Panax notoginseng saponins influence on transplantation of neural stem cell-derived dopaminergic neurons in a rat model of Parkinson’s disease *☆

BACKGROUND： Dopaminergic neurons differentiated from neural stem cells have been successfully used in the treatment of rat models of Parkinson＇s disease; however, the survival rate of transplanted cells has been low. Most cells die by apoptosis as a result of overloaded intracellular calcium and the formation of oxygen free radicals.
OBJECTIVE： To observe whether survival of transplanted cells, transplantation efficacy, and dopaminergic differentiation from neural stem cells is altered by Panax notoginseng saponins （PNS） in a rat model of Parkinson＇s disease.
DESIGN, TIME AND SETTING： Cellular and molecular biology experiments with randomized group design. The experiment was performed at the Animal Experimental Center, First Hospital of Sun Yat-sen University from April to October 2007.
MATERIALS： Thirty-two adult, healthy, male Sprague Dawley rats, and four healthy Sprague Dawley rat embryos at gestational days 14-15 were selected. The right ventral mesencephalon was injected with 6-hydroxydopamine to establish a model of Parkinson＇s disease. 6-hydroxydopamine and apomorphine were purchased from Sigma, USA.
METHODS： Neural stem cells derived from the mesencephalon of embryonic rats were cultivated and passaged in serum-free culture medium. Lesioned animals were randomly divided into four groups （n = 8）： dopaminergic neuron, dopaminergic neuron ＋ PNS, PNS, and control. The dopaminergic neuron group was injected with 3 μL cell suspension containing dopaminergic neurons differentiated from neural stem cells. The dopaminergic neurons ＋ PNS group received 3 μ L dopaminergic cell suspension combined with PNS （250 mg/L）. The PNS group received 3 μL PNS （250 mg/L）, and the control group received 3 μL DMEM/F12 culture medium.
MAIN OUTCOME MEASURES： The rats were transcardially perfused with 4% paraformaldehyde at 60 days post-grafting for immunohistochemistry. The rats were intraperitoneally injected with apomorphine （0.5 mg/kg） to induce rotational behavior. RESU     

骨髓间充质干细胞对1-甲基-4-苯基吡啶离子损伤的离体纹状体-黑质脑片多巴胺能神经元的保护作用

BACKGROUND： To date, the use of bone marrow-derived mesenchymal stem cells （MSCs） for the treatment of Parkinson’s disease have solely focused on in vivo animal models. Because of the number of influencing factors, it has been difficult to determine a consistent outcome.
OBJECTIVE： To establish an injury model in brain slices of substantia nigra and striatum using 1-methyl-4-phenylpytidinium ion （MPP＋）, and to investigate the effect of MSCs on dopaminergic neurons following MPP＋ induced damage.
DESIGN, TIME AND SETTING： An in vitro, randomized, controlled, animal experiment using I mmunohistochemistry was performed at the Laboratory of the Department of Anatomy, Fudan University between January 2004 and December 2006.
MATERIALS： Primary MSC cultures were obtained from femurs and tibias of adult Sprague Dawley rats. Organotypic brain slices were isolated from substantia nigra and striatum of 1-day-old Sprague Dawley rat pups. Monoclonal antibodies for tyrosine hydroxylase （TH, 1：5 000） were from Santa Cruz （USA）; goat anti-rabbit IgG antibodies labeled with FITC were from Boster Company （China）.
METHODS： Organotypic brain slices were cultured for 5 days in whole culture medium supplemented with 50% DMEM, 25% equine serum, and 25% Tyrode’s balanced salt solution. The medium was supplemented with 5 μg/mL Ara-C, and the culture was continued for an additional 5 days. The undergrowth of brain slices was discarded at day 10. Eugonic brain slices were cultured with basal media for an additional 7 days. The brain slices were divided into three groups： control, MPP＋ exposure, and co-culture. For the MPP＋ group, MPP＋ （30 μmol/L） was added to the media at day 17 and brain slices were cultured for 4 days, followed by control media. For the co-culture group, the MPP＋ injured brain slices were placed over MSCs in the well and were further cultured for 7 days.
MAIN OUTCOME MEASURES： After 28 days in culture, neurite outgrowth was examined in the brain slices under phase-contrast microscopy. The percent of area containing dead cells in each brain slice was calculated with the help of propidium iodide fluorescence. Brain slices were stained with antibodies for TH to indicate the presence of dopaminergic neurons. Transmission electron microscopy was applied to determine the effect of MSCs on neuronal ultrastructure.
RESULTS： Massive cell death and neurite breakage was observed in the MPP＋ group. In addition, TH expression was significantly reduced, compared to the control group （P 〈 0.01）. After 7 days in culture with MSCs, the co-culture group presented with less cell damage and reduced neurite breakage, and TH expression was increased. However, these changes were not significantly different from the MPP＋ group （P 〈 0.01）. Electron microscopy revealed reduced ultrastructural injury to cells in the brain slices. However, vacuoles were present in cells, with some autophagic vacuoles.
CONCLUSION： Bone marrow-derived MSCs can promote survival of dopaminergic neurons following MPP＋-induced neurotoxicity in co-cultures with substantia nigra and striatum brain slices.     

