铁离子对黑质纹状体多巴胺神经元毒性作用的实验研究

目的 探讨铁离子对黑质纹状体多巴胺神经元的毒性作用。方法 采用立体定向偏侧大鼠黑质内注入50μg FeCl3和FeCl2，4周后用阿朴吗啡诱导动物行为学变化，高效液相色谱(HPLC)检测纹状体内多巴胺、去甲肾上腺素、肾上腺素递质含量的变化，免疫组化观察黑质多巴胺神经元和胶质细胞的改变。结果 FeCL3和FeCL2均可引起注射侧纹状体内DA含量选择性降低，而NA、A含量无显著改变；注射侧黑质内DA神经元显著缺失、胶质细胞显著增生；FeCL3组阿朴吗啡诱导大鼠向同侧旋转行为，FeCL2组大鼠于术后即出现特征性自发性对侧旋转行为，阿朴吗啡不能诱发其旋转。结论 铁离子对黑质纹状体多巴胺神经元具有毒性作用，Fe^3 作用最强，胶质细胞的增生可能参与了这一毒性作用过程。     

铜离子引起大鼠黑质多巴胺能神经元的退行性变

目的探讨黑质（substantia nigra,SN）内注射不同剂量的CuSO45H2O对大鼠黑质纹状体系统多巴胺能神经元的影响。方法实验用Wistar大鼠,分成对照组和左侧SN内分别注射10nmol、50nmol、200nmol CuSO。组,7天后采用高效液相色谱法（high performance lipid chromotophotography,HPLC）检测纹状体内多巴胺（dopamine,DA）及其代谢产物的含量;酪氨酸羟化酶（tyrosine hydroxylase,TH）免疫组织化学法检测纹状体内TH免疫阳性纤维的改变;半定量RT-PCR法检测黑质内TH,Caspase-3mRNA的表达量：用生化试剂盒分析大鼠中脑内超氧化物岐化酶（superoxide dismutase,SOD）活性的改变。结果在10nmol CuSO4注射组中,DA及其代谢产物的含量与对照组相比没有统计学差别。但是从50nmol组开始,损毁侧纹状体内DA含量随注射CuSO4剂量的增加而逐渐减少,显示出明显的剂量依赖关系（F=34．16,P〈0．01）。注射50nmol CuSO4组大鼠纹状体内TH免疫阳性纤维明显少于对照组和未损毁侧（F=121．9,P〈0．01）。注射50nmol CuSO4组大鼠SN内THmRNA的表达与对照组相比下降（t=3．12,P〈0．01）,但Caspase-3mRNA的表达量与对照组相比却明显增加（t=8．96,P〈0．01）。在注射50nmolCuSO4组中,大鼠损伤侧中脑内SOD的活性与对照组相比下降（t=2．33,P〈0．01）。结论铜离子可以导致大鼠黑质内多巴胺能神经元的损伤,该损伤作用可能是通过破坏抗氧化保护系统和促进细胞凋亡而实现的。     

蛋白酶体功能与帕金森病相关性实验研究

目的 观察蛋白酶体抑制剂诱导大鼠黑质多巴胺能神经元α-突触核蛋白（α-synuclein，α-Syn）的表达及聚集．探讨蛋白酶体功能在帕金森病（PD）发病中的作用机制。方法采用立体定向将蛋白酶体抑制剂Lactacystin注射至大鼠黑质部位。以免疫荧光法观察黑质区多巴胺能神经元变性缺失，并应用免疫荧光双标法观察多巴胺能神经元内蛋白聚集的包涵体及其主要成分α-Syn的表达．然后通过原位杂交分析α-Syn mRNA表达及Western印迹法检测黑质α-Syn表达量改变。结果注射Lactacystin第7天大鼠开始出现自发性活动减少．阿扑吗啡尚可诱导出旋转行为；3周后患侧黑质部位酪氨酸羟化酶（TH）阳性细胞明显减少。TH与硫磺素、硫磺素与α-Syn复合染色呈阳性。α-Syn mRNA表达量升高，蛋白表达水平增加。结论Lactacystin诱导大鼠黑质细胞α-Syn表达升高并出现蛋白聚集可能是导致PD发病的机制之一。     

