Intranigral ventral mesencephalic grafts and nigrostriatal injections of glial cell line-derived neurotrophic factor restore dopamine release in the striatum of 6-hydroxydopamine-lesioned rats

We have previously reported that grafting of fetal ventral mesencephalic (VM) tissue to the nigral region of unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats, in conjunction with glial cell line-derived neurotrophic factor (GDNF) injection between nigra and striatum, restores nigrostriatal tyrosine hydroxylase (TH) immunoreactivity. In this study, we investigated the electrochemical indices of dopamine (DA) release in these grafted animals in the striatum and nigra. Adult Sprague-Dawley rats were anesthetized and unilaterally injected with 6-OHDA into the medial forebrain bundle. The completeness of lesions was tested by measuring methamphetamine-induced rotations. One to two months after 6-OHDA administration, fetal VM tissues were grafted in the lesioned nigral area followed by injection of GDNF, brain-derived neurotrophic factor (BDNF), or phosphate-buffered saline (PBS), along a tract from nigra to striatum. Animals receiving transplantation and GDNF, but not BDNF or PBS, injection showed a significant decrease in rotation 1–3 months after grafting. High-speed chronoamperometric recording techniques, using Nafion-coated carbon fiber electrodes, were used to evaluate DA overflow in the striatum. We found that 6-OHDA lesions resulted in a loss of KCl-induced DA overflow in the urethane-anesthetized rats. Three months after GDNF-bridged grafting, application of KCl elicited DA release both in nigra and striatum. The KCl-evoked DA release area was limited to the GDNF-bridging tract in the striatum. On the other hand, KCl did not induce DA release in the BDNF- or PBS-bridged grafts. Immunocytochemical studies indicated that TH-positive neurons and fibers were found in the nigra and striatum after GDNF-bridged grafting. Taken together, our data suggest that fetal nigral transplantation and GDNF injection may restore the nigrostriatal DA pathway and DA release in these hemiparkinsonian animals and support the hypothesis of trophic activity of GDNF on fiber outgrowth from midbrain DA neurons. Received: 8 August 1997 / Accepted: 9 August 1997     

GDNF激活星形胶质细胞保护PD模型大鼠黑质多巴胺能神经元

探索在胶质细胞源性神经营养因子(GDNF)保护受损多巴胺(DA)能神经元过程中,星形胶质细胞的可能作用。成年大鼠右侧纹状体内注射6-羟多巴胺(6-OHDA)制备Parkinson病(PD)模型。动物模型右侧黑质内注射GDNF,于注射后第7、14和21d进行动物行为学分析,行黑质节段连续冠状石蜡切片,以抗酪氨酸羟化酶(TH)、胶质原纤维酸性蛋白(GFAP)抗体免疫组化染色,分别显示DA能神经元和激活的星形胶质细胞,光镜观察并进行细胞计数。用免疫印迹法分析注射后第21d黑质内TH、GFAP蛋白表达量,蛋白条带用图像处理仪扫描,LabWorks软件分析处理。结果显示:动物在注射GDNF后第21d,行为学功能出现显著性恢复;TH阳性神经元数量显著增加;在检测的各时间点均可观察到大量的GFAP阳性细胞;TH、GFAP的蛋白表达量显著高于对照组。以上结果提示,黑质内注射GDNF可能通过激活了的星形胶质细胞保护PD模型大鼠黑质DA能神经元。     

GDNF reduces oxidative stress in a 6-hydroxydopamine model of Parkinson's disease

Many current theories of Parkinson's disease (PD) suggest that oxidative stress is involved in the neurodegenerative process. Potential neuroprotective agents could protect neurons through inherent antioxidant properties or through the upregulation of the brain's antioxidant defenses. Glial cell line-derived neurotrophic factor (GDNF) has been shown to protect and restore dopamine neurons in experimental models of PD and to improve motor function in human patients. This study was designed to investigate GDNF's effect on oxidative stress in a model of PD. GDNF or vehicle was injected into the right striatum of male Fischer-344 rats. Three days later 6-OHDA or saline was injected into the same striatum. The striatum and substantia nigra from both sides of the brain were removed 24h after 6-OHDA or saline injection and analyzed for the oxidative stress markers protein carbonyls and 4-hydroxynonenal. Both markers were significantly reduced in GDNF+6-OHDA treated animals compared to vehicle+6-OHDA treated animals. In addition, in animals allowed to recover for 3.5-4 weeks after the 6-OHDA administration, the GDNF led to significant protection against loss of striatal and nigral tissue levels of dopamine. These results suggest that the protective effects of GDNF against 6-OHDA involve a reduction in oxidative stress.     