Rapid differentiation of NT2 cells in Sertoli-NT2 cell tissue constructs grown in the rotating wall bioreactor

Cell replacement therapy is of great interest as a long-term treatment of neurodegenerative diseases such as Parkinson's disease (PD). We have previously shown that Sertoli cells (SC) provide neurotrophic support to transplants of dopaminergic fetal neurons and NT2N neurons, derived from the human clonal precursors cell line NTera2/D1 (NT2), which differentiate into dopaminergic NT2N neurons when exposed to retinoic acid. We have created SC-NT2 cell tissue constructs cultured in the high aspect ratio vessel (HARV) rotating wall bioreactor. Sertoli cells, NT2, and SC plus NT2 cells combined in starting ratios of 1:1, 1:2, 1:4 and 1:8 were cultured in the HARV in DMEM with 10% fetal bovine serum and 1% growth factor reduced Matrigel for 3 days, without retinoic acid. Conventional, non-HARV, cultures grown in the same culture medium were used as controls. The presence of tyrosine hydroxylase (TH) was assessed in all culture conditions. Sertoli-neuron-aggregated-cell (SNAC) tissue constructs grown at starting ratios of 1:1 to 1:4 contained a significant amount of TH after 3 days of culture in the HARV. No TH was detected in SC HARV cultures, or SC, NT2 or SC-NT2 conventional co-cultures. Quantitative stereology of immunolabled 1:4 SNAC revealed that approximately 9% of NT2 cells differentiate into TH-positive (TH+) NT2N neurons after 3 days of culture in the HARV, without retinoic acid. SNAC tissue constructs also released dopamine (DA) when stimulated with KCl, suggesting that TH-positive NT2N neurons in the SNAC adopted a functional dopaminergic phenotype. SNAC tissue constructs may be an important source of dopaminergic neurons for neuronal transplantation.     

Calretinin and calbindin-D28k in dopaminergic neurons of the rat midbrain: a triple-labeling immunohistochemical study