同型半胱氨酸对多巴胺神经元的影响及其机制研究

目的研究同型半胱氨酸（homocysteine，Hcy）对帕金森病（PD）模型动物的影响及其机制。方法将63只大鼠随机分成3组，即吡咯烷二硫代氨基甲酸盐（PDTC）组27只、生理盐水对照组27只、假手术组9只。分别在实验前1h腹腔注射PDTC或生理盐水，以后每天1次，连续注射7d，通过脑立体定向注射6-羟多巴胺（6-OHDA）建立大鼠PD模型，2h后同侧脑立体定向注射Hcy或生理盐水，采用TUNEL法、免疫组化技术，选择实验后1d、7d及14d为研究时点，观察黑质多巴胺神经元数量、形态改变，黑质细胞凋亡数，以及黑质细胞NF-κB p65的阳性细胞数的变化。结果（1）局部注射Hcy能明显增加6-OHDA引起的黑质多巴胺神经元变性；（2）局部注射Hcy能明显增加6—OHDA引起的黑质细胞凋亡；（3）局部注射Hcy能明显增加黑质细胞NF—κB p65阳性细胞数；（4）PDTC可抑制Hcy和（或）6-0HDA引起的NF—κB p65活化，减少黑质细胞凋亡，增加多巴胺神经元数目。结论NF—κB p65的激活是Hcy促进6-OHDA引起的黑质多巴胺神经元变性及黑质细胞凋亡机制中的重要因素之一，PDTC可显著抑制NF—κB p65的活化，多巴胺神经元变性和黑质细胞凋亡。     

6-羟基多巴胺单侧纹状体注射对大鼠双侧多巴胺能神经元的影响

目的探讨帕金森病大鼠模型中6-羟基多巴胺（6-OHDA）单侧纹状体注射对双侧黑质纹状体多巴胺能神经元的影响。方法大鼠随机分成模型组和对照组，模型组自一侧纹状体注射6-OHDA，对照组注射PBS；用免疫组织化学方法分别检测大鼠双侧黑质和纹状体区酪氨酸羟化酶（TH）阳性细胞和纤维的表达；高效液相色谱检测双侧纹状体多巴胺（DA）及其代谢产物3，4-二羟基苯乙酸（DOPAC）和高香草酸（HVA）含量。结果模型组大鼠双侧（毁损侧与其对侧）黑质致密区TH阳性细胞数量均少于对照组（P〈0．01），模型组双侧纹状体区TH阳性纤维密度均低于对照组；模型组大鼠双侧纹状体区DA含量均低于对照组（P〈0．01）；双侧DOPAC和HVA含量也降低。结论6-羟多巴胺单侧纹状体注射制作的帕金森病大鼠模型的对侧黑质纹状体也有损伤。     

阿扑吗啡对6-羟基多巴损伤的黑质纹状体大鼠的保护

摘要：目的本研究旨在评定6-羟基多巴损伤纹状体后黑质和腹侧被盖区的变化,并探讨阿扑吗啡在该模型中可能存在的神经保护作用。方法 6-羟基多巴损伤纹状体模型：在造模前15分钟,连续22天皮下注射阿扑吗啡10mg／kg．d。在第5周时,观察大鼠行为学（安啡他明诱导的旋转数目）、组织化学（TH／Nissl染色后黑质和腹侧被盖区的多巴胺细胞）、神经化学（高压液相法测定纹状体的多巴胺含量）改变。结果阿扑吗啡不仅消弱安啡他明诱导的旋转数日,而且,显著减少黑质的损伤（与对照组相比,多巴胺细胞达到69％,细胞形态恢复正常）和腹侧被盖区的损伤（多巴胺细胞增加了60％,细胞形态恢复正常）;并且,阿扑吗啡显著消弱6-羟基多巴损伤纹状体的多巴胺含量,使DOPAC／DA率恢复正常。结论在6-羟基多巴损伤纹状体模型中,阿扑吗啡不仅有神经保护作用,而且有神经营养作用。但神经营养作用局限于腹侧被盖区,并随着用药时间延长而增加。     

