ascribed to the suggested lack of presynaptic autoreceptors in this region.     

The effects of D1 or D2 dopamine receptor antagonism in the medial preoptic area, ventral pallidum, or nucleus accumbens on the maternal retrieval response and other aspects of maternal behavior in rats

The medial preoptic area (MPOA), ventral pallidum (VP), and nucleus accumbens (NA) receive dopaminergic afferents and are involved in maternal behavior. Experiments investigated whether dopamine (DA) receptor antagonism in NA disrupts maternal behavior, determined the type of DA receptor involved, and investigated the involvement of drug spread to VP or MPOA. Injection of SCH 23390 (D1 DA receptor antagonist) into NA of postpartum rats disrupted retrieving at dosage levels that were ineffective when injected into MPOA or VP. Motor impairment was not the cause of the deficit. Injection of eticlopride (D2 DA receptor antagonist) into NA or VP was without effect. Results emphasize the importance of DA action on D1 receptors in NA for retrieval behavior.     

Complex Analysis of Efficiency of Transplantation of Embryonic Nerve Tissue to Rats with Hemiparkinsonism

Effect of transplantation of embryonic ventral mesencephalon preparation containing dopaminergic neurons on repair of the dopaminergic nigrostriatal system was studied in rats with hemiparkinsonism induced by 6-hydroxydopamine. Transplantation of embryonic ventral mesencephalon into denervated striatum led to a more than 50% decrease in apomorphine-induced rotation, recovery of dopamine and DOPAC levels in the brain, and to an increase in DOPAC excretion and the DOPAC-dopamine ratio in daily urine of rats with hemiparkinsonism. Dopaminergic neurons of the transplant survived, forming a network of tyrosine hydroxylase-positive processes growing beyond the transplant and reinnervating the adjacent compartments of the striatum. A positive correlation between urinary excretion of DOPAC and brain concentration of dopamine was revealed in denervated rats after transplantation of ventral mesencephalon. Intrastriatal transplantation of cell preparations of embryonic striatum containing no dopaminergic neurons and isolated local injury to the striatum did not affect regeneration of the denervated nigrostratal system.     

氯化锰对大鼠中脑多巴胺能神经元毒性的研究

本文通过锰诱导多巴胺能神经元凋亡及其可能的神经化学机制的研究 ,进而探讨锰中毒与帕金森病发病的相互关系。分离培养大鼠中脑黑质多巴胺能神经元用不同剂量 Mn Cl2 处理后 ,用荧光染料进行染色 ,观察了凋亡神经元数量。用腹腔注射及脑内单侧注射 Mn Cl2 染毒方法处理大鼠 ,并采用脑内微透析技术和高效液相色谱 -电化学方法 (HPLC-ECD)在活体检测了术后不同时间的纹状体细胞外液中 DA及其代谢产物 DOPAC、HVA以及 5 -HT的代谢产物 5 -HIAA等的含量 ;同时作丙二醛含量和过氧化物歧化酶活性检测。结果发现 ,凋亡神经元的细胞核缩小、不规则、染色质呈块状深染 ,凋亡细胞数量随 Mn Cl2 剂量升高而增多。Mn Cl2 脑内注射侧与注射对侧相比 ,术后 4、7、10、2 0 d的 DA、DOPAC、HVA和 5 -HIAA含量均有不同程度的降低。腹腔染毒高、低剂量组 2 0 d后大鼠整体纹状体匀浆的上述指标也明显降低。此外 ,染毒大鼠纹状体中丙二醛水平随染毒剂量增高而增高 ,过氧化物歧化酶活性随染毒剂量增高却下降。以上结果表明 ,锰中毒可能是引起帕金森病发病的原因之一     

Comparisons between brain dopaminergic neurons of juvenile and aged rats: sex-related differences.

