GDNF促进大鼠背根神经元的存活和突起生长

探讨胶质细胞源性神经营养因子(GDNF)对正常胚胎大鼠背根神经节(DRGn)的存活及突起生长的作用。本实验采用神经组织原代分离培养的方法建立体外胚胎大鼠背根神经节单细胞培养体系，从细胞形态学及应用MTT。法观察1μg／L、10μg／L、50μg／L和100μg／L GDNF对体外培养的正常感觉神经元生长的影响。结果表明：GDNF能明显促进体外培养的正常大鼠背根神经节感觉神经元的存活及突起生长。提示GDNF对正常大鼠胚胎发育期感觉神经元具有神经营养作用。     

胶质细胞源性神经营养因子体外对脊髓运动神经元的作用

目的 :观察不同浓度的胶质细胞源性神经营养因子 (GDNF) ,对大鼠胚胎脊髓运动神经元生长活性的作用。方法 :取大鼠胚胎脊髓腹侧组织体外分离 ,进行原代细胞培养 ,应用抗神经微丝单克隆抗体 (mAb)SMI32进行运动神经元的免疫细胞化学染色 ,从细胞形态学及应用MTT比色法 ,研究GDNF对大鼠脊髓运动神经元的影响。结果 :GDNF能明显促进体外培养的大鼠脊髓运动神经元存活及突起的生长 (P <0 .0 5 ) ,且具有剂量依赖的趋势。结论 :不同浓度的GDNF对体外培养的大鼠胚胎脊髓运动神经元 ,有不同程度的促生长作用     

神经再生素促大鼠背根神经节生长作用的研究

目的研究神经再生素（NRF）对体外培养新生大鼠背根神经节生长及NF—H表达的影响。方法体外大鼠背根神经节（DRG）培养;免疫荧光细胞化学、实时荧光定量PCR和Western blot等方法。结果免疫荧光细胞化学结果提示,NRF能促进背根神经节神经突起的生长,浓度为2．0mg／L时生长状况最佳;实时荧光定量PCR和Western blot结果提示,NRF能增加体外培养的背根神经节NF-H mRNA和蛋白的表达,在浓度为2．0mg／L时表达最高。结论NRF能促进体外培养背根神经节神经突起的生长和NF—H的表达,表明NRF对发育期感觉神经元具有神经营养作用。     

神经生长颗粒含药血清对大鼠背根神经节生长的影响

目的: 研究神经生长颗粒(NGG)含药血清对体外培养新生大鼠背根神经节生长及高分子量神经丝蛋白(NF-H)和生长相关蛋白43(GAP43)表达的影响.方法: 采用体外大鼠背根神经节(DRG)植块培养,通过免疫荧光细胞化学法,观察不同剂量的含药血清对DRG神经突起生长的影响;采用DRG单细胞分离培养,通过实时荧光定量PCR和免疫印迹法分别观察不同剂量的含药血清对DRG细胞NF-H和GAP43基因及蛋白表达的影响.结果: 免疫荧光细胞化学法提示NGG含药血清能促进DRG神经突起的生长;实时荧光定量PCR和免疫印迹法结果提示NGG含药血清能增加体外培养的DRG细胞NF-H、 GAP43 mRNA和蛋白的表达.结论: NGG含药血清能促进体外培养DRG神经突起的生长并促进NF-H和GAP43的表达,表明NGG对发育期感觉神经元具有一定的神经营养作用.     

细胞外基质对培养神经元细胞存活和轴突生长的影响

目的：探讨不同的细胞外基质对体外培养胚胎大鼠脊髓运动神经元和背根神经节感觉神经元生长的影响。方法：取大鼠胚胎脊髓腹侧组织和背根神经节体外分离培养，选取不同生长底物包括多聚赖氮酸(PLL)、Ⅰ型胶原(CoⅠ)、层粘连蛋白(LN)、PLL联合LN进行包被培养板，观察运动神经元和感觉神经元的体外生长状况。结果：PLL和LN联合包被时，神经元存活率高，细胞分散良好。CoⅠ包被时细胞聚集成团．突起粗长。结论：不同的细胞外基质影响神经元的生长方式，PLL联合LN包被是体外研究单一神经元胞体和突起的较好方法。     

GDNFR-〆在体外培养的大鼠脊髓和背根节神经元中分布的免疫组织化学研究

目的　观察胶质细胞源性神经营养因子受体 - α(GDNFR- α)在体外培养的大鼠脊髓和背根节神经元中的分布 ,探讨 GDNF对脊髓运动神经元和感觉神经元的作用。　方法　原代培养脊髓和背根节神经元 ,5 d后行抗 GDNFR- α多克隆抗体免疫组织化学 SP法染色。　结果　 GDNF免疫反应存在于体外培养的脊髓神经元、背根节神经元以及胶质细胞中。　结论　 GDNF可能对脊髓神经元、背根节神经元和胶质细胞的生理功能具有一定的调节作用     

