基因修饰的神经干细胞脑内移植后TH表达和神经递质的变化

目的：探讨经TH基因修饰的神经干细胞脑内移植后，PD模型动物脑内TH表达和神经递质的变化。方法：构建pN2ATH逆转录病毒载体质粒，用PA317细胞包装，G418筛选阳性克隆，病毒上清感染神经干细胞，将神经干细胞和表达TH的神经干细胞植入PD大鼠纹状体内，观察移植后TH的表达情况以及DA和DOPAC含量变化。结果：TH基因修饰的神经干细胞移植2月内，TH在纹状体内的表达增加，纹状体DA和DOPAC含量增高，好于单纯神经干细胞移植组和对照组，但比正常水平低，结论：TH基因修饰的神经干细胞，脑内移植能增加纹状体内TH的表达以及DA和DOPAC含量，为其脑内移植治疗PD提供了理论基础。     

GFP转基因小鼠神经干细胞移植治疗大鼠帕金森病的实验研究

目的观察GFP(绿色荧光蛋白)转基因小鼠胚胎神经干细胞植入帕金森病大鼠纹状体后的存活、分化情况及治疗作用。方法建立PD模型大鼠及体外培养神经干细胞,然后将GFP转基因小鼠神经干细胞定向植入帕金森病大鼠毁损侧纹状体内,于移植后不同时间诱发旋转行为,并与对照组相比,观察症状的改善,并用酪氨酸羟化酶(TH)免疫组织化学染色方法检测移植GFP转基因小鼠神经干细胞的存活及分化状况。结果 GFP转基因小鼠神经干细胞脑内移植后,帕金森病大鼠的旋转行为明显改善。移植后2至4周时可检测到成片或散在的TH免疫阳性细胞。结论 GFP转基因小鼠神经干细胞移植至帕金森病大鼠纹状体后,可分化为多巴胺能神经元并能改善旋转症状。     

胚胎干细胞分化的神经前体细胞移植治疗帕金森病的实验研究

目的探讨胚胎干细胞（embryonic stemcell,ESC）来源的神经前体细胞（Neural precursorcell）移植治疗帕金森病的可能性。方法将胚胎干细胞诱导分化到神经前体细胞阶段后移植到大鼠帕金森病模型纹状体中,并设生理盐水组做对照研究,观察两组移植后行为学改变及检查纹状体内DA、DOPAC的含量。结果移植组在2～4周后与对照组相比行为学上有明显改善（P〈0．01）,纹状体内DA、DOPAC的含量显著提高（P〈0．01）。结论胚胎干细胞经诱导分化成神经前体细胞可用于帕金森病的修复治疗,胚胎干细胞是良好的干细胞移植治疗用细胞来源。     

小鼠胚胎干细胞诱导的神经前体细胞纹状体移植对PD大鼠的治疗作用

目的 观察来源于小鼠胚胎干细胞的神经前体细胞移植PD大鼠纹状体后的存活、分化以及细胞移植对PD大鼠的治疗作用。方法 采用无血清方法将小鼠胚胎干细胞定向诱导为神经前体细胞，免疫组化技术观察移植细胞的存活、分化。结果 胚胎体在N2选择性培养基选择生长5d后，85％以上的小鼠胚胎干细胞分化为nestin阳性的神经前体细胞。移植到PD大鼠纹状体后大部分神经前体细胞存活良好，移植细胞分别保持为未分化的nestin阳性的神经前体细胞和TH阳性的神经元。移植后3周，PD大鼠的旋转次数明显减少。结论 胚胎干细胞来源的神经前体细胞移植PD大鼠纹状体后能分化为TH阳性的神经细胞，对PD有治疗作用。     