脑源性神经营养因子基因治疗对大鼠脑内多巴胺含量的影响

采用脑源性神经营养因子（BDNF）基因工程成肌细胞纹状体内移植观察其对帕金森病（PD）大鼠脑内纹状体多巴胺含量的影响，结果发现：基因工程成肌细胞脑内移植可使纹状体多巴胺含量明显增加，并可维持达两月之久，这为帕金森病的治疗提供了一种新的有效的治疗方法。     

重组人促红细胞生成素预处理对帕金森病大鼠胶质细胞源性炎症因子表达的影响

目的探讨重组人促红细胞生成素(rhEPO)预处理对帕金森病(PD)大鼠胶质细胞源性炎症因子表达的影响。方法 40只SD大鼠随机分为4组,A组:右侧纹状体内注射rhEPO 24 h后,同侧黑质内注射6-羟基多巴胺(6-OHDA);B组:右侧纹状体内立体定向注射与rhEPO等量的生理盐水,24 h后同侧黑质内立体定向注射6-OHDA;C组:右侧黑质内立体定向注射6-OHDA;D组:右侧黑质内立体定向注射与6-OHDA等量的生理盐水。4周后采用酶联免疫吸附法检测血清诱导型一氧化氮合酶(iNOS)和肿瘤坏死因子(TNF)-α含量;逆转录(RT)-PCR法检测黑质iNOS和TNF-αmRNA的表达。结果与D组比较,A、B、C组大鼠血清iNOS、TNF-α含量增多,黑质iNOS、TNF-αmRNA表达增高(均P<0.05);与B组和C组比较,A组大鼠血清iNOS、TNF-α含量显著减少,黑质iNOS、TNF-αmRNA表达显著降低(均P<0.05)。结论 rhEPO可能通过抑制黑质TNF-α、iNOS表达,减轻6-OHDA对多巴胺能神经元的毒性损害,具有神经保护作用。     

粒细胞集落刺激因子对百草枯干预小鼠黑质纹状体通路的影响

目的探讨粒细胞集落刺激因子(G-CSF)对百草枯干预小鼠脑内黑质纹状体通路的影响。方法 C57BL/6小鼠随机分为百草枯组、G-CSF干预组和对照组(均n=8)。高效液相色谱分析测定纹状体多巴胺含量,同时采用免疫组化ABC法观察黑质部酪氨酸羟化酶(TH)阳性表达神经元。结果腹腔注射百草枯20 mg·kg~(-1)·d~(-1),连续5 d后,小鼠自发性活动明显减少,纹状体多巴胺含量明显减少,黑质部TH阳性表达明显减少,与对照组比较差异有统计学意义(P0.05);给予G-CSF干预后纹状体多巴胺含量和黑质部TH阳性表达明显增加,与百草枯组比较差异有统计学意义(P0.05)。结论 G-CSF干预后小鼠纹状体的多巴胺含量升高,黑质部TH阳性表达增加,提示G-CSF对小鼠黑质纹状体通路多巴胺能神经元可能具有一定的保护作用。     

拟抽动秽语综合征大鼠模型的复制及再评价

目的复制拟抽动秽语综合征大鼠模型并对其进行较为全面的评价,以建立治疗此征的新型实验平台。方法将45只SD大鼠,用亚氨基二丙腈腹腔注射的方法制备拟抽动秽语综合征模型,观察动物刻板行为,应用旷场分析测定动物空间认知力和中枢兴奋性,应用攀网实验测定大鼠肌力和运动协调性,应用高效液相色谱法测定大鼠纹状体内多巴胺含量,用放射性配基法测定大鼠纹状体内Ⅱ型多巴胺受体亲和力。结果模型组大鼠出现明显刻板行为;中央格时间延长,跨格次数增多;纹状体内多巴胺(dopa-mine,DA)含量无明显改变,但高香草酸(homovanillic acid,HVA)含量显著增加(P0.01),Ⅱ型多巴胺受体亲和力明显升高(P0.05)。结论此大鼠模型能够较好模拟抽动秽语综合征患者的行为学和神经生化改变,可以作为新型实验动物平台。     