GDNFR-α在体外培养的大鼠脊髓和背根节神经元中分布的免疫组织化学研究

目的观察胶质细胞源性神经营养因子受体-α(GDNFR-α)在体外培养的大鼠脊髓和背根节神经元中的分布,探讨GDNF对脊髓运动神经元和感觉神经元的作用. 方法原代培养脊髓和背根节神经元,5d后行抗GDNFR-α多克隆抗体免疫组织化学SP法染色. 结果 GDNF免疫反应存在于体外培养的脊髓神经元、背根节神经元以及胶质细胞中. 结论 GDNF可能对脊髓神经元、背根节神经元和胶质细胞的生理功能具有一定的调节作用.     

激活素A与NGF协同刺激鸡胚背根神经节神经突起生长

目的:探讨激活素A(Activin A)与神经生长因子(Nerve growth factor,NGF)共同刺激鸡胚背根神经节(DRG)神经突起生长作用。方法:采用8 d的鸡胚DRG原代培养法,通过Activin A与NGF联合刺激,观察DRG神经突起生长和DRG神经元存活情况。采用RT-PCR检测钙基因相关肽(CGRP)。结果:Activin A与NGF体外联合培养3 d时DRG神经突起生长比单纯NGF组更明显,DRG神经元存活数量也明显增加,Activin A与NGF联合作用可以明显促进CGRP mRNA表达。结论:Activin A对NGF诱导的鸡胚DRG神经突起生长和维持DRG神经元存活具有增强作用,提示二者的联合应用可能为治疗神经元损伤及变性疾病的应用提供了新的数据和实验依据。     

NGF、CNTF和GDNF对感觉和运动神经元的协同作用研究

为探讨神经生长因子(NGF)、睫状神经营养因子(CNTF)和胶质细胞源性神经营养因子(GDNF)对感觉和运动神经元的协同作用机制,本研究采用免疫组织化学技术对脊髓背根节感觉神经元和脊髓运动神经元经特异性烯醇化酶(NSE)和胆碱乙酰转移酶(ChAT)染色后,通过图像分析测量阳性神经元数量、胞体直径、突起数量及长度。结果表明:NGF能明显促进感觉神经元的存活,对突起发育有轻微作用,对胞体发育的作用不显著,对运动神经元的存活无明显作用;CNTF对感觉和运动神经元的胞体发育均有很强的作用,对感觉和运动神经元的存活有一定的作用;GDNF对感觉和运动神经元的突起发育和延伸作用最强,对运动神经元的存活有很强的促进作用,对胞体发育的作用不如CNTF显著。本研究结果提示:联合应用上述三种神经营养因子,可克服单一因子功能的局限,全面促进感觉和运动神经元的存活和生长。     

内源性GDNF、NT-4对针刺猫脊髓备用根背根节神经元的作用

为了解部分背根切除和针刺及内源性GDNF和NT-4对体外培养备用背根节(DRG)的作用,本研究对5只成年猫进行双侧备用根手术(切除双侧L1~L5和L7~S2DRG,其中L6DRG作为备用背根)。术后当日开始针刺一侧L6脊神经后肢分布区的两组穴位,即足三里和悬钟、伏兔和三阴交,每天一次,每次30min,连续针刺7d后无菌条件下取出双侧L6DRG进行体外培养,24h后全量换液,并将针刺侧的一部分培养孔的培养液分别用含有200ng/ml抗GDNF和NT-4抗体培养液替换,分别作为抗GDNF和NT-4抗体封闭组。7d后终止培养,于显微镜下用显微测微尺测量神经突起的长度;并用抗NSE抗体行免疫细胞化学ABC法染色进行神经元鉴定。结果显示:(1)免疫细胞化学染色可见体外培养的细胞95%以上为NSE阳性细胞,且为典型的体外培养的DRG神经元;(2)体外培养备用根组和抗GDNF抗体组神经突起的平均长度比针刺组的短(P<0.05);(3)而针刺组神经突起的平均长度与抗NT-4抗体组间无差异(P>0.05);两抗体组平均突起长度比备用根组的突起长(P<0.05)。本研究结果提示,针刺可促进体外培养DRG神经元突起的生长,进而可能与脊髓可塑性密切相关;内源性GDNF有促进DRG神经元突起生长的作用;而内源性NT-4在DRG神经元突起生长中发挥的作用却不明显。     

Glial cell line-derived neurotrophic factor (GDNF) enhances sympathetic neurite growth in rat hearts at early developmental stages

