应用6-羟基多巴胺建立帕金森病大鼠模型的稳定性评价

背景：应用6-羟基多巴胺(6-Hydroxydopamine，6-OHDA)损毁黑质多巴胺神经元(nigral dopaminergic neuron，NDN)制作的大鼠模型，在目前帕金森病的研究中应用最广，但由于黑质致密部体积狭小，技术难度大，故模型制备成功率一般仅为30％-40％左右。目的：探讨提高6-羟基多巴胺帕金森病大鼠模型成功率的方法，并对模型进行评价。设计：完全随机的实验研究。地点和材料：实验在安徽中医学院第一附属医院中心实验室完成，实验材料包括SD大鼠，6-OHDA，阿朴吗啡，兔抗TH血清，ABC试剂盒，SR-6N大鼠脑立体定向仪，JEM-100CX电镜。干预：取SD大鼠90只，将6-OHDA立体定向微量注射于左侧黑质区及黑质纹状体通路，观察大鼠行为及黑质细胞形态学变化。主要观察指标：旋转行为，免疫组织化学观察多巴胺能神经元数量，电镜观察多巴胺能神经元形态改变。结果：90只大鼠中经阿朴吗啡诱导后有64只(占71％)恒定转向右侧且结果稳定，旋转圈数30min&;gt;210r，被视为成功大鼠模型；免疫组化观察发现注射侧黑质区多巴胺能神经元较对侧明显减少，电镜观察发现其普遍存在凋亡及坏死样改变。结论：应用本方法可较快建立稳定的成功率较高的大鼠模型，但在病理和行为学等方面同自然患者仍有较多差异。     

帕金森病大鼠模型的行为学及神经元形态学评价

背景：由于黑质致密部体积狭小，应用6-羟基多巴胺(6-Hydroxydopamine，6-OHDA)损毁黑质多巴胺神经元(Nigral dopaminergic neuron，NDN)制作帕金森病大鼠模型的成功率较低，一般仅为30％～40％左右。目的：探讨帕金森病大鼠模型建立后的行为学及神经元形态学改变。设计：完全随机的实验研究。地点和材料：安徽省药物研究所，SD大鼠，6-OHDA，阿朴吗啡，兔抗TH血清，SR-6N大鼠脑立体定向仪等。干预：将6-OHDA立体定向注射于90只SD大鼠的左侧黑质区及黑质纹状体通路，观察大鼠行为及多巴胺神经元形态学变化。主要观察指标：旋转行为，震颇及其他异常行为，免疫组织化学观察，电镜观察。结果：经阿朴吗啡诱导共筛选出64只成功大鼠模型(占71％)；免疫组化观察发现注射侧黑质区NDN较对侧明显减少，电镜观察发现其普遍存在凋亡及坏死样改变。结论：本方法是建立帕金森病大鼠模型的有效方法，在行为学和神经元形态学等方面同帕金森病人有许多相似之处。     

帕金森病大鼠模型的行为学及神经元形态学评价

背景由于黑质致密部体积狭小,应用6-羟基多巴胺(6-Hydroxydopamine,6-OHDA)损毁黑质多巴胺神经元(Nigral dopaminergic neuron,NDN)制作帕金森病大鼠模型的成功率较低,一般仅为30%～40%左右.目的探讨帕金森病大鼠模型建立后的行为学及神经元形态学改变.设计完全随机的实验研究.地点和材料安徽省药物研究所,SD大鼠,6-OHDA,阿朴吗啡,兔抗TH血清,SR-6N大鼠脑立体定向仪等.干预将6-OHDA立体定向注射于90只SD大鼠的左侧黑质区及黑质纹状体通路,观察大鼠行为及多巴胺神经元形态学变化.主要观察指标旋转行为,震颤及其他异常行为,免疫组织化学观察,电镜观察.结果经阿朴吗啡诱导共筛选出64只成功大鼠模型(占71%);免疫组化观察发现注射侧黑质区NDN较对侧明显减少,电镜观察发现其普遍存在凋亡及坏死样改变.结论本方法是建立帕金森病大鼠模型的有效方法,在行为学和神经元形态学等方面同帕金森病人有许多相似之处.     