人羊膜上皮细胞侧脑室移植对帕金森病大鼠模型行为、多巴胺能神经元、神经递质影响的实验研究

目的 观察人羊膜上皮细胞(HAECs)移植入帕金森病(PD)大鼠模型侧脑室后的存活及分化情况,及其对PD大鼠模型旋转行为、纹状体区多巴胺及其代谢产物的影响.方法 采用6-羟多巴立体定向脑内注射制作PD大鼠模型,将制模成功大鼠随机分成3组:人羊膜上皮细胞移植组(HAECs组)、磷酸缓冲组(PBS组)和帕金森组(PD组),1w后腹腔注射阿朴吗啡观察各组大鼠旋转行为的变化,连续观察10w,HAECs组5w后用人特异性抗体Nestin和Vimentin检测人羊膜细胞的存活情况,10w后酪氨酸羟化酶(TH)染色观察各组PD大鼠模型黑质部TH阳性神经元的变化情况及HAECs的分化情况,高效液相色谱--电化学仪测定纹状体多巴胺(DA)、高香草酸(HVA)、3,4-二羟基苯乙酸(DOPAC)等神经递质的水平.结果 HAECs在PD大鼠侧脑室内移植可以长期存活达10w,并且可以分化为DA能神经元,HAECs组大鼠旋转数较PBS组及PD组明显降低(P<0.01),黑质部TH阳性神经元数量较PD组及PBS组升高(P<0.01),HAECs组大鼠纹状体区DA及其代谢产物DOPAC、HVA含量较PBS组明显升高(P<0.05).结论 人羊膜上皮细胞移植入PD大鼠侧脑室可以改善PD大鼠的旋转行为,其机制可能与增加纹状体区DA等神经递质有关.     

过表达多巴胺脱羧酶、酪氨酸羟化酶和三磷酸鸟苷环化水解酶1重组慢病毒构建及其在帕金森病大鼠模型治疗作用的研究

目的构建同时表达3种多巴胺(DA)合成相关酶的重组慢病毒(rLV),验证其在6-羟基多巴胺(6-OHDA)帕金森病(PD)大鼠模型中治疗作用,为PD临床基因治疗提供实验基础。方法构建过表达多巴胺脱羧酶(DDC)、酪氨酸羟化酶(TH)和三磷酸鸟苷环化水解酶1(GCH1)rLV及对照rLV。6-OHDA制备的PD模型大鼠,随机分为生理盐水组(n=6)、阴性病毒组(n=6)和阳性病毒组(n=12),分别经立体定向向大鼠纹状体注射生理盐水、对照rLV和DDC+TH+GCH1-rLV。观察构建病毒对PD大鼠旋转行为的影响,免疫荧光法检测病毒转染情况,同时使用高效液相色谱-串联质谱联用法(HPLC-MS/MS)检测纹状体内DA及其代谢产物改变情况。结果 PD大鼠纹状体注射后,阳性病毒组行为改变与生理盐水组、阴性病毒组比较,差异有显著统计学意义(P0.01);阳性病毒组呈现显著的旋转行为改善。免疫荧光显示阳性病毒转染表达有TH活性的阳性细胞。阳性病毒组纹状体内DA及其代谢产物与生理盐水组、阴性病毒组比较,显著增高(P0.01)。结论成功构建过表达DDC+TH+GCH1-rLV,并且在PD模型大鼠中呈现显著的行为改善及DA水平增加,为PD的基因治疗提供实验基础。     

微囊化牛视网膜色素上皮细胞移植治疗伴Lewy小体形成帕金森病的实验研究

目的观察微囊化牛视网膜色素上皮细胞(RPE)移植对伴Lewy小体形成帕金森病(PD)大鼠的治疗作用。方法用蛋白酶体选择性抑制剂lactacystin制作伴Lewy小体形成的PD模型;将微囊化牛RPE植入大鼠纹状体,移植分生理盐水对照组(NS)、RPE、空微囊(APA)及微囊化RPE(RPE-APA)四组;观察移植后旋转行为变化、纹状体中多巴胺(DA)和高香草酸(HVA)及酪氨酸羟化酶(TH)含量的变化。结果微囊化RPE的旋转行为在移植后第1周开始改善(改善程度为21.3%,与空微囊组相比P<0.05),第4周改善更加明显(改善程度为57.89%,与第1周相比P<0.05),并一直持续到第14周(改善程度为59.47%,与空微囊组相比P<0.05)。行为学改善的大鼠,纹状体内DA和HVA含量的变化同其行为学改善相符合。行为学有改善大鼠囊内细胞TH染色呈弱阳性,微囊周边的纹状体可见TH阳性纤维密度较空微囊组高。结论微囊化牛RPE脑内移植对伴Lewy小体形成PD大鼠有治疗作用。     