Long-term restoration of nigrostriatal system function by implanting GDNF genetically modified fibroblasts in a rat model of Parkinson’s disease

The motor behavior and levels of dopamine and its metabolites in the striatum were studied in rats that received a unilateral injection of 6-OHDA and underwent grafting of rat-derived primary fibroblasts that had been genetically modified to express lacZ and human glial cell line-derived neurotrophic factor (GDNF). Rotation behavior tests were performed each week and striatal levels of DA and its metabolites were measured every 4 weeks after grafting of fibroblasts that expressed lacZ, with or without additional transfection of the GDNF transgene. Rats grafted with GDNF-producing fibroblasts showed a significant improvement in motor behavior as determined by the rotation test, with a less pronounced reduction in the levels of dopamine and its metabolites in the striatum as compared with those in the control animals or brain parts. In addition, there was a lower decrease in the number of TH immunoreactive neurons in the substantia nigra ipsilateral to the lesion in rats with GDNF-producing fibroblasts than in rats with lacZ-expressing fibroblasts. These results support the notion that intracerebral grafting of fibroblasts that express GDNF is a potentially useful therapeutic strategy for treating Parkinsons disease.     

Expression of the fibroblast growth factor-2 isoforms and the FGF receptor 1-4 transcripts in the rat model system of Parkinson's disease

Basic fibroblast growth factor (FGF)-2 occurs in different isoforms representing different translation products of a single mRNA. We have previously shown that the high molecular weight FGF-2 isoforms (21, 23 kD) stimulated survival- and neurite-promoting activities and protective effects on cultured embryonic dopaminergic (DA) neurons of the substantia nigra (Neuroscience 100 (2000) 73). In this study the expression of FGF-2 isoforms in the striatum and substantia nigra was analyzed by Western blot in adult intact rats and following complete unilateral 6-hydroxydopamine (6-OHDA) lesion. In intact rats, all three FGF-2 isoforms (18, 21, 23 kD) are expressed. Neurotoxin-mediated lesion of nigral DA neurons revealed no change of the FGF-2 isoform expression pattern in the nigrostriatal system. Additionally, the FGF receptors 1, 2 and 3 are expressed in these tissues and displayed no alterations after 6-OHDA injection as demonstrated by RT-PCR. The presence of all three FGF-2 isoforms and the FGFR 1–3, together with the previous demonstrated neurotrophic effects of FGF-2 on dopaminergic neurons, suggest a physiological function of the FGF-2 isoforms in the nigrostriatal system.     

6-Hydroxydopamine induces the loss of the dopaminergic phenotype in substantia nigra neurons of the rat

Intraparenchymal injections of the neurotoxin 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle in rats destroys the dopaminergic neurons in the pars compacta of the substantia nigra. In other transmitter systems it has been found that axotomy or neurotoxin exposure produces an initial loss of neurotransmitter phenotype, with cell death occurring over a much slower time course. To determine whether this also occurs in dopamine neurons after 6-OHDA, two approaches were utilized. First, the effect of injections of 6-OHDA into the medial forebrain bundle on nigral dopaminergic neurons was studied using combined fluorogold and immunocytochemical labeling. Four weeks after the 6-OHDA injection, there was an 85% reduction in the number of tyrosine hydroxylase (TH)-immunoreactive cells on the lesioned side. In contrast, there was only a 50% reduction in the number of fluorogold-labeled cells on the lesioned side. Second, the time course of the rescue of dopaminergic neurons after 6-OHDA by glial cell line-derived neurotrophic factor (GDNF) was determined using TH immunocytochemistry. Greater numbers of dopamine neurons were rescued 9 weeks after GDNF, compared with counts made 5 weeks after GDNF. Taken together, these results suggest loss of dopaminergic phenotype is greater than cell loss following 6-OHDA injections, and that GDNF restores the phenotype of affected cells.     