We used triple-labeling immunohistochemistry in rat midbrain sections to identify dopaminergic neurons that contain either one or both of the calcium-binding proteins, calretinin (CR) and calbindin-D28k (CB). Midbrain dopaminergic neurons were immunohistochemically labeled for tyrosine hydroxylase (TH), CR, and CB. In the substantia nigra pars compacta (SNC), TH+/CR+/CB+ cells were clustered in two regions: the dorsal tier of the rostral SNC and the medial part of the intermediate SNC. The ventral tier of the rostral SNC mainly comprised both TH+/CR+/CB- and TH+/CR-/CB- cells. The lateral part of the intermediate SNC and the caudal SNC primarily consisted of TH+/CR-/CB- cells. Throughout the extent of the SNC, approximately half of the TH+ neurons were stained for neither CR nor CB, while the remaining TH+ populations were labeled for CR and/or CB. Throughout the ventral tegmental area, TH+/CR+/CB+ cells, TH+/CR+/CB- cells, TH+/CR-/CB+ cells, and TH+/CR-/CB- cells were found generally scattered, though the TH+/CR-/CB- cells were dominant in number. In the substantia nigra pars lateralis, interfascicular nucleus, and caudal linear nucleus, more than half of the TH+ cells were stained for both CR and CB. In the retrorubral field, two-thirds of the TH+ neurons contained neither protein. The present findings suggest that the SNC can be divided into subcompartments based on the distribution of dopaminergic neurons that contain calcium-binding proteins. Furthermore, because CR and CB likely contribute to calcium homeostasis by buffering intracellular calcium concentrations, midbrain dopaminergic neurons containing one or both of these calcium-binding proteins may have a higher calcium-buffering capacity than those lacking the two proteins.     

Mesencephalic dopaminergic neurons in primary cultures express functional neurotensin receptors.

The cellular distribution and functional aspects of neurotensin (NT) binding sites in rat mesencephalic cells in primary culture were investigated by an original approach combining anatomical and biochemical studies. Using a double-labeling protocol combining 125I-NT receptor radioautography and tyrosine hydroxylase (TH) immunocytochemistry, we obtained the first direct visualization of NT binding sites on TH-immunoreactive neurons. Eighty percent of the TH neurons were endowed with NT binding sites, which can be observed on both cell bodies and processes. TH-immunoreactive neurons were characterized as dopaminergic neurons by their ability to take up dopamine in a benztropine- and nomifensine-sensitive manner. In the mesencephalic cultures, NT increased potassium-evoked release of tritiated dopamine, and the relative potencies of various NT-related peptides to increase dopamine release were in good agreement with their abilities to bind to NT sites. These results show for the first time that cultured rat mesencephalic dopaminergic cells express functional NT receptors. Finally, the specificity and distribution of NT receptors on dopaminergic neurons in primary culture are quite similar to what was observed in the adult rat brain using pharmacological and radioautographic approaches. These data indicate that NT can influence the activity of dopaminergic neurons at very early stages of the rat brain development.     

碱性成纤维细胞生长因子预诱导对骨髓基质干细胞向多巴胺能神经元分化的影响

目的探讨碱性成纤维细胞生长因子（bFGF）预诱导对骨髓基质干细胞（MSCs）向多巴胺（DA）能神经元分化的影响。方法取雄性Wistar大鼠股骨和胫骨骨髓,进行MSCs的体外培养、传代扩增及纯化。bFGF预诱导24h后,依据加入的神经营养因子不同分为单唾液酸四己糖神经节苷脂（GMl）组、胶质源性神经营养因子（GDNF）组和GDNF＋GMl组,以及对照组。倒置显微镜下观察细胞形态变化,分别在预诱导第3d、7d进行神经元特异性烯醇化酶（NSE）、神经胶质酸性蛋白（GFAP）、酪氨酸羟化酶（TH）免疫细胞化学检测。计数NSE和TH阳性细胞数,并计算阳性细胞百分比。结果对照组见少量NSE阳性细胞。实验组于诱导第3d、7d见较多数量的NSE、TH阳性细胞,GFAP阴性。bFGF预诱导各组中GDNF＋GMl组NSE、TH阳性细胞率最高,GDNF组次之,GMl组最低,组间比较差异有统计学意义（均P〈0．01）。结论bF—GF预诱导不仅可明显促进GDNF、GMl诱导MSCs向神经元样细胞分化,表达神经元细胞标志物——NSE;还可促进MSCs向DA能神经元分化,表达DA能神经元标志物——TH。     