SPIO、EGFP双标GDNF基因修饰中脑神经干细胞移植治疗帕金森病

目的探讨超顺磁氧化铁(SPIO)、增强型绿色荧光蛋白(EGFP)双标胶质细胞源性神经营养因子(GDNF)基因修饰中脑神经干细胞(mNSCs)移植对帕金森病(PD)大鼠的治疗作用。方法将PD大鼠随机分为对照组、GFP基因修饰mNSCs移植组和GDNF基因修饰mNSCs移植组,将相应细胞移植到PD大鼠纹状体。阿朴吗啡(APO)诱导PD大鼠旋转行为评估细胞移植的治疗作用。磁共振成像、免疫荧光组织化学研究移植细胞的存活、迁移和分化。结果GDNF基因修饰mNSCs移植能显著改善APO诱导PD大鼠的异常旋转行为;大多数移植细胞停留于移植原位,移植8w后,GDNF基因修饰mNSCs移植组有更多的细胞存活并分化为多巴胺神经元。结论MR I成像可对体内移植的SPIO标记细胞进行活体示踪。GDNF基因修饰胚胎mNSCs移植可显著改善PD大鼠的运动障碍,其分子机制有待进一步研究。     

深部脑刺激丘脑底核治疗帕金森病大鼠脑中钙调蛋白的变化

目的 研究慢性高频电刺激帕金森病(Parkinson's disease,PD)大鼠丘脑底核治疗PD的机制.方法 通过6-羟基多巴立体定向注射至大鼠中脑前脑束建立偏侧PD动物模型,对造模成功的PD大鼠模型丘脑底核进行高频电刺激,观察高频电刺激对PD大鼠行为的影响,并用蛋白印迹法检测纹状体酪氨酸羟化酶(TH)、钙调蛋白Calbindin-28和突触前膜囊泡蛋白(synaptophysin)的表达情况.此外,对接受慢性高频电刺激的6-羟基多巴损伤纹状体双侧PD大鼠的脑片进行免疫组化染色,检测黑质Calbindin-28和突触前膜囊泡蛋白的表达情况.结果 (1)高频电刺激偏侧PD大鼠丘脑底核可使阿朴吗啡诱导的旋转行为减少31%;(2)偏侧PD大鼠损伤侧纹状体内已无TH表达,慢性高频电刺激同侧丘脑底核对纹状体TH缺失无逆转,但增加健纹状体内TH表达量78.6%±9.5%;(3)偏侧PD大鼠损伤侧纹状体内Calbindin-28表达量增加75.4%±15.0%,慢性高频电刺激使其表达量减少43.0%±7.1%;(4)慢性高频电刺激亦显著抑制双侧PD大鼠黑质Calbindin-28表达:假手术大鼠、PD大鼠以及慢性高频电刺激后PD大鼠黑质致密部Calbindin-28阳性神经元分别为74.5±10.2、75.7±15.6和33.1±7.8;(5)慢性高频电刺激未影响黑质和纹状体内突触前膜囊泡蛋白的表达.结论 深部脑刺激治疗PD的机制与调节黑质和纹状体内钙调蛋白Calbindin-28和TH表达量有关.     