阿扑吗啡对6-羟基多巴胺毁损纹状体鼠的神经保护和神经营养作用

目的在6-羟基多巴胺毁损纹状体鼠模型上,探讨阿扑吗啡的神经保护和神经营养作用。方法用6-羟基多巴胺毁损大鼠单侧纹状体,在毁损前15 m in注射阿扑吗啡(10 mg/kg,s.c.),连续注射11 d。毁损2周后,分别进行行为学(苯丙胺引起的旋转数目)、组织学(黑质和腹侧被盖区的酪氨酸羟化酶阳性细胞计数)的观察。结果阿扑吗啡降低苯丙胺引起的向损伤侧旋转的次数,显著降低黑质神经元的损伤(从50%降至30%)。且多巴胺细胞形状可恢复到类似正常组;阿扑吗啡对正常鼠黑质细胞数无影响,但可使腹侧被盖区细胞数显著增高37%。结论阿扑吗啡对6-羟基多巴胺毁损纹状体模型鼠的黑质和腹侧被盖区神经细胞具有显著的保护作用,且改善运动功能。同时,对正常鼠的腹侧被盖区也显示神经营养作用。     

Constitutive Ret signaling is protective for dopaminergic cell bodies but not for axonal terminals

Ret is the canonical signaling receptor for glial cell line-derived neurotrophic factor (GDNF), which has been shown to have neuroprotective effects when administered prior to neurotoxic challenge. A missense Meth918Thr mutation causes the constitutive activation of Ret, resulting in multiple endocrine neoplasia type 2 B (MEN2B). To clarify the role of Ret signaling in neuroprotection, we studied the effects of the neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) on the dopaminergic system of mice carrying the MEN2B mutation. We found that MEN2B mice were significantly more resistant to nigral tyrosine hydroxylase (TH)-positive cell loss induced by unilateral striatal 6-OHDA than Wt mice. However, 6-OHDA caused profound dopamine (DA) depletion in the striatum of both MEN2B and Wt mice. Systemic MPTP caused similar DA depletion and a decrease in TH-immunostaining in the striatum of MEN2B and Wt mice. Neither neurotoxin induced a compensatory increase in striatal metabolite/DA ratios in the MEN2B mice, possibly contributing to an increased amphetamine-induced turning behavior observed in behavioral assessments of these mice. Thus, our data suggest that activated Ret protects DA cell bodies in the substantia nigra pars compacta, but does not protect DA axons in the striatum.     

The restorative effect of intramuscular injection of tetanus toxin C-fragment in hemiparkinsonian rats

The C-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) is a peptide that has a neuroprotective action against dopaminergic damage by MPP⁺, both in vitro and in vivo. The trophic effects of Hc-TeTx have been related to its ability to activate the pathways of the tropomyosin receptor kinase, which are crucial for survival process. Our group had previously shown neuroprotective effect of intramuscular Hc-TeTx treatment on animals with a dopaminergic lesion; however, there is no evidence indicating its restorative effects on advanced dopaminergic neurodegeneration. The aim of our study was to examine the restorative effects of an intramuscular injection of the Hc-TeTx fragment on the nigrostriatal system of hemiparkinsonian rats. The animals were administered with a vehicle or Hc-TeTx (20 μg/kg) in the gastrocnemius muscle for three consecutive days post-dopaminergic lesion, which was made using 6-hydroxydopamine. Post-Hc-TeTx treatment, the hemiparkinsonian rats showed constant motor asymmetry. Moreover, the ipsilateral striatum of the post-Hc-TeTx group had a lower number of argyrophilic structures and a major immunorreactivity to Tyrosine Hydroxylase in the striatum and the substantia nigra pars compacta compared to the 6-OHDA group. Our results show the restorative effect of the Hc-TeTx fragment during the dopaminergic neurodegeneration caused by 6-OHDA.     