神经营养因子对体外培养中脑多巴胺能神经元存活和分化的影响

目的观察不同神经营养因子对体外培养中脑多巴胺能神经元(DN)存活和分化的作用。方法选取14d孕鼠,无菌条件下取出胎鼠,采用酶消化法培养中脑DN神经元,在培养过程中,分别加入不同浓度的胶质细胞源性神经营养因子(GDNF)、神经营养因子3(NT3)、脑源性神经营养因子(BDNF)和神经生长因子(NGF),通过细胞形态学和免疫荧光方法进行细胞纯度鉴定,观察不同作用条件下TH阳性细胞率确定细胞存活。结果以10~60ng/L的GDNF或BDNF持续培养10d,中脑多巴胺能神经元的存活率明显高于NGF和NT3作用组,浓度为20ng/m l的GDNF作用最强,能够维持60%的DN神经元存活。此外BDNF和GDNF能够增加DN神经元的数目,但未发现明显的剂量依赖效应,当GDNF与BDNF联合应用时,未见DN神经元的保护作用增强。结论GDNF和BDNF对原代培养的多巴胺能神经元存活具有较强的促进作用,并能诱导神经前体细胞分化为DN神经元。     

Toxicity of Dieldrin for Dopaminergic Neurons in Mesencephalic Cultures

Dieldrin can be retained for decades in lipid-rich tissue and has been measured in some postmortem PD brains. Dieldrin has been reported to deplete brain monoamines in several species and has been shown to inhibit mitochondrial respiration. To further investigate the possibility that it may be involved in the pathogenesis of parkinsonism, its toxicity for dopaminergic (DA) neurons was assessed in a mesencephalic cell culture model. Primary neuronal cultures of mesencephalic neurons were prepared from fetal rats or fetal mice, grown for 1 week and incubated with Dieldrin (0.01–100 μM) for 24 or 48 h. Toxicity for DA neurons was determined by measuring density of surviving tyrosine hydroxylase immunoreactive (TH-ir) cells. Toxicity for gamma-aminobutyric acid (GABA)-ergic neurons was determined by measuring survival of glutamate decarboxylase (GAD)-ir neurons. General, nonselective cytotoxicity was determined by counting cells visualized by phase contrast microscopy or by DAPI-stained cells with fluorescence microscopy. Dieldrin exposure for 24 h resulted in a dose-dependent decrease in survival of TH-IR cells (DA neurons) with a 50% decrease (EC₅₀) produced by 12 μM in rat mesencephalic cultures. Dieldrin also produced a dose- and time-dependent decrease in mouse DA-ergic and GABA-ergic neurons in mouse mesencephalic cultures. GABA-ergic neurons were less sensitive to the toxin compared to DA-ergic neurons. Cellular uptake of³H-DA was also affected by lower concentrations of Dieldrin (EC₅₀ = 7.98 μM) than uptake of³H-GABA (EC₅₀ = 43 μM). Thus, Dieldrin appears to be a relatively selective DA-ergic neurotoxin in mesencephalic cultures. Dieldrin, which may be ubiquitous in the environment, is proposed as an agent which can initiate and promote dopaminergic neurodegeneration in susceptible individuals.     

17β-雌二醇对6-OH多巴胺损伤的多巴胺能神经细胞的影响

目的 研究17β-雌二醇(17β-E2)对6-羟基多巴胺(6-OHDA)所致的黑质多巴胺(DA)能神经细胞损伤的影响,探讨17β-E2是否具有神经保护作用.方法 新生2～3 d的雄性SD大鼠行常规中脑脑片培养,6-OHDA(200 μmol/L)诱导损伤后分为实验对照组和17β-E2组,实验对照组继续常规培养,17β-E2组又分为1.0× 10-8 mol/L、1.0×10-9 mol/L和1.0×1 0-10 mol/L 3个亚组,分别按上述浓度添加17β-E2进行干预.Western blotting方法 检测每组细胞干预15min、30 min、60 min后酪氨酸羟化酶(TH)的表达.结果 1.0×10-9 mol/L 173-E2组TH的表达最高,与实验对照组比较差异有统计学意义(P＜0.05);增加或减少17β-E2的浓度均不能上调TH的表达;3个不同时间点中,17β-E2作用15min时,TH的表达最高,延长作用时间并不能增加TH的表达量.结论 适当浓度(1.0× 10-9 mol/L)17β-E2作用一定时间(15 min)可以对受损的DA能神经细胞起到保护作用.     