慢病毒介导的三种神经元谱系相关转录因子脑内转移治疗帕金森病的实验研究

目的探讨慢病毒介导的三种神经元谱系相关转录因子(Ascl1-Brn2-Ngn2,ABN)脑内转移治疗帕金森病(PD)的疗效。方法采用SD大鼠单侧纹状体注射6-羟基多巴胺(6-OHDA)法建立PD模型并分为高剂量治疗组(n=11)、低剂量治疗组(n=11)和空载病毒PD模型组(空载病毒组,n=8),另设PBS阴性对照组(n=6)。应用立体定向仪将携带ABN基因的慢病毒(LV)注射至PD模型大鼠纹状体,观察行为学变化并用Western blot方法检测黑质内酪氨酸羟化酶(TH)表达;利用高效液相色谱法检测多巴胺(DA)及二羟苯乙酸(DOPAC)含量。结果 PBS阴性对照组大鼠未出现明显旋转行为;所有PD模型大鼠自术后第7～10d左右开始出现偏侧PD症状,动物自发向右侧旋转,肌内注射APO后出现快速向右侧旋转(10～17次/min)。基因治疗前空载病毒组大鼠与两治疗组相比,平均旋转圈数差异无统计学意义(均P0.05);空载病毒组大鼠在空载慢病毒注射前后未见有明显旋转行为改善(P0.05);而两ABN基因治疗组在治疗后3周有显著的旋转行为改善(均P0.05),其中高剂量治疗组在治疗后2周出现平均旋转圈数减少,趋势较低剂量治疗组更加明显。PBS阴性对照组术侧黑质内TH表达最高,空载病毒组大鼠TH蛋白表达明显降低;经ABN基因治疗后的两组大鼠TH蛋白表达明显高于空载病毒组,但仍低于PBS阴性对照组。移植后12周,空载病毒组DA及DOPAC水平均显著低于PBS阴性对照组(均P0.05);而ABN基因高低剂量组中DA及DOPAC含量均明显高于空载病毒组(均P0.05),但仍低于PBS阴性对照组。结论 ABN基因脑内转移可显著改善PD大鼠的旋转行为,其作用机制可能与促进多巴胺能神经元再生、增加纹状体多巴胺表达相关。     

An experimental study on intracerebroventricular transplantation of human amniotic epithelial cells in a rat model of Parkinson's disease

Abstract

Objectives: Human amniotic epithelial (HAE) cells are formed from amnioblasts, separated from the epiblast at about the eighth day after fertilization. In the present study, we attempt to investigate the effects of intracerebroventricular transplantation of HAE cells on Parkinson's disease (PD) rats.

Methods: A PD rat model was induced by 6-OHDA injections. Then the rats were transplanted intracerebroventricularly with HAE cells. Apomorphin-induced turns were used to assess the neurobehavioral deficit in rats. Immunofluorescence cytochemistry was used to track the survival of HAE cells. Tyrosinehydroxylase (TH) immunohistochemistry was used to determine the density of TH-positive cells in rat substantia nigra and the differentiation of HAE cells. High performance liquid chromatography (HPLC) was used to measure the levels of dopamine (DA) and its metabolites 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in rat striatum. HVA levels in the cerebrospinal fluid of rats were also determined by HPLC.

Results: The results showed that transplanted HAE cells can survive for at least 10 weeks and differentiate into TH-positive cells in PD rats. The grafts significantly ameliorated apomorphine-induced turns in PD rats. TH immunohistochemistry showed that HAE cells attenuated the loss of TH-positive cells in rat substantia nigra. In addition, HAE cells prevented the fall of DA and its metabolites DOPAC and HVA in PD rats. Increased HVA levels in the cerebrospinal fluid of PD rats were also observed.

Conclusion: These results demonstrate that HAE cells have beneficial effect on 6-OHDA-induced PD rats, which may be due to the neurotrophic factors secrete by HAE cells.     

人羊膜上皮细胞移植治疗帕金森病鼠的研究

目的 观察人羊膜上皮细胞在帕金森病鼠移植后的存活情况,以及它对帕金森病鼠旋转行为的改善作用.方法 采用6-羟多巴胺立体定向纹状体注射制作帕金森病鼠模型;51只大鼠随机分三组:人羊膜上皮细胞移植组、假手术PBS对照组以及空白模型对照组.制模成功后第5周用人特异性抗体Nestin和Vimentin检测人羊膜细胞的存活情况,第10周切片观察黑质部TH阳性神经元的变化情况,高效液相色谱--电化学仪测定纹状体多巴胺(DA),高香草酸(HVA),3,4-二羟基苯乙酸(DOPAC)等浓度以及脑脊液DA的含量.结果 人羊膜上皮细胞帕金森病鼠侧脑室内移植可以存活达10周;移植组大鼠旋转数较对照组明显降低(P≤0.01);黑质部TH阳性神经元数量较对照组升高(P≤0.01),纹状体区DA、HVA和DOPAC含量较PBS对照组明显升高(P＜0.01～0.05),移植组脑脊液DA含量较PBS对照组也显著增加(P＜0.01).结论 人羊膜上皮细胞侧脑室移植可以改善帕金森病鼠的旋转行为,其机制可能与其增加纹状体区多巴胺等递质水平有关.     