Effect of nigrostriatal damage induced by 6-hydroxydopamine on the expression of glial cell line–derived neurotrophic factor in the striatum of the rat

Several types of brain injuries have been associated with alterations in the striatal expression of neurotrophic factors, including glial cell line–derived neurotrophic factor (GDNF). However, contradictory results on the striatal expression of GDNF have been reported in different animal models of Parkinson's disease. For this reason, we examined the effect of nigrostriatal damage on the mRNA and protein expression levels of GDNF in the striatum as a function of time following a striatal or medial forebrain bundle 6-hydroxydopamine lesion. At different time points after the administration of 6-hydroxydopamine, striatal expression levels of GDNF were analyzed with semi-quantitative Western blotting. No significant changes in GDNF expression levels were observed within the 35-day observation period, either between the denervated and the intact striatum of medial forebrain bundle and striatally lesioned rats or between the striata of lesioned animals and those of control animals. In order to reinforce these results, striata of lesioned rats, sacrificed 18 days after lesioning, were analyzed with enzyme-linked immunosorbent assay and real-time polymerase chain reaction. At this time point, both techniques confirmed the results of the Western blot analysis, detecting no changes in striatal expression of GDNF, either at the protein level, or at the mRNA level. These data show that nigrostriatal damage induced by 6-hydroxydopamine has no effect on the striatal expression of GDNF.     

The industrial chemical Tinuvin 123 does not induce dopaminergic neurotoxicity in C57Bl/6 mice

We compared in rats with 6-hydroxydopamine (6-OHDA)-induced hemiparkinsonism the content of tumor necrosis factor (TNF)-alpha in the nigrostriatal dopaminergic region of the control side with that of the 6-OHDA-injected experimental side, and explored the effects of 6-OHDA injection combined with the immunosuppressant FK506 treatment (0.5 or 4 mg/kg per day for 2 weeks). The ratios of the concentration of TNF-alpha in the striatum and substantia nigra on the 6-OHDA injection side to that on the control side in the 6-OHDA hemiparkinsonism rats were significantly higher than those in the control rats without 6-OHDA treatment, whereas those in the rats treated with 6-OHDA and FK506 were not significantly different from those in the control rats. Thus FK506 attenuated increased TNF-alpha level in the nigrostriatal dopaminergic region injured by 6-OHDA treatment.     

Effects of intra-striatal GDNF on motor coordination and striatal electrophysiology in aged F344 rats

The purpose of this study was to examine whether improvement in motor function could be demonstrated in old rats, and to see if GDNF affected post-synaptic DA function. Aged (20 month old) versus young rats were tested following GDNF treatment for postural control by using an inclined balance beam and a wire grip strength test. Rats were also examined electrophysiologically for spontaneous striatal cell firing rate alone and in the presence of DA receptor agonists, and histologically for the intensity of striatal TH staining, and number of DA containing nigral cells. Behavior was significantly improved in the aged animals who received central GDNF infusions, although the extent of improvement was less than what has been observed in 16-month-old rats. There was no effect of GDNF treatment in the aged animals on spontaneous firing rate in the striatum, or on the post synaptic response to locally applied D(1) and D(2) receptor family agonists. However, there was an effect of age alone on firing rate, and on the response to locally applied SKF 38393 and quinpirole. By using unbiased cell counting we observed no age-related decline in the number of TH positive cells in the substantia nigra. There was no effect of GDNF on the number of TH positive cells in the substantia nigra in either young or aged rats, although there were morphological improvements in DA neurons of the GDNF treated aged rats. These results replicate earlier studies showing an effect of age on striatal firing rate and dopamine receptor function, and suggest that the GDNF mediated improvement in behavior may be located other than post synaptically within the striatum.     

Molecular mechanism of the neurotrophic effect of GDNF on DA neurons: role of protein kinase CK2