The glutamate antagonist, MK-801, does not prevent dopaminergic cell death induced by the 1-methyl-4-phenylpyridinium ion (MPP) in rat dissociated mesencephalic cultures

The neuroprotective effects of MK-801, a non-competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor/channel, were assessed in a culture model which reproduces in vitro the selective degeneration of mesencephalic dopaminergic neurons seen in parkinsonian brains. Dissociated mesencephalic cells derived from rat embryonic brains were subjected for 24 h to intoxication by the 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPP+ at 3 and 10 microM produced selective and dose-dependent damages to dopaminergic neurons as quantified by the loss of the number of TH immunoreactive cells and the loss of [3H]DA uptake whereas other cell types remained unaffected. MK-801 at 3 and 10 microM failed to rescue degenerating dopaminergic neurons in presence of MPP+. At 50 microM, i.e. the highest concentration that is not toxic by itself in this culture system, MK-801 was also found ineffective. Furthermore, degree of dopaminergic cell damage was not reduced when repeated additions of the glutamate antagonist (10 microM/6 h for 24 h) were performed during exposure to MPP+ or when mesencephalic cultures were left after intoxication for up to 2 days in a culture medium still supplemented with MK-801 but free of toxin. In accordance with these results, MK-801 did not affect significantly the uptake of [3H]DA in control cultures, thereby suggesting that this compound cannot prevent intracellular accumulation of MPP+ within dopaminergic neurons. At higher concentrations of MPP+ (100 microM) tested, toxic effects were seen toward dopaminergic neurons and non-dopaminergic cells as quantified by Trypan blue dye accumulation and loss of [3H]GABA uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

栀子甙在炎症诱导的多巴胺能神经元损伤中的作用及机制

目的 研究栀子甙在脂多糖(LPS)介导的星形胶质细胞(AC)过度激活时对多巴胺能神经元保护作用的影响及可能机制.方法 建立高纯度多巴胺能神经元培养体系、多巴胺能神经元和AC混合培养体系并分别经栀子甙预处理后再予以LPS作用24 h,同时设立对照组,观察多巴胺能神经元的生存率,TH mRNA的表达及培养基中TNF-α、NO、IL-6、基质金属蛋白酶-9(MMP-9)和胶质源性神经生长因子(GDNF)含量的变化.结果 AC促进多巴胺能神经元的存活.栀子甙不能增加高纯度多巴胺能神经元培养体系中多巴胺能神经元的存活率,却可剂量依赖地增加混合培养体系中多巴胺能神经元存活率,同对照组相比,40 mg/L组增加细胞存活率从203.0%±17.4%升高到256.7%±15.2%(F=17.22,P=0.001).LPS作用24 h后混合培养体系中TNF-α、NO、GDNF含量并无显著变化,IL-6、MMP-9含量显著上升,栀子甙可明显下调这种反应.同对照组相比,40 mg/L组IL-6含量下降到原来的67.2%±6.4%(F=12.89,P=0.001),MMP-9下降到原来的77.3%±9.8%(F=8.27,P=0.001).结论 LPS通过过度激活AC而增加炎性因子的分泌,从而削弱其对多巴胺能神经元的保护作用,栀子甙通过抑制AC分泌炎性介质,不增加GDNF的分泌,上调AC对多巴胺能神经元的保护作用.     