Regulation of neurotensin-containing neurons in the rat striatum and substantia nigra. Effects of unilateral nigral lesion with 6-hydroxydopamine on neurotensin content and its binding site density

The effect of unilateral lesion of the rat substantia nigra with 6-hydroxydopamine (6-OHDA) was investigated on the endogenous contents of neurotensin (NT) and its binding site densities in the striatum and substantia nigra. Tyrosine hydroxylase (T-OH) activity, γ-aminobutyric acid (GABA) content, binding site densities of dihydrotetrabenazine (TBZOH), a marker of dopaminergic synaptic vesicles, and of iodosulpride, a ligand for dopamine D₂ receptors, were also determined. Fourteen days following nigral lesions, these markers were analyzed by means of radioimmunoassay for NT levels, fluoremetric method for GABA content, radiochemical method for T-OH activity and quantitative autoradiography for NT, TBZOH and iodosulpiride binding site densities. Unilateral nigral lesion with 6-OHDA provoked only ipsilateral modifications in dopamine markers. T-OH activity and TBZOH binding site densities significantly decreased in both the ipsilateral striatum and substantia nigra. Iodosulpiride binding sites decreased in the substantia nigra and increased in the striatum on the ipsilateral side. In contrast to these unilateral changes observed for dopamine markers, dramatic increases in NT contents were found in both the ipsi- and contralateral striata. No change was found in nigral NT levels on either side. On the other hand, NT binding sites decreased in the ipsilateral striatum and substantia nigra, which reflected the destruction of dopaminergic elements in these regions. The present results strongly suggest a dopaminergic control of striatal NT systems and demonstrate that a unilateral loss of this control may lead to strong bilateral alterations in NT levels.     

Intrastriatal quinolinic acid injections protect against 6-hydroxydopamine-induced lesions of the dopaminergic nigrostriatal system

We tested the effect of intrastriatal quinolinic acid (QA) injections 2 weeks before subsequent intrastriatal injections of 6-hydroxydopamine (6-OHDA). Levels of DA and its metabolites were measured 2 days and 21 days after lesioning the dopaminergic nigrostriatal system with 6-OHDA. Intrastriatal 6-OHDA injections in the absence of prior treatment of QA significantly decreased dopamine (DA) and its metabolite levels in striatum but not in substantia nigra at day 2, and in striatum and substantia nigra at day 21, a clear indication of a time-dependent retrograde axonal degeneration of substantia nigra cell bodies. Intrastriatal QA injections 2 weeks before subsequent intrastriatal injection of 6-OHDA partially prevented the 6-OHDA-depleting effect on DA and its metabolite levels in both striatum and substantia nigra 21 days after 6-OHDA injection. However, no statistically significant differences were found between QA + 6-OHDA- and 6-OHDA-treated animals at day 2. Our results suggest that intrastriatal QA injections partially prevent the naturally-occurring retrograde axonal degeneration of substantia nigra cell bodies caused by 6-OHDA, and illustrate a target-derived interaction between dopaminergic nerve endings and cell bodies. We suggest that the protective effect found in the QA-injected animals against the neurotoxic action of 6-OHDA is mediated by neurotrophic agents released by activated astroglia.     

Characterization and autoradiographic distribution of neurotensin binding sites in the human brain

The characteristics and topographical distribution of monoiodo¹²⁵I-Tyr₃-neurotensin (NT) binding sites in normal human brain tissue were studied on brain sections and by quantitative autoradiography. Sections at the level of the substantia nigra show a dissociation constant and maximal binding capacity of4.8 ± 0.8nM and 70 ± 7fmol/mg protein, respectively. High density of¹²⁵I-NT binding sites were mainly found in dopaminergic (DA)-rich areas such as the substantia nigra, the ventral tegmental area, the striatum and the nucleus accumbens, further supporting an interaction between NT and DA neurons in human brain.     

Specificity of dopaminergic neuronal degeneration induced by intracerebral injection of 6-hydroxydopamine in the nigrostriatal dopamine system.