It is known that several aspects of dopaminergic neurotransmission deteriorate with advanced age. In the present report, we have studied the possible existence of sexual differences in these aging-induced changes. Thus, we measured several pre- and postsynaptic biochemical parameters, indicative of the activity of dopaminergic neurons, in striatum, limbic forebrain and hypothalamic-anterior pituitary area of aged (24-26 months) and young (2 months) rats of both sexes. Tyrosine hydroxylase (TH) activity, as well as the number of D2-dopaminergic receptors, decreased in the striatum of aged rats, especially in the males in which the decrease in the number of receptors was associated with an increase in their affinity. In addition, the ratio between dopamine (DA) and its intraneuronal metabolite, L-3,4-dihydroxyphenyl-acetic acid (DOPAC), which can be used as an index of neurotransmitter turnover, was increased in aged females in parallel with a decreased DA content. In the limbic forebrain, TH activity was also decreased during aging, but only in males, whereas the DOPAC/DA ratio was increased in females, although in parallel with an increased DOPAC production. Finally, in the hypothalamic-anterior pituitary area, aging only affected the females, in which increased plasma prolactin levels were observed. This effect might be the result of a low responsiveness of pituitary lactotrophs to DA released from hypothalamic neurons, in spite of high prolactin levels producing a constant, although ineffective, stimulation of the activity of these neurons, as reflected by the high DOPAC content and DOPAC/DA ratio observed in the medial basal hypothalamus. In summary, these data allow us to suggest that the activity of brain dopaminergic neurons is modified with aging and there are significant differences as a function of sex and brain area.     

Effect of Brain Lesions and Chlorpromazine on Accumulation and Disappearance of Catecholamines Formed in vivo from 14C-Tyrosine

Rats were subjected to a unilateral stereotaxic lesion at the level of the mesencephalic-hypothalamic junction where the nigro-striatal dopamine (DA) pathway is known to pass. Between 6 and 18 h after the lesion the level of DA in the striatum on the same side was increased by about 50 per cent. Between 18 and 48 h after the lesion the content of DA declined to 15 per cent of that of the contralateral striatum and stayed at this low level during the following 6 days. The content of noradrenaline (NA) tended to be increased 24 h after the lesion but no change was found on the following days. The accumulation and disappearance of ¹⁴C-DA formed from ¹⁴C-tyrosine in the striatum 12–24 h after the nigral lesion were decreased on the lesion side in comparison with the contralateral side. The accumulation and disappearance of ¹⁴C-NA did not differ between the 2 sides. Chlorpromazine accelerated both accumulation and disappearance of ¹⁴C-DA in the striatum on the side contralateral to the lesion but not in the striatum of the lesion side. In animals with a unilateral lesion above the DA pathway, in the posterior thalamus, chlorpromazine accelerated the accumulation of ¹⁴C-DA on both sides. The accumulation of ¹⁴C-XA in the striatum was increased by chlorpromazine on the intact side following both types of lesions. The disappearance of ¹⁴C-NA, however, was not influenced by the drug. The results are taken as evidence that chlorpromazine accelerates DA turnover in the striatum by activating the nerve impulse flow in the nigro-striatal DA pathway.     

Complex analysis of efficiency of transplantation of embryonic nerve tissue to rats with hemiparkinsonism

Effect of transplantation of embryonic ventral mesencephalon preparation containing dopaminergic neurons on repair of the dopaminergic nigrostriatal system was studied in rats with hemiparkinsonism induced by 6-hydroxydopamine. Transplantation of embryonic ventral mesencephalon into denervated striatum led to a more than 50% decrease in apomorphine-induced rotation, recovery of dopamine and DOPAC levels in the brain, and to an increase in DOPAC excretion and the DOPAC-dopamine ratio in daily urine of rats with hemiparkinsonism. Dopaminergic neurons of the transplant survived, forming a network of tyrosine hydroxylase-positive processes growing beyond the transplant and reinnervating the adjacent compartments of the striatum. A positive correlation between urinary excretion of DOPAC and brain concentration of dopamine was revealed in denervated rats after transplantation of ventral mesencephalon. Intrastriatal transplantation of cell preparations of embryonic striatum containing no dopaminergic neurons and isolated local injury to the striatum did not affect regeneration of the denervated nigrostratal system. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 130, No. 12, pp. 665–670, December, 2000