构建大鼠类似于人类帕金森病中晚期行为模型的实验研究

目的：探讨建立帕金森病模型过程中的关键步骤。摸索提高模型成功率的方法。方法：选择SD大鼠作为实验动物。随机分组为模型组和空白对照组。使用特异性的神经毒素6-羟多巴胺(6-OHDA)损伤一侧的黑质纹状体纤维。建立帕金森病动物模型。损伤后10d起，用阿朴吗啡可诱发大鼠向健侧旋转。结果：模型组诱发成功率达51％；病理学观察，损伤侧黑质致密部TH免疫阳性细胞、TH免疫阳性纤维严重缺失。空白对照组无旋转现象，病理学观察无异常现象。结论：用6-OHDA注射建立的大鼠帕金森病模型类似于人类帕金森病中晚期行为病理，制作方法科学、可靠、成功率高，可为医学研究提供依据。     

构建大鼠类似于人类帕金森病中晚期行为模型的实验研究

目的:探讨建立帕金森病模型过程中的关键步骤,摸索提高模型成功率的方法。方法:选择SD大鼠作为实验动物,随机分组为模型组和空白对照组。使用特异性的神经毒素6-羟多巴胺(6-OHDA)损伤一侧的黑质纹状体纤维,建立帕金森病动物模型,损伤后10d起,用阿朴吗啡可诱发大鼠向健侧旋转。结果:模型组诱发成功率达51%;病理学观察,损伤侧黑质致密部TH免疫阳性细胞、TH免疫阳性纤维严重缺失。空白对照组无旋转现象,病理学观察无异常现象。结论:用6-OHDA注射建立的大鼠帕金森病模型类似于人类帕金森病中晚期行为病理,制作方法科学、可靠、成功率高,可为医学研究提供依据。     

6-羟基多巴胺两点注射法建立帕金森病模型大鼠的行为学与组织病理学特征

目的:探讨提高6-羟基多巴胺(6-hydroxydopamine,6-OHDA)诱发帕金森病大鼠模型成功率,并能缩短其成模周期的方法。方法:实验于2004-05-08/07-12在暨南大学医学院解剖教研室进行。采用SD雄性大鼠24只,随机分为实验组(18只),对照组(6只),通过脑内立体定向术,将6-OHDA注入实验组大鼠左侧黑质致密部(substantianigraparscompacta,SNC)和中脑腹侧被盖区(ventraltegmentalarea,VTA)以建立帕金森病模型,观察大鼠行为变化及黑质细胞形态学改变。结果:实验组大鼠经APO诱导后,有15只(83.3%)向右侧旋转速度>7r/min(30min旋转>210r),术后2周与术后3,4周之间大鼠旋转行为差异无显著性意义(P>0.05)。成功模型鼠经Nissl染色可见左侧SNC和VTA区神经元数目较对侧明显减少,对照组无旋转行为和形态学改变。结论:应用6-OHDA于单侧SNC,VTA两点注射可明显提高帕金森病大鼠模型成功率,加大6-OHDA的注射剂量可以缩短其成模周期。同时,注射部位的准确性和注射速度及留针时间都至关重要。     