Dopaminergic neurotoxins require excitotoxic stimulation in organotypic cultures

We have investigated the properties of the dopaminergic neurotoxins 6-hydroxydopamine, 1-methyl-4-phenylpyridinium and rotenone using an organotypic culture that included slices of substantia nigra, striatum and cortex maintained for about 20 days in vitro. At this age, the organotypic culture contains dopaminergic neurons, visualized using tyrosine hydroxylase (TH) immunohistochemistry, that project into the striatal slice and extend up to 1 mm into the cortical slice. Using TH immunohistochemistry to assess survival of dopaminergic neurons, we found that the three dopaminergic toxins alone were not selectively neurotoxic. However, the addition of a low concentration of N-methyl-d-aspartate together with each individual toxin resulted in profound injury to the dopaminergic neurons, reflected by the loss of cell bodies and the fragmentation of processes. The combined toxicity was completely blocked by MK801. To assess the specificity of the injury, we measured the diameter of cell nuclei in the organotypic culture stained with Hoechst 33342 because the nucleus shrinks when neurons are injured. These measurements showed that the combined toxin treatment selectively injured only the TH immunoreactive cells. Thus, in a model culture system where dopaminergic neurons innervate appropriate targets, excitotoxicity appears to be essential for the manifestation of the toxic actions of 6-hydroxydopamine, 1-methyl-4-phenylpyridinium and rotenone.     

A muscle-derived factor(s) induces expression of a catecholamine phenotype in neurons of cultured rat cerebral cortex

We sought to determine the source of the signal(s) that promotes expression of the catecholamine (CA) enzyme tyrosine hydroxylase (TH) in cultured neurons of embryonic rat cerebral cortex, a tissue which is not thought to contain CA cells in vivo. Cortical neurons were cultured with their non-neuronal constituents and 48 hr later immunostained for TH. Fibroblasts or glia had no effects, however, blood vessels increased the numbers of TH neurons nearly 4-fold. Coculture with either perinatal aorta, skeletal or cardiac muscle, clonal muscle cell lines 1440 (smooth) and L6 (skeletal), conditioned media from L6 cells, or a soluble extract of L6 cells increased the number of TH neurons up to 20-fold. The induction of TH by muscle extract was (1) dose dependent; (2) paralleled by a proportional increase in the steady-state levels of TH mRNA; (3) greatly reduced by the RNA synthesis inhibitor alpha-amanitin or the protein synthesis inhibitor cycloheximide; and (4) unassociated with change in the survival of neurons in culture. The response was not replicated by treatment with other established neurotrophic substances, including NGF, EGF, FGF, PDGF, neuroleukin, insulin, pyruvate, KCI, adenosine, or inosine. We conclude that muscle contains a potentially novel substance, muscle-derived differentiation factor (MDF) that promotes differentiation but not survival of neurons of cerebral cortex by de novo synthesis of TH mRNA and TH protein. Thus, neurons of the CNS, as in periphery, may undergo phenotypic interconversion in response to biologically derived molecules in their environment.     

Nurr1对小胶质细胞联合神经干细胞共培养促进神经干细胞向多巴胺神经元分化作用研究

目的探讨联合过表达核受体相关因子1(Nurr1)基因的小胶质细胞(MG)和神经干细胞(NSC)共培养对神经干细胞向多巴胺神经元分化的影响。方法原代培养SD大鼠神经干细胞和小胶质细胞,并过表达Nurr1基因。CCK-8法检测Nurr1过表达对神经干细胞以及小胶质细胞活率的影响。Transwell系统共培养神经干细胞和小胶质细胞,实验分为NSC组、NSC+MG组和N(NSC+MG)组。ELISA检测共培养后第3天、第6天和第9天各组脑源性神经营养因子(BDNF)、血小板源性神经营养因子(PDNF)和胶质细胞源性神经营养因子(GDNF)表达变化;RT-PCR和Western Blot检测各组第9天酪氨酸羟化酶(TH)、多巴胺转运蛋白(DAT)DAT和Nurr1的表达变化;细胞免疫荧光鉴定神经干细胞的分化,并对TH和DAT阳性细胞计数,计算各组神经干细胞向多巴胺神经元的分化效率。结果原代培养小胶质细胞以及神经干细胞并成功过表达Nurr1基因。CCK-8法检测结果表明,Nurr1过表达对神经干细胞以及小胶质细胞活率无明显影响。ELISA检测结果表明,N(NSC+MG)组在不同时间点神经营养因子(BDNF、PDNF和GDNF)表达量明显高于其他各组(P0.05)。RT-PCR和Westen Blot检测结果表明,N(NSC+MG)组TH、DAT和Nurr1的表达水平明显高于其他各组(P0.05)。细胞免疫荧光鉴定结果表明,N(NSC+MG)组TH阳性细胞率明显高于其他各组(P0.05)。结论Nurr1基因可促进神经干细胞和小胶质细胞共培养系统神经营养因子的分泌。过表达Nurr1基因的神经干细胞和小胶质细胞共培养可促进神经干细胞向多巴胺神经元的分化。     