The ability of grafted human sympathetic neurons to synthesize and store dopamine: a potential mechanism for the clinical effect of sympathetic neuron autografts in patients with Parkinson's disease

We have investigated the potential of autologous sympathetic neurons as a donor for cell therapy of Parkinson's disease (PD). Our recent study demonstrated that sympathetic neuron autografts increase the duration of levodopa-induced “on” periods with consequent reduction in the percent time spent in “off” phase. We also found that human sympathetic neurons grown in culture have the ability to convert exogenous levodopa to dopamine and to store the synthesized dopamine. This may explain the clinically observed prolongation in the duration of levodopa effects. To further analyze the mechanism for the graft-mediated effect, the present study investigated the metabolic function of human sympathetic ganglionic neurons xenografted into the dopamine (DA)-denervated striatum of rats by monitoring striatal levels of DA and its primary metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), after systemic administration of levodopa. We also explored whether the graft-mediated effect above may last in four PD patients who had been given the grafts and followed for 12-36 months postgrafting. Clinical evaluations showed that an increase in the duration of levodopa-induced “on” phase is detected during a follow-up period of 12-36 months postgrafting in all the four patients tested. Accordingly, the percent time spent in “off” phase exhibited a 30-40% reduction as compared to the pregrafting values. The animal experiment showed that a significant increase in striatal DA levels is noted after systemic levodopa treatment, and that the DA levels remain high for longer periods of time in the grafted rats than in control animals. When given reserpine pretreatment, the levodopa-induced rise of striatal DA levels was significantly attenuated with concomitant increase in DOPAC levels. Histological examinations demonstrated that the grafts contain some tyrosine hydroxylase (TH)-positive cells. These cells were also found to express aromatic-l-amino acid decarboxylase (AADC) and vesicular monoamine transporter-2 (VMAT), both of which are important molecules for the synthesis and the storage of DA, respectively. These results indicate that grafted sympathetic neurons can provide a site for both the conversion of exogenous levodopa to DA and the storage of the synthesized DA in the DA-denervated striatum, explaining a mechanism by which sympathetic neuron autografts can increase the duration of levodopa-induced “on” phase in PD patients.     

Glial cell line‐derived neurotrophic factor–secreting genetically modified human bone marrow‐derived mesenchymal stem cells promote recovery in a rat model of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disease characterized by progressive degeneration of nigrostriatal dopaminergic (DA) neurons. The therapeutic potential of glial cell line‐derived neurotrophic factor (GDNF), the most potent neurotrophic factor for DA neurons, has been demonstrated in many experimental models of PD. However, chronic delivery of GDNF to DA neurons in the brain remains an unmet challenge. Here, we report the effects of GDNF‐releasing Notch‐induced human bone marrow‐derived mesenchymal stem cells (MSC) grafted into striatum of the 6‐hydroxydopamine (6‐OHDA) progressively lesioned rat model of PD. Human MSC, obtained from bone marrow aspirates of young, healthy adult volunteers, were transiently transfected with the intracellular domain of the Notch1 gene (NICD) to generate SB623 cells. SB623 cells expressing GDNF and/or humanized Renilla green fluorescent protein (hrGFP) following lentiviral transduction or nontransduced cells were stereotaxically placed into rat striatum 1 week after a unilateral partial 6‐OHDA striatal lesion. At 4 weeks, rats that had received GDNF‐transduced SB623 cells had significantly decreased amphetamine‐induced rotation compared with control rats, although this effect was not observed in rats that received GFP‐transduced or nontransduced SB623 cells. At 5 weeks, rejuvenated tyrosine hydroxylase‐immunoreactive (TH‐IR) fibers that appeared to be host DA axons were observed in and around grafts. This effect was more prominent in rats that received GDNF‐secreting cells and was not observed in controls. These observations suggest that human bone‐marrow derived MSC, genetically modified to secrete GDNF, hold potential as an allogeneic or autologous stem cell therapy for PD. © 2010 Wiley‐Liss, Inc.     