6-羟基多巴胺两点注射法建立帕金森病模型大鼠的行为学与组织病理学特征

目的：探讨提高6-羟基多巴胺(6-hydroxy dopamine，6-OHDA)诱发帕金森病大鼠模型成功率，并能缩短其成模周期的方法。方法：实验于2004-05-08／07-12在暨南大学医学院解剖教研室进行。采用SD雄性大鼠24只，随机分为实验组(18只)，对照组(6只)，通过脑内立体定向术，将6．OHDA注入实验组大鼠左侧黑质致密部(substantia nigra pars compacta,SNC)和中脑腹侧被盖区(ventral tegmental area，VTA)以建立帕金森病模型，观察大鼠行为变化及黑质细胞形态学改变。结果：实验组大鼠经APO诱导后，有15只(83．3％)向右侧旋转速度&;gt;7r／min(30min旋转&;gt;210r)，术后2周与术后3，4周之间大鼠旋转行为差异无显著性意义(|P&;gt;0．05)。成功模型鼠经Nissl染色可见左侧SNC和VTA区神经元数目较对侧明显减少，对照组无旋转行为和形态学改变。结论：应用6-OHDA于单侧SNC，VTA两点注射可明显提高帕金森病大鼠模型成功率，加大6-OHDA的注射剂量可以缩短其成模周期。同时，注射部位的准确性和注射速度及留针时间都至关重要。     

烟草成份保护多巴胺神经元作用的研究

目的：探讨烟草成份保护多巴胺神经元对抗6－羟基多巴胺（6－OHDA）的神经毒性作用。方法：采用大鼠脑内立体注射6－OHDA建立帕金森病模型，连续观察术前4周开始分别给予被动吸烟和腹腔注射尼古丁（每次0.1mg/kg或0.4mg/kg,bid，持续6周）对阿朴吗啡诱发的旋转行为，纹状体多巴胺（dopamine,DA)的含量和黑质酪氨酸羟化酶（Tyrosine Hydroxylase,TH）阳性神经细胞数目的影响。结果：被动吸烟和腹腔注射尼古丁的大鼠旋转行为明显减少，受损侧纹状体DA含量和黑质TH阳性神经元的数目较对照组增高（P＜0．01），高剂量尼古丁作为更为显著。结论：烟草成份可减轻6－OHDA对黑质DA神经元的损伤。     

改良帕金森病大鼠模型的建立及评价

目的：建立一种改进的帕金森病大鼠模型制作方法，并对模型进行综合评价。 方法：实验于2004—02／2005—06在军事医学科学院干细胞研究中心完成。（1）选取Wistar大鼠60只，随机分为3组，传统组20只。改良组30只。对照组10只。（2）传统组向右侧中脑黑质致密部和中脑腹侧被盖区两点各注射6-羟基多巴胺8μg制作传统帕金森病大鼠模型。改良组向中脑腹侧被盖区单点6-羟基多巴胺12μg制作改良帕金森病大鼠模型。对照组向右侧中脑黑质致密部和中脑腹侧被盖区两点各注射含2g／L抗坏血酸的生理盐水4μL。（3）检测各组大鼠行为学变化及黑质多巴胺能神经元变化。 结果：对照组和传统组术后分别有1只和4只动物在1周内死亡，改良组无动物死亡。行为学和免疫组化检测显示：（1）改良组30只大鼠有22只旋转稳定。平均转数〉7r／min。成功率超过70％，与传统组相当。改良组大鼠死亡率低于传统组（20％）和对照组（10％）。（2）术后传统组和改良组均有部分动物出现震颤、活动迟缓、易激惹、竖毛、尾僵等异常行为。（3）成功帕金森病模型大鼠中脑黑质注射侧神经元数量明显减少．达90％以上，改良组与传统组比较差异无显著性（P〉0．05），与对照组比较注射侧黑质多巴胺能神经元显著减少（P〈0．001）。 结论：改良帕金森病大鼠模型方法简便实用，动物死亡率低，模型成功率高，可以较快建立稳定的帕金森病大鼠模型。     