Glial cell line-derived neurotrophic factor (GDNF) is suggested as a specific neurotrophic factor for midbrain dopamine (DA) neurons, but the molecular mechanism underlying the neuroprotective action of GDNF is not well known. In the present study, we have shown that GDNF increased protein kinase CK2 activity in rat substantia nigra (SN) in a dose-dependent and time-dependent manner. This effect is prevented by prior treatment of the receptor Ret blocker K-252b. Immunostaining results also revealed that CK2 is expressed in TH-positive neurons in mesencephalon culture. Transfection of the wildtype CK2alpha DNA increased, whereas transfection of the catalytically inactive CK2alphaA156 mutant DNA decreased CK2 activity in the SN. CK2alphaA156 mutant DNA also antagonized the enhancing effect of GDNF on CK2 activity. It also antagonized the enhancing effects of GDNF on tyrosine hydroxylase (TH) protein level in the SN, DA turnover in the striatum and rotarod performance in rats. Further, CK2alpha wildtype DNA increased, whereas CK2alphaA156 mutant DNA decreased TH activity in the SN without altering the TH protein level. On the other hand, the DA neuron toxin 1-methyl-4-phenylpyridinium iodide (MPP+) markedly decreased the number of TH-positive neurons and TH protein level in the SN, decreased DA level in the striatum and impaired rotarod performance in rats. Over-expression of the CK2alpha wildtype DNA partially, but significantly, prevented the deteriorating effect of MPP+ on these measures. Prior administration of MPP+ also antagonized the enhancing effect of GDNF on CK2 activity. These results together suggest that the CK2 signaling pathway contributes to the neuroprotective action of GDNF on DA neurons.     

单唾液酸神经节苷脂(GM_1)对经MPTP破坏的小鼠黑质纹状体多巴胺神经元的保护作用

将1一甲基一4苯基一1.2.3.6四氢吡啶(MPTP)经腹腔注入小鼠,连续注射6天,一组动物注射后次日处死;另一组动物存活二周后处死;第三组动物在给予1—甲基一4苯基一1.2.3.6四氢吡啶的同时,经腹膜腔注射神经节苷脂持续三周;第四组为对照组.各组动物脑用酪氨酸羟化酶抗体进行免疫组化观察.结果发现1一甲基一4苯基一1.2.3.6四氢吡啶连续注射后次日处死的动物黑质致密部酪氨酸羟化酶阳性神经元数量明显减少,纹状体中酪氨酸羟化酶阳性终末亦明显稀疏.损害后存活二周组,不论黑质或纹状体的损害均略轻.单唾液酸神经节苷脂组黑质致密部酪氨酸羟化酶阳性神经元及纹状体中酪氨酸羟化酶阳性终末均较上两组明显增多.实验表明,单唾液酸神经节苷脂对经1一甲基一4苯基一1.2.3.6四氢吡啶破坏后的小鼠黑质纹状体系多巴胺神经元有保护作用,防止多巴胺神经元因损害引起的继发性退变.     

Differential effects of glial cell line-derived neurotrophic factor on A9 and A10 dopamine neuron survival in vitro

Glial cell-line derived neurotrophic factor (GDNF) enhances dopamine (DA) cell survival and fiber outgrowth, and may be beneficial in enhancing cell restorative strategies for Parkinson's disease (PD). However, GDNF may have different roles for transplanted DA cell sub-types. The present in vitro study investigated the effect of GDNF on the survival of rat DA cells displaying a phenotype consistent with either the substantia nigra [A9 cells immunopositive for tyrosine hydroxylase (TH) and G-protein-gated inwardly rectifying potassium channel subunit 2 (GIRK2)] or with the ventral tegmental area [A10 cells immunopositive for TH and calbindin]. It was found that a single exposure of GDNF enhanced the number of DA cells of an A9 phenotype, without affecting DA cells of an A10 phenotype. Conversely, repeated GDNF exposure did not alter the survival of A9 phenotypic cells, but doubled the percentage of A10 cells. It was concluded that GDNF administration may affect dopaminergic cells differently depending on time and degree of GDNF exposure. For cell transplantation in PD, long-term GDNF administration may result in detrimental effects for transplanted A9 TH+ cells as this may introduce competition with A10 TH+ cells for survival and fiber outgrowth into the host striatum. These results may have important implications for clinical neural transplantation in PD.     