Intranigral transplants of GABA-rich striatal tissue induce behavioral recovery in the rat Parkinson model and promote the effects obtained by intrastriatal dopaminergic transplants

Intrastriatal transplantation of fetal ventral mesencephalon (VM) is currently explored as a potential clinical therapy in Parkinson's disease (PD). Although providing substantial benefit for the patient, behavioral recovery so far obtained with intrastriatal VM grafts is not complete. Using the 6-hydroxydopamine lesion model of PD, we show here that near-complete restoration of the striatal dopamine (DA) innervation can be achieved by multiple intrastriatal microtransplants of fetal DA cells; nevertheless, complete recovery in complex sensorimotor behaviors was not obtained in these animals. In line with the current model of basal ganglia function, this suggests that the lesion-induced overactivity of the basal ganglia output structures, i.e., the substantia nigra (SN) and the entopeduncular nucleus, may not be completely reversed by intrastriatal VM grafts. In the present study, we have transplanted fetal VM tissue or fetal striatal tissue, as a source of DA and GABA neurons, respectively, into the SN of DA-depleted rats. Intranigral VM grafts induced behavioral recovery in some sensorimotor behaviors (forelimb akinesia and balance tests), but the effect did not exceed the recovery observed after intrastriatal VM grafts. Intranigral grafts of striatal tissue induced a pattern of functional recovery which was distinctly different from that observed after intranigral VM grafts, and recovery in coordinated forelimb use in the paw-reaching test was even more pronounced than after intrastriatal transplantation of VM cells. Combined transplantation of DA neurons into the striatum and GABA-rich striatal neurons into the SN induced additive effects of behavioral recovery observed in the forelimb akinesia test. We propose that intranigral striatal transplants, by a GABA-mediated inhibitory action, can reduce the overactivity of the host SN projection neurons and can induce significant recovery in complex motor behavior in the rat PD model and that such grafts may be used to increase the overall functional efficacy of intrastriatal VM grafts.     

Neuroprotective and neurorescue effect of black tea extract in 6-hydroxydopamine-lesioned rat model of Parkinson's disease

In the present study, an attempt has been made to explore the neuroprotective and neuroreparative (neurorescue) effect of black tea extract (BTE) in 6-hydroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD). In the neuroprotective (BTE + 6-OHDA) and neurorescue (6-OHDA + BTE) experiments, the rats were given 1.5% BTE orally prior to and after intrastriatal 6-OHDA lesion respectively. A significant recovery in d-amphetamine induced circling behavior (stereotypy), spontaneous locomotor activity, dopamine (DA)-D2 receptor binding, striatal DA and 3-4 dihydroxy phenyl acetic acid (DOPAC) level, nigral glutathione level, lipid peroxidation, striatal superoxide dismutase and catalase activity, antiapoptotic and proapoptotic protein level was evident in BTE + 6-OHDA and 6-OHDA + BTE groups, as compared to lesioned animals. BTE treatment, either before or after 6-OHDA administration protected the dopaminergic neurons, as evident by significantly higher number of surviving tyrosine hydroxylase immunoreactive (TH-ir) neurons, increased TH protein level and TH mRNA expression in substantia nigra. However, the degree of improvement in motor and neurochemical deficits was more prominent in rats receiving BTE before 6-OHDA. Results suggest that BTE exerts both neuroprotective and neurorescue effects against 6-OHDA-induced degeneration of the nigrostriatal dopaminergic system, suggesting that possibly daily intake of BTE may slow down the PD progression as well as delay the onset of neurodegenerative processes in PD.     