6-羟基多巴胺制备帕金森病大鼠模型的神经行为学特点

目的:采用在大鼠脑右侧黑质注射6-羟基多巴胺制备帕金森病模型,观察帕金森病模型大鼠神经行为学的特点。方法:实验于2001-03/12在上海中医药大学病理学实验室完成。将62只Wistar大鼠随机分为3组。模型组:采用6-羟基多巴胺(溶于含0.2g/L抗坏血酸的生理盐水中,浓度为2g/L,以1.0mm/min速度缓慢进针,3μL/孔,注射速度为1μL/min,注射完毕后留针5min,然后以1.0mm/min速度缓慢退针)。注射于大鼠脑右侧黑质造成偏侧帕金森病模型。假手术组:注射含0.2g/L抗坏血酸的等量生理盐水,其余条件与模型组相同。正常对照组:不进行任何处理。在模型制备后第10天,以腹腔注射阿朴吗啡0.5mg/kg诱发大鼠向一侧旋转,记录开始旋转至30min内的旋转圈数,以旋转圈数平均每分钟超过7次者为合格的帕金森病模型。并观察各组大鼠在第45天的旋转行为。结果:62只大鼠中有23只大鼠造模不成功,39只大鼠进入结果分析。①正常对照组、假手术组无旋转行为。模型组在腹腔注射阿朴吗啡后0.5～4min,均开始出现旋转行为,即以左侧后肢为支点原地旋转,头尾相接,身体成环状,同时还伴有觅食、嗅探动作。②42只模型组大鼠中,造模成功19只,成功率为45%(19/42)。向左旋转的大鼠显著多于向右旋转及向左一圈,右一圈旋转的大鼠[14只,4只,1只,(P<0.05)]。③模型组在第45天的旋转圈数与第10天时接近[(526.10±173.96),(538.52±187.88)圈/h,(t=1.448,P>0.05)]。结论:①模型大鼠具有典型的神经行为学特点,主要表现为以健侧后肢为支点原地旋转。②该模型不能自行恢复,是研究帕金森病的稳定、可靠模型。     

6- 羟基多巴不同部位毁损大鼠帕金森氏病模型的建立及比较

目的比较 6- OHDA毁损黑质 , 黑质 + 内侧前脑束 , 纹状体而制作 PD模型的异同及可靠性 . 方法在脑立体定位技术下 , 将 20 μ g 6- OHDA分两点注入大鼠上述脑区 , 术后不同时间用阿扑吗啡诱发大鼠异常旋转行为 , 并进行检测比较 . 结果 6- OHDA毁损黑质及黑质 + 内侧前脑束 , PD鼠于术后一周即出现稳定旋转行为 , 两部位模型成功率均为 50% ; 纹状体毁损后一周 PD鼠可出现轻微旋转 , 逐渐增强 , 模型成功率可达 80% . 结论 6- OHDA毁损大鼠黑质 , 黑质 + 内侧前脑束 , 纹状体均可制作成功的 PD模型 .     

A proteomic approach in the study of an animal model of Parkinson's disease

BACKGROUND: The aetiology of Parkinson's disease (PD), an age-related disorder characterized by a progressive degeneration of dopaminergic neurons of the substantia nigra (SN) pars compacta, remains unclear. Current treatments, such as administration of L-DOPA, are only symptomatic and do not stop or delay the progressive loss of neurons. In fact, it has been suggested that the dopamine precursor L-DOPA, increases generation of reactive oxygen species (ROS) leading to further neuronal damage. A similar loss in nigrostriatal dopaminergic neurons is produced on intracerebral administration of the catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA). In an animal model of PD, termed 'the hemiparkinsonian rat', unilateral injection of 6-OHDA into the nigrostriatal pathway results in extensive loss of dopaminergic cells in the ipsolateral SN. In an attempt to identify some of the proteins that are involved in dopaminergic neuronal death, we used the proteomic methods to analyze this animal model of PD. METHODS: Five hemiparkinsonian rats were obtained by intranigral stereotaxic injection of 6-OHDA.The right 6-OHDA-lesioned substantia nigra and striatum tissues along with the left, unlesioned controlateral tissues, were excised and homogenized, using urea-based buffer, to extract the tissues protein. The separation of the protein mixtures and the visualization of the protein patterns obtained were performed using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Protein profiles of control and treated tissues were compare by the PDQuest 2D-gel analysis software (BIO-Rad laboratory). The protein spots showing differential expression were analysed by matrix assisted laser desorption/ionizing time of flight (MALDI-TOF) mass spectrometry. RESULTS: The brain protein extraction and solubilization protocol was validated obtaining a satisfactory protein profile. In comparison to the normal rats, hemiparkinsonian animals exhibited a different expression in alpha-enolase and beta-actin in substantia nigra and striatum, respectively. CONCLUSION: The proteomic study of 6-OHDA-induced lesions in the nigrostriatial pathway allowed us to identify two proteins, alpha-enolase and beta-actin, showing increased levels in the 6-OHDA-lesioned brain tissues compared to control. Previous studies described the same proteins as oxidized and proteins in Alzheimer's disease (AD) brain. Our preliminary data could mirror those results pointing out a common mechanism of neurodegenerative diseases.     