The neurotoxic specificity of injections of 6-hydroxydopamine (6-OHDA) into areas containing either dopamine (DA) cell bodies (substantia nigra) or DA axon terminals (striatum) was studied. This selective effect was compared to the unspecific effects of copper sulfate (CuSO4) injection and electrocoagulation. One to two days after unilateral nigral injection of 2 mug of either 6-OHDA or CuSO4 into the nigra the volume of the unspecific lesions around the tip of the cannula was very similar. Only the 6-OHDA-induced lesions were associated with elective degeneration of the nigral DA neurons. Ten days after the administration of the same compounds the gliosis in the substantia nigra was much more extensive in CuSO4-than in 6-OHDA-treated rats; however, the reduction of DA concentrations in the ipsilateral striatum was only noticeable after 6-OHDA (-62%). A somewhat similar decrease of striatal DA levels (-52%) was observed after large electrocoagulation of the substantia nigra. Ten days after 6-OHDA (8mug) or electrolytic lesion of the striatum the Km for DA, serotonin and choline uptakes were similar in the striata of both sides, suggesting that the uptake process in the non-damaged neurons of the lesioned side was functionally normal. Following electrolytic lesion of the striatum, serotonin and choline Vmax values were decreased to about the same extent as the striatal reduction in weight and DA levels. When directly administered into the striatum 6-OHDA also produced a decline in DA concentration and Vmax but in contrast did not affect serotonin and choline uptake (Vmax), suggesting that the drug specifically destroyed dopaminergic neurons. The present data confirm that selective DA denervation can be achieved when appropriate amounts of the drug are injected into brain tissue in order to limit the unspecific lesion.     

Effects of KCl-induced depolarization on the GABA concentration in the corpus striatum and in the substantia nigra

Summary The effects of potassium ion depolarization on the concentration, the synthesis and the utilization of GABA as well as the effects on the concentrations of glutamine, dopamine (DA) and DOPAC in the substantia nigra and in the corpus striatum of the rat were investigated.An intranigral KCl injection did not influence the GABA concentration or the synthesis and utilization of GABA in the substantia nigra. Following an intrastriatal KCl injection the GABA concentration increased and the glutamine concentration decreased in the corpus striatum. A marked increase in the DA concentration in the corpus striatum was seen following an intranigral KCl injection, whereas the DA concentration decreased and the DOPAC concentration increased following an intrastriatal KCl injection. It is concluded that the GABA in the substantia nigra and in the corpus striatum is affected differently by potassium ions, perhaps due to the different cellular localization of GABA in the two structures. The increase of GABA in the corpus striatum was accompanied by a decreased glutamine concentration indicating that glutamine might be a precursor of GABA.     

Effect of chronic haloperidol on dopamine release following microinjection of GABA into the substantia nigra zona reticulata in the rat

To investigate the influence of the striatonigral gamma-aminobutyric acid (GABA) system on the nigrostriatal dopamine (DA) system, the release of DA and/or 3,4-dihydroxyphenylacetic acid in the striatum ipsilateral to the injection side was examined by in vivo voltammetry following microinjection of GABA into the substantia nigra zona reticulata (SNR). The microinjection of GABA (100-300 micrograms/2 microliters) into the SNR produced a dose-dependent increase in the electrochemical signals recorded from the caudate nucleus ipsilateral to the injection side. Following chronic treatment with haloperidol, microinjection of GABA into the SNR produced only a slight (non-significant) increase in the electrochemical signals recorded from the caudate nucleus ipsilateral to the injection side. These results provide additional evidence to support the concept that DA cells in the substantia nigra zona compacta are regulated by the SNR non-DA neurons in an inhibitory manner. It appears, furthermore, that chronic treatment with haloperidol reduces the release of DA in the striatum ipsilateral to the injection side and that this effect may be due to a gradual development of depolarization block of DA cells by chronic administration of haloperidol.     

Inhibition of striatal energy metabolism produces cell loss in the ipsilateral substantia nigra

This study examined whether damage to dopamine (DA) nerve terminals via inhibition of energy metabolism in the striatum would result in the retrograde loss of cell bodies in the substantia nigra. Infusion of 2 μmol malonate into the left striatum of rats resulted in a 67% loss of striatal DA and a 40% loss of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra. No change in the number of Nissl-positive-TH-negative neurons was observed. These findings demonstrate the retrograde destruction of DA cell bodies in the substantia nigra resulting from energy impairment at their terminal projection site.     