Motor function,graft survival and gliosis in rats with 6-OHDA lesions and foetal ventral mesencephalic grafts chronically treated with L-DOPA and carbidopa

Summary In rats with a unilateral 6-OHDA lesion of the nigrostriatal pathway, foetal ventral mesencephalic grafts implanted into the 6-OHDA-lesioned striatum produced a reduction in apomorphine-induced contralateral rotation, and complete abolition of (+)-amphetamine-induced ipsilateral rotation. The graft-induced reduction of apomorphine and (+)-amphetamine-induced rotation was not affected by chronic 27 week administration of L-DOPA and carbidopa to rats receiving foetal grafts.TH-immunohistochemistry revealed >96% loss of dopamine cells in the substantia nigra ipsilateral to the 6-OHDA lesion in all animals, but cell loss in the ipsilateral ventral tegmental area was more variable (21–46% of the intact side). TH-positive cells in the intact substantia nigra and ventral tegmental area were not affected by chronic treatment with L-DOPA and carbidopa. In the lesioned striatum of rats receiving sham grafts, no TH-positive cells or fibres were seen. In the 6-OHDA-lesioned striatum of animals receiving foetal grafts, many TH-positive cells were seen in the grafts and chronic treatment with L-DOPA and carbidopa did not reduce cell survival.GFA-P immunohistochemistry revealed that a unilateral 6-OHDA lesion followed by a sham graft was not associated with a reactive gliosis reaction in the striatum at the time of study (38 weeks after lesion surgery and 30 weeks after sham-graft), and treatment of such rats with L-DOPA and carbidopa was also without effect on glia. In contrast there was a marked gliosis in the striatum surrounding foetal grafts which was unaffected by chronic treatment with L-DOPA and carbidopa. The grafts themselves were surrounded by a rim of glial cells, and the glial density within the grafts was higher in animals receiving chronic L-DOPA and carbidopa treatment. However, there was no obvious relationship between the number of TH-positive cells within the grafts, or graft volume, and glial cell density within the grafts.These results suggest that long-term treatment with L-DOPA and carbidopa does not impair either the behavioural recovery produced by foetal ventral mesencephalic grafts in rats or the long-term survival of grafts as revealed by TH-immunohistochemistry. The presence of a foetal graft is associated with a reactive gliosis in the implanted striatum, which was not altered by long-term treatment with L-DOPA and carbidopa. However such treatment did result in an increase in glial density within the grafts themselves.     

Immunohistochemical changes of nigrostriatal tyrosine hydroxylase and dopamine transporter in the golden hamster after a single intrastriatal injection of 6-hydroxydopamine

One of the most important models for analyzing the pathomorphological aspects of Parkinson's disease (PD) is the 6-hydroxydopamine (6-OHDA) model where lesions of the nigrostriatal axis are observed when 6-OHDA is intrastriatally injected.Despite the widespread use in rats, only few studies about the toxicity of 6-OHDA have been carried out in other species. In the present study, we evaluated for the first time the effects of a single intrastriatal injection of 6-OHDA (20 μg dissolved in 2 μl of vehicle) in the young-adult golden hamster (GH). Significant decreases in tyrosine hydroxylase (TH)-positive area and dopamine transporter (DAT)-positive area were found in the ipsilateral striatum 3 days after the injection. These decreases in immunoreactivity continued for 7 days and a recovery trend was found at days 15 and 21 post injection. On the other hand, no effect of injection was found on the contralateral side. In the substantia nigra pars compacta (SNpc), a significant decrease in the number of TH-positive cells appeared one week after the injection with the peak in the loss of TH-positive immunoreactivity being recorded two weeks post-injection.On the basis of the results herein reported, we believe that the GH is a suitable model for studying the patterns of spontaneous recovery of striatal axons following the 6-OHDA intrastriatal injection.     

Assessment of recovery in the hemiparkinson rat: drug-induced rotation is inadequate

Recovery from apomorphine-induced rotational behavior was compared to sensorimotor and motor function in hemiparkinsonian rats receiving intrastriatal grafts of astrocytes expressing recombinant tyrosine hydroxylase (TH) or control beta-galactosidase (beta-gal). Rats received unilateral intranigral infusions of 6-hydroxydopamine (6-OHDA). Animals with large lesions, as determined by apomorphine-induced rotation, received grafts of astrocytes into the denervated striatum. Behavioral recovery was assessed on days 14-16 post-transplantation using apomorphine-induced rotation, somatosensory neglect, and reaching for pellets using the Montoya staircase method. Rats that received transplants of TH-transfected astrocytes showed a 34% decrease in rotational behavior, but no consistent recovery of somatosensory neglect or skilled reaching. Post-mortem histological analyses revealed survival of grafted astrocytes in host striatum and expression of TH at 17 days post-transplantation. We suggest that TH-expressing astrocytes may reverse post-synaptic dopamine (DA) receptor supersensitivity; however, sensorimotor and motor abilities are not restored due to a failure by TH-expressing astrocytes to reestablish dopaminergic circuitry. The present results demonstrate the need to utilize a variety of sensory and motor behavioral tests that cohesively provide greater interpretability than a single behavioral measure used in isolation, such as drug-induced rotational behavior, to assess the efficacy of experimental gene therapies.     