基因修饰骨髓源性神经元样干细胞治疗帕金森大鼠的研究

目的 观察大鼠酪氨酸羟化酶(tyrosinehydroxylase,TH)修饰的骨髓基质源性神经元样干细胞(neuronoid stem cells derived from bone marrow stem cells,NdSCs-D-BMSCs)在脑室移植途径下对帕金森病(Parkinson disease,PD)大鼠的治疗作用.方法 将酶切鉴定后的新构建质粒pEGFP-C2-TH经电穿孔法转染培养第8天NdSCs-D-BMSCs,注射到PD大鼠模型右侧脑室,观察大鼠行为学变化,移植细胞在大鼠脑组织内的迁移,以及高效液相方法检测脑内DA含量.结果 质粒pEGFP-C2-TH转染NdSCs-D-BMSCs移植后10周,PD大鼠症状显著改善,DA恢复至正常水平33.0%,移植细胞可以在PD大鼠脑内存活,并出现远处迁移.结论 TH修饰的大鼠NdSCs-D-BMSCs经脑室移植对PD大鼠具有明显的治疗作用,为临床中腰椎穿刺干细胞移植的应用提供实验依据.     

Relationships between various behavioural abnormalities and nigrostriatal dopamine depletion in the unilateral 6-OHDA-lesioned rat

While rotational asymmetry is used as a characteristic behavioural sign of striatal dopamine (DA) loss in unilateral animal models of Parkinson's disease (PD), there is relatively little analysis of how other common behavioural deficits relate to nigrostriatal DA depletion. We analysed the relationships between several deficits induced by unilateral 6-OHDA lesions and striatal neurochemistry, as well as neuronal loss in the dopaminergic substantia nigra (SN). Behaviour was evaluated from before until 6 weeks after surgery and abnormalities appeared in body axis, head position and sensorimotor performance as well as apomorphine-induced rotation. As expected, rotational behaviour correlated with striatal DA loss and not with other striatal neurotransmitters measured. Similar observations were found for sensorimotor deficits ('disengage task'). Both deficits were observed in rats with >70% loss of TH+ nigral neurons and >80% loss of striatal DA. Additional postural abnormalities appeared with mean losses of 87% of nigral DA neurons and 97% striatal DA, consistent with observations in patients with advanced PD. The data show that the repertoire of behavioural abnormalities manifested by hemiparkinsonian rats relate directly to the degree of nigrostriatal DA loss and, therefore, mimic features of PD. Analysis of such behaviours are relevant for chronic therapeutic studies targeting PD.     

Co-grafted embryonic striatum increases the survival of grafted embryonic dopamine neurons

To enhance the current therapeutic benefit of dopamine (DA) neuron grafts in Parkinson's disease, strategies must be developed that increase both DA neuron survival and fiber outgrowth into the denervated striatum. Previous work in our laboratory has demonstrated that dopaminergic neurons grow to greater size when co-grafted with striatal cell suspensions and display extensive tyrosine hydroxylase-positive (TH+) projections, but no conclusion could be reached concerning enhancement of survival of grafted DA neurons. The aim of the present study was to characterize further the potential trophic effects of striatal co-grafts on grafted mesencephalic DA neuron survival. Unilaterally lesioned male Fischer 344 rats were grafted with either a suspension of mesencephalic cells or with both mesencephalic and striatal cell suspensions. Co-grafts were either mixed together or placed separately into the striatum. Lesioned rats receiving no graft served as controls. Rotational behavior was assessed following amphetamine challenge at 2 weeks prior to grafting and at 4 and 8 weeks following grafting. Only rats receiving co-grafts of nigral and striatal suspensions separated by a distance of 1 mm showed significant behavioral recovery from baseline rotational asymmetry. Both mixed and separate striatal co-grafts were associated with a doubling of DA neuron survival compared with solo mesencephalic grafts. In the mixed co-graft experiment, DA neurite branching appeared enhanced and TH-rich patches were observed, whereas with co-grafts that were separated, TH+ innervation of the intervening host striatum was increased significantly. These results provide the first evidence suggesting that nigral-striatal co-grafts, particularly those placed separately and in proximity to each other, increase both DA neuron survival and neurite extension from the mesencephalic component of the grafts. J. Comp. Neurol. 399:530–540, 1998. © 1998 Wiley-Liss, Inc.