Serotonergic sprouting is induced by dopamine-lesion in substantia nigra of adult rat brain

We have previously extracted a serotonin (5-HT) neurotrophic supernatant from the 5,7-DHT lesioned hippocampus. The current study shows that a new 5-HT neurotrophic signal was monitored in the striatum and nigra after DA-denervation. Such a signal may be involved in the heterotypic sprouting. Dopaminergic neurotoxin, 6-hydroxydopamine (6-OHDA), was injected directly into the substantia nigra of adult rats. Two months after surgery, immunocytochemical staining showed that tyrosine hydroxylase (TH)-positive cell bodies had mostly disappeared in the substantia nigra, and TH-positive terminals in the striatum were almost completely depleted. Meanwhile, the 5-HT fibers, which exist in the same areas with low density, sprouted in the nigra as well as in the striatum and became dense. Normally 5-HT fibers innervate the striatum sparsely and the globus pallidus densely with sharp delineation (in the control side), and become dense across both areas with no appreciable delineation (in the lesion side). The increase of 5-HT fibers was more prominent in the posterior than in the anterior striatum. A significant increase in 5-HT and 5-HIAA levels was also evident in the posterior striatum when the decrease in DA level exceeded 90% in the nigra and striatum. In addition, we found that induction of 5-HT sprouting requires a greater than 90% decrease of DA level. Current data support that 6-OHDA injection in the substantia nigra of adult rats triggered a trophic signal or removed an inhibition for the growth of 5-HT neurons which responded with sprouting in the nigra as well as in the striatum.     

Administration of prostaglandin E2 into the striatum induces hyperthermia in rats

Changes in thermoregulatory function were assessed in unanesthetized rats after a unilateral injection of prostaglandin E2 (PGE2) into the striatum or the substantia nigra. Intrastriatal injection of PGE2, but not vehicle solution, induced a dose-dependent rise in rectal temperature. The hyperthermia in response to intrastriatal injection of PGE2 was due to increased metabolism and/or cutaneous vasoconstriction. Furthermore, the PGE2-induced hyperthermia was antagonized by prior intrastriatal injection of kainic acid (to destroy cell bodies in the striatum) but not by pretreatment with 6-hydroxydopamine (to destroy the dopaminergic nerve fibers in the striatum). On the other hand, administration of PGE2 into the substantia nigra induced no significant change in thermoregulatory functions. The data showed that PGE2, when injected into the striatum, may act on cell bodies in the striatum to induce hyperthermia by promoting an increase in heat production and/or vasoconstriction.     

Effects of intraventricular 6-hydroxydopamine on the dopaminergic innervation of striatum: Histochemical and neurochemical analysis

The impact of intracerebroventricular administration of 6-hydroxydopamine (6-HDA) on the dopamine (DA)-containing nigrostriatal projection was determined by regional histochemical and biochemical analyses. One week postinjection, we observed that tyrosine hydroxylase (TH)-positive terminals were almost completely absent from the medial portion of striatum but gradually increased in density toward the lateral margin of this structure. A similar gradient was indicated by fluorescence histochemistry and biochemical analyses of DA. In contrast, the 6-HDA-induced changes in TH activity and in dihydroxyphenylacetic acid content were less severe and showed little or no medial-to-lateral gradient. These high levels of TH activity and DOPAC content, relative to local DA concentrations, suggest an increase in the synthesis and release of DA from residual terminals that may serve to compensate for the brain damage. By 4 months postlesion, both histochemical and biochemical analyses indicated the presence of more DA terminals in striatum than there had been one week postlesion. This change was most markedly obvious in the medial striatum, which had been almost completely devoid of terminals at one week postlesion. Retrograde tracing experiments revealed that terminals appearing in the medial striatum at 4 months postlesion arise from the same region of the substantia nigra that innervates the medial striatum in the intact animal. Thus, no change in the topographic relation between substantia nigra and striatum occurred as a result of the lesion.