肌酸对帕金森病模型大鼠黑质多巴胺能神经元保护作用的研究

目的:观察肌酸对帕金森病模型大鼠黑质多巴胺能神经元的保护作用及其机制。方法:30只雄性SD大鼠随机分成3组:对照组(A组),6-羟多巴胺(6-OHDA)组(B组),肌酸+6-OHDA组(C组),每组各10只。A组不注射6-OHDA,B组进行6-OHDA造模,C组在造模前5天每天给予肌酸(100 mg/kg)灌胃,连续两周,B组给予生理盐水4 ml灌胃。分别观察各组动物行为学、黑质多巴胺能神经元数量及氧化应激指标谷胱甘肽(GSH)和丙二醛(MDA)水平变化。结果:6-OHDA立体定向注射后,实验大鼠出现阿朴吗啡诱导的旋转行为,黑质致密部多巴胺能神经元显著减少,GSH含量降低、MDA水平升高;肌酸能够减轻动物行为学异常(P0.01)、显著增加黑质致密部多巴胺神经元数量(P0.01),拮抗6-OHDA诱导的氧化应激水平升高(P0.01)。结论:肌酸对6-OHDA诱导的多巴胺神经元损伤具有明显保护作用,其机制与抑制氧化应激损伤有关。     

Cellular immune response to intrastriatally implanted allogeneic bone marrow stromal cells in a rat model of Parkinson's disease

Background

Marrow stromal cells (MSC), the non-hematopoietic precursor cells in bone marrow, are being investigated for therapeutic potential in CNS disorders. Although in vitro studies have suggested that MSC may be immunologically inert, their immunogenicity following transplantation into allogeneic recipients is unclear. The primary objective of this study was to investigate the cellular immune response to MSC injected into the striatum of allogeneic recipients (6-hydroxydopamine [6-OHDA]-hemilesioned rats, an animal model of Parkinson's disease [PD]), and the secondary objective was to determine the ability of these cells to prevent nigrostriatal dopamine depletion and associated motor deficits in these animals.

Methods

5-Bromo-2-deoxyuridine (BrdU) – labeled MSC from two allogeneic sources (Wistar and ACI rats) were implanted into the striatum of adult Wistar rats at the same time as 6-OHDA was administered into the substantia nigra. Behavioral tests were administered one to two weeks before and 16–20 days after 6-OHDA lesioning and MSC transplantation. Immunocytochemical staining for T helper and T cytotoxic lymphocytes, microglia/macrophages, and major histocompatibility class I and II antigens was performed on post-transplantation days 22–24. MSC were detected with an anti-BrdU antibody.

Results

Tissue injury due to the transplantation procedure produced a localized cellular immune response. Unexpectedly, both sources of allogeneic MSC generated robust cellular immune responses in the host striatum; the extent of this response was similar in the two allograft systems. Despite these immune responses, BrdU⁺ cells (presumptive MSC) remained in the striatum of all animals that received MSC. The numbers of remaining MSC tended to be increased (p = 0.055) in rats receiving Wistar MSC versus those receiving ACI MSC. MSC administration did not prevent behavioral deficits or dopamine depletion in the 6-OHDA-lesioned animals.

Conclusion

MSC, when implanted into the striatum of allogeneic animals, provoke a marked immune response which is not sufficient to clear these cells by 22–24 days post-transplantation. In the experimental paradigm in this study, MSC did not prevent nigrostriatal dopamine depletion and its associated behavioral deficits. Additional studies are indicated to clarify the effects of this immune response on MSC survival and function before initiating trials with these cells in patients with PD or other neurodegenerative disorders.