Induction of Dopaminergic Neurotransmitter Phenotype in Rat Embryonic -Cerebrocortex by the Synergistic Action of Neurotrophins and Dopamine

Neurotrophins, including nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/51 (NT-4/5), have been shown to enhance survival and differentiation of a variety of central neuronal populations, such as those with the dopaminergic, cholinergic, GABAergic phenotype during development. In this paper we present evidence that BDNF, NT-3 and NT-4/5 acting synergistically with dopamine (DA) can artificially induce the full dopaminergic phenotype in rat fetal cerebral cortex which normally has very few dopaminergic neurons in adulthood. Thus, BDNF/DA, NT-3/DA, NT-4/DA elicited a great increase in the number of tyrosine hydroxylase (TH)-immunoreactive cells, which was up to 57% of total neuronal population in cultures of fetal rat cortical cells. This stimulatory effect was not dependent on glial proliferation, or on addition of serum to the culture. Pharmacological studies showed that dopamine receptors D, and D2 were involved in this effect. The TH’cortical cells possessed other biochemical phenotypic features of dopaminergic neurons. Thus, high-affinity DA uptake was elevated in cortical cultures treated with neurotrophin/DA. Also DA and 3,4–dihydroxyphenlacetic acid production was detected (5.42 ± 1.24 and 13.72 ? 2.84 pmol/dish respectively, zero in controls). This shows the presence of functionally active TH, aromatic acid decarboxylase and monoamine oxidase. Neurotrophins/DA had no effect on noradrenergic phenotype expression by cortical fetal cells. Taken together, these results support the long-standing view that development of the central nervous system is determined not only by intrinsic genetic programmes, but also involves environmental influences such as the action of growth factors and extracellular neurotransmitters. In this case we report the effect of specific DA phenotype-inducing agents.     

Tyrosine hydroxylase expression is unstable in a human immortalized mesencephalic cell line--studies in vitro and after intracerebral grafting in vivo

We have studied the stability of the dopaminergic phenotype in a conditionally immortalized human mesencephalic cell line, MESC2.10. Even though MESC2.10 cells exhibit features of dopaminergic neurons in vitro, none of the cells expressed tyrosine hydroxylase (TH) after transplantation into a rat model of Parkinson's disease. We examined whether this is caused by cell death or loss of transmitter phenotype. Cells were cultured in differentiation medium, then harvested and replated into the same medium where they continued to express TH, whereas replated cells fed medium lacking differentiation factors (dibutyryl cAMP and glial cell line-derived neurotrophic factor) did not. Interestingly, cultures grown in the absence of differentiation factors could regain TH expression once exposed to differentiation medium. Our data suggest that TH expression in vitro is inducible in neurons derived from the MESC2.10 cell line and that the dopaminergic phenotype of these cells in vivo might be unstable.     