转录因子EN1在帕金森病模型小鼠中脑表达中的变化

目的:研究小鼠帕金森病(PD)模型急性期中脑转录因子EN1表达变化的特点及小鼠的自发性活动改变。方法:应用6-羟多巴胺(6-OHDA)纹状体注射制备小鼠PD模型;旷场试验观察其行为学变化;免疫组织化学/荧光染色检测注药后24h内中脑EN1、酪氨酸羟化酶(TH)的表达变化。结果:与对照组相比,模型组运动方式明显异常,7d内基本恢复正常;运动总量随注药后时间延长呈恢复趋势,但与对照组对比无显著差异;中央区域活动时间逐渐减少,反映了焦虑指数增高。与对照侧相比,实验侧中脑EN1免疫阳性细胞数在3—9h时已出现减少,12—18h时已较显著,18h为65.2±22.3%(P<0.05),21h时减少非常显著。TH阳性的多巴胺能(DA)神经元数目也随时间逐渐减少,但时间上略晚于EN1,15h才可见减少,18h时减少明显,为68.3±1.2%(P<0.05)。EN1在中脑主要定位于细胞核,但也可见于细胞浆。结论:注射6-OHDA后,小鼠出现行为学改变,焦虑指数增高;实验侧中脑EN1和TH阳性细胞数均随时间逐渐减少且EN1变化早于TH,提示转录因子EN1的减少可能是诱导DA神经元凋亡及PD部分症状出现的重要原因。     

Differential effects of glial cell line-derived neurotrophic factor (GDNF) in the striatum and substantia nigra of the aged Parkinsonian rat

Injection of an adenoviral (Ad) vector encoding human glial cell line-derived neurotrophic factor (GDNF) protects dopaminergic (DA) neurons in the substantia nigra (SN) of young rats. As Parkinson's disease occurs primarily in aged populations, we examined whether chronic biosynthesis of GDNF, achieved by adenovirus-mediated delivery of a GDNF gene (AdGDNF), can protect DA neurons and improve DA-dependent behavioral function in aged (20 months) rats with progressive 6-OHDA lesions of the nigrostriatal projection. Furthermore, the differential effects of injecting AdGDNF either near DA cell bodies in the SN or at DA terminals in the striatum were compared. AdGDNF or control vector was injected unilaterally into either the striatum or SN. One week later, rats received a unilateral intrastriatal injection of 6-OHDA on the same side as the vector injection. AdGDNF injection into either the striatum or SN significantly reduced the loss of FG labelled DA neurons 5 weeks after lesion (P 相似文献   

白藜芦醇对神经干细胞诱导分化为多巴胺能神经元移植治疗帕金森模型鼠的影响

背景:目前神经干细胞体外诱导分化的多巴胺能神经元移植治疗帕金森病仍面临细胞存活率低的问题,大部分细胞因氧自由基形成及脂质过氧化而发生程序性凋亡.目的:探讨白藜芦醇对神经干细胞体外诱导分化的多巴胺能神经元移植至帕金森模型鼠后的细胞存活及移植效果的影响.设计、时间及地点:随机对照动物实验,于2007-10/2008-06在中山大学动物实验中心完成.材料:成年健康雄性SD大鼠32只,随机分为模型对照组、多巴胺能神经元组、白藜芦醇组、联合组,8只/组;孕十四五天的健康SD大鼠4只,取胎鼠用于神经干细胞的分离培养.白藜芦醇为深圳晶美生物工程有限公司产品.方法:取体外分离培养的胎鼠中脑神经干细胞,在含表皮生长因子、碱性成纤维细胞生长因子的无血清培养液中传代扩增后,在分化液中诱导向多巴胺能神经元方向分化.各组大鼠均应用6-羟基多巴胺制备帕金森病模型.采用两点移植法,多巴胺能神经元组向大鼠毁损同侧纹状体注入多巴胺能神经元细胞悬液3μL(1×105个/μL):白藜芦醇组注入40 mg/L白藜芦醇3 μL;联合组注入40 mg/L白藜芦醇3 μL+多巴胺能神经元细胞悬液3 pL(1×105个/μL).模型对照组注入DMEM/F12细胞培养液3 μL.主要观察指标:胎鼠神经干细胞诱导分化的酪氨酸羟化酶染色阳性细胞率,细胞移植后帕金森模型鼠不对称旋转行为的变化,纹状体移植区酪氨酸羟化酶染色阳性细胞存活情况.结果:流式细胞仪检测诱导分化6 d的细胞中酪氨酸羟化酶染色阳性细胞率为(17.8±4.2)%.与模型对照组比较,联合组大鼠移植后10d不对称旋转行为有显著性改善(P<0.01),移植后20d不对称旋转圈数开始明显下降(P<0.01).移植后10～60 d,联合组大鼠的不对称旋转圈数明显低于多巴胺能神经元组(P<0.01).白藜芦醇组、模型对照组均未见酪氨酸羟化酶染色阳性细胞,联合组酪氨酸羟化酶染色阳性细胞数明显多于多巴胺能神经元组(P<0.01).结论:胎鼠神经干细胞诱导分化的多巴胺能神经元移植至帕金森模型鼠后,白藜芦醇可提高纹状体移植区植入细胞的存活率,改善大鼠不对称旋转行为.     

骨髓基质多能成体祖细胞移植治疗大鼠帕金森病

背景:帕金森病是一种常见的神经系统变性疾病,胚胎干细胞移植治疗能缓解帕金森病的症状,但受技术和伦理学方面制约。与胚胎干细胞比较,骨髓基质多能成体祖细胞的许多特性也使其逐渐成为细胞移植治疗中理想的细胞资源之一。目的:探讨骨髓基质分离的多能成体祖细胞通过系统移植方式进入大鼠脑组织内并修复受损的神经功能。设计:随机对照实验。单位:武汉市第一医院神经内科。材料:实验于2003-10/2005-03在华中科技大学同济医学院附属协和医院神经内科完成。选用健康SD大鼠80只,体质量180～200g,由华中科技大学同济医学院实验动物中心提供。方法:制作实验性帕金森病大鼠模型,将在体外纯化、增殖和已用5-溴-2-脱氧尿苷处理过的骨髓基质多能成体祖细胞通过尾静脉注入帕金森病大鼠体内。3个月后,对移植大鼠采用免疫组化技术、RT-PCR、免疫电镜、行为学评定等方法鉴定移行入大鼠脑组织内的骨髓基质多能成体祖细胞、其分化的神经元样细胞以及神经功能的修复。主要观察指标:①行为学观察结果。②免疫组织化学染色结果。结果:骨髓基质多能成体祖细胞能移行入大鼠脑组织内并在中脑黑质和纹状体区分化为神经元样细胞;6-羟多巴诱导的大鼠行为损伤有明显恢复;多巴胺-β-羟化酶、神经生长因子和多巴胺转运体mRNA的表达水平明显升高;电镜下观察到骨髓基质多能成体祖细胞所分化的神经细胞与其它神经细胞形成突触联系。结论:骨髓基质多能成体祖细胞能在大鼠脑组织内分化为多巴胺能神经细胞并发挥相应的神经功能。     

Parkin protects against neurotoxicity in the 6-hydroxydopamine rat model for Parkinson's disease.