小胶质细胞活化刺激骨髓间充质干细胞释放胶质细胞源性神经营养因子保护多巴胺能神经元的实验☆

背景：目前尚未见骨髓间充质干细胞对活化的小胶质细胞特异性反应的报道，且有关骨髓间充质干细胞在特定微环境下如何维持多巴胺能神经元的存活也缺乏相应的实验证据。 目的：观察骨髓间充质干细胞在活化的小胶质细胞刺激下保护多巴胺能神经元存活的作用。 方法：取Wistar大鼠，贴壁法分离培养骨髓间充质干细胞，体外培养并活化小胶质细胞，酶消化法培养中脑多巴胺能神经元。实验分为5组：骨髓间充质干细胞组；小胶质细胞组；脂多糖+小胶质细胞组；骨髓间充质干细胞+脂多糖+小胶质细胞组；分别取各实验组的培养上清，对中脑多巴胺神经元进行培养。单纯多巴胺能神经元组采用体积分数为10%胎牛血清+DMEM/F12进行培养。采用免疫荧光技术检测不同微环境对多巴胺能神经元存活的影响及不同微环境对骨髓间充质干细胞释放胶质细胞源性神经营养因子的影响。 结果与结论：含有骨髓间充质干细胞的实验组胶质细胞源性神经营养因子的释放量均较相应的对照组高。酪氨酸羟化酶免疫荧光染色结果发现，单纯多巴胺能神经元组神经元的存活率为15%；小胶质细胞组多巴胺能神经元的存活率为10%；骨髓间充质干细胞组多巴胺能神经元的存活率为35%；脂多糖+小胶质细胞组多巴胺能神经元的存活率为5%；而骨髓间充质干细胞+脂多糖+小胶质细胞组多巴胺能神经元的存活率达到了28%，高于除骨髓间充质干细胞组外的其他各组(P < 0.05)。此外体外培养多巴胺能神经元存活率随培养时间延长下降，但含有骨髓间充质干细胞实验组的多巴胺能神经元存活率明显高于相应对照组。提示小胶质细胞活化刺激骨髓间充质干细胞上调胶质细胞源性神经营养因子表达，使得多巴胺能神经元免受毒素的损害，抑制了多巴胺能神经元的延迟性死亡。     

GAPDH knockdown rescues mesencephalic dopaminergic neurons from MPP+ -induced apoptosis.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) has a number of diverse functions apart from glycolytic function. We explored the possible involvement of GAPDH in 1-methyl-4-phenylpyridinium (MPP+)-induced death of mesencephalic dopaminergic neurons (MDNs) in culture. MPP+ (10 and 20 microM, 24 h) exposure selectively decreased the survival of tyrosine hydroxylase positive (TH+) MDNs, which manifested apoptotic features including shrinkage of the cell body, chromatin condensation and nuclear fragmentation. Two types of GAPDH antisense oligonucleotides almost completely rescued MDNs from MPP+ toxicity. GAPDH was strongly expressed in apoptotic TH+ neurons, and MPP+ exposure significantly increased the percentage of TH+ neurons in which GAPDH is over-expressed. Confocal microscopic analysis demonstrated the nuclear accumulation of GAPDH in neurons undergoing MPP+-induced apoptosis. These results suggest that MPP+ causes apoptosis of MDNs, concomitant with the over-expression and nuclear accumulation of GAPDH.     

Expression of tyrosine hydroxylase in neurons of cultured cerebral cortex: evidence for phenotypic plasticity in neurons of the CNS

In vivo, neurons of the cerebral cortex of rat embryos did not stain with antibodies to the catecholamine (CA) biosynthetic enzyme tyrosine hydroxylase (TH) even when examined using a highly sensitive technique for radioimmunocytochemistry. However, when embryonic day (E) 13 cortex was grown 1 d in culture, several thousand cells expressed immunoreactive and catalytically active TH. All TH cells simultaneously labeled with the neuronal enzyme, neuronal specific enolase, indicating that the TH was exclusively localized in neurons. Moreover, all TH neurons were postmitotic since they did not incorporate 3H-thymidine. With time in culture, the number of TH cells selectively declined from nearly 3000 cells at 2 d to several cells at 14 d. Similarly, the number of neurons competent to express TH in culture declined with advancing age of the donor embryo. Thus, by E18, very few cortical neurons had the capacity to express TH. We conclude that during a critical period of development, postmitotic cerebral cortical neurons can express catecholamine traits in vitro but not in vivo. Thus, the neurotransmitter phenotype of certain classes of central neurons is not fixed but can be influenced by epigenetic factors found in their environment, thereby providing evidence of phenotypic plasticity in the central nervous system (CNS).