Loss-of-function mutations in the PARK2 gene are the major cause of early onset familial Parkinson's disease. The gene product, parkin, is an E3 ligase of the ubiquitin-proteasome pathway involved in protein degradation. Dopaminergic neuron loss may result from the toxic accumulation of parkin substrates, suggesting a key role for parkin in dopaminergic neuron survival. In this study, we have investigated the neuroprotective capacity of parkin in the 6-OHDA rat model for Parkinson's disease. 6-OHDA induces the generation of reactive oxygen species leading to the degeneration of catecholaminergic neurons, but may also impair proteasome activity. Lentiviral vectors encoding human wild-type parkin or green fluorescent protein were stereotactically injected into the substantia nigra 2 weeks prior to a striatal 6-OHDA lesion. Histological analysis 1 and 3 weeks after lesioning showed a significant preservation of dopaminergic cell bodies and nerve terminals. Moreover, lesioned rats overexpressing parkin displayed a corresponding behavioral improvement as measured by the amphetamine-induced rotation test and the cylinder test. The improved performance in the amphetamine-induced rotation test lasted until 20 weeks after lesioning. Our results demonstrate that parkin acts as a potent neuroprotective agent in vivo against 6-OHDA toxic insults. These data support the therapeutic potential of parkin for the treatment of not only familial but also sporadic Parkinson's disease.     

Prevention of onset of Parkinson's disease by in vivo gene transfer of human hepatocyte growth factor in rodent model: a model of gene therapy for Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra (SNi). As neurotrophic factors support the survival and enhance the function of dopaminergic neurons, gene therapy using neurotrophic factors has become the center of interest. Thus, we focused on hepatocyte growth factor (HGF) as a neurotrophic and angiogenic growth factor. At 7 days before injection of 6-hydroxydopamine into the SNi, stereotaxic transfection of human HGF or lacZ plasmid was performed into the unilateral striatum of rats. Expression of human HGF in the injected sites could be detected in rats transfected with HGF plasmid DNA, using immunohistochemical staining. Consistently, human immunoreactive HGF protein could be detected at least up to 12 days after transfection. Interestingly, PD rats transfected with lacZ demonstrated amphetamine-induced rotational asymmetry. However, transfection of HGF plasmid DNA resulted in significant inhibition of abnormal rotation up to 24 weeks in a dose-dependent manner. Over 90% of dopaminergic neurons were lost in PD rats transfected with lacZ, whereas over 70% survived in rats transfected with HGF, as assessed by immunohistochemical staining. Overall, the present study demonstrated that overexpression of HGF prevented neuronal death in a PD rat model, providing a potential novel therapy for PD.     

Nonviral glial cell-derived neurotrophic factor gene transfer enhances survival of cultured dopaminergic neurons and improves their function after transplantation in a rat model of Parkinson's disease

Transplantation of dopaminergic fetal mesencephalic tissue into the striatum is currently being developed for treatment of patients with advanced Parkinson's disease. Ethical concerns regarding the use of human fetal tissue, and the limited availability as well as poor survival and differentiation of dopaminergic neurons after transplantation have reduced the extent and outcome of this approach so far. With the purpose of finding means to increase the yield of dopaminergic neurons in transplants, and to reduce the amount of fetal tissue needed for each transplanted patient, we transfected rat fetal ventral mesencephalic (VM) tissue grown as organotypic free-floating roller tube (FFRT) cultures with a vector encoding human glial cell-derived neurotrophic factor (hGDNF). For transfer of an episomal expression vector (pRep7-GDNF8) a nonviral, nonliposomal cationic transfection technique was applied and optimized. Recombinant hGDNF expression resulted in a higher number of TH-positive neurons in the cultures as measured 6 days after transfection. Ventral mesencephalic cultures expressing hGDNF were then grafted into the striatum of unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats. Grafting of genetically modified VM cultures resulted in earlier functional recovery compared with grafting nontransfected cultures. We conclude that organotypic free-floating roller tube cultures can be successfully transfected to produce hGDNF with effects on TH-expressing neurons in vitro and functional effects after grafting in a rat Parkinson's disease model.