Molecular signaling of sympathetic innervation of myocardium is an unresolved issue. The purpose of this study was to investigate the effect of neurotrophic factors on sympathetic neurite growth towards cardiomyocytes. Cardiomyocytes (CMs) and sympathetic neurons (SNs) were isolated from neonatal rat hearts and superior cervical ganglia, and were co-cultured, either in a random or localized way. Neurite growth from SNs toward CMs was assessed by immunohistochemistry for neurofilament M and α-actinin in response to neurotrophic factors-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF) and a chemical repellent, semaphorin 3A. As a result, GDNF as well as NGF and BDNF stimulated neurite growth. GDNF enhanced neurite outgrowth even under the NGF-depleted culture condition, excluding an indirect effect of GDNF via NGF. Quantification of mRNA and protein by real-time PCR and immunohistochemistry at different developmental stages revealed that GDNF is abundantly expressed in the hearts of embryos and neonates, but not in adult hearts. GDNF plays an important role in inducing cardiac sympathetic innervation at the early developmental stages. A possible role in (re)innervation of injured or transplanted or cultured and transplanted myocardium may deserve investigation.     

Integrin alphav and NCAM mediate the effects of GDNF on DA neuron survival,outgrowth, DA turnover and motor activity in rats

Glial cell line-derived neurotrophic factor (GDNF) is a specific neurotrophic factor for midbrain dopamine (DA) neurons, but the mechanism underlying the neurotrophic action of GDNF is not well known. The cell adhesion molecules integrin and Neural cell adhesion molecule (NCAM) play important roles in neurite outgrowth and fasciculation. In the present study, we found that subchronic GDNF administration to the pars compacta of substantia nigra in rats increased the expression of integrin alphav and NCAM. Immunostaining results demonstrated the wide distribution of integrin alphav and NCAM in all mesencephalic neurons. The results also demonstrated the co-expression of TH with integrin alphav and NCAM in the same neurons of mesencephalic culture. Further, GDNF significantly increased integrin alphav expression in single TH-positive neurons. Function-blocking anti-integrin alphav and anti-NCAM antibodies antagonized the effects of GDNF on DA neuron survival, outgrowth, DA turnover, and locomotor activity in rats. These results demonstrate that integrin alphav and NCAM mediate the effects of GDNF on DA neuron survival and outgrowth during development and on DA turnover and motor function during adulthood.     

Glial cell line-derived neurotrophic factor (GDNF) is required for differentiation of pontine noradrenergic neurons and patterning of central respiratory output

Genetic mutations affecting signaling by glial cell line-derived neurotrophic factor (GDNF) perturb development of breathing in mice and are associated with congenital central hypoventilation syndrome in humans. However, the role of GDNF in development of brainstem neurons that control breathing is largely unknown. The present study demonstrates that genetic loss of GDNF decreases the number of tyrosine hydroxylase (TH) neurons in the pontine A5 noradrenergic cell group, a major source of inhibitory input to the medullary respiratory pattern generator. This phenotype is associated with a significant increase in the frequency of central respiratory output recorded from the fetal medulla-spinal cord in vitro. In dissociate cultures of the A5 region from rat embryos, GDNF increases TH cell number and neurite growth without affecting total neuronal survival or proliferation of TH neurons. These effects of GDNF are inhibited by function blocking antibodies against endogenous brain-derived neurotrophic factor (BDNF), indicating that GDNF requires BDNF as a cofactor to stimulate differentiation of A5 neurons. Our findings demonstrate that GDNF is required for development of pontine noradrenergic neurons in vivo and indicate that defects in the A5 cell group may contribute to the effects of genetic disruption of GDNF signaling on respiratory control.     

Spinal cord contains neurotrophic activity for spinal nerve sensory neurons. Late developmental appearance of a survival factor distinct from nerve growth factor

Several tissues of the developing chick embryo have been reported to contain neurotrophic activity which can sustain the survival of sensory neurons maintained in culture. In a previous study, however, we noted that such nerve growth promoting activity was exceptionally low, if not absent, from extracts of spinal cord from chick embryos of up to 16 days incubation. Since then the combined results from a number of tissue culture studies have suggested that the central nervous system may be the source of a neurotrophic growth factor essential during the late development of sensory neurons. We have therefore carried out an extended range study of the neurotrophic properties of avian spinal cord. Extracts of spinal cord tissue prepared from chicks at stages between the last wk of embryogenesis and 12 wks after hatching were tested for their ability to promote survival and neurite outgrowth from both explant and dissociated neuron-enriched cultures of dorsal root, trigeminal, nodose and paravertebral chain sympathetic ganglia from chick embryos between 8 and 16 days old. We conclude from our results that spinal cord is a potent source of neurotrophic activity for sensory neurons, although this activity appears relatively late in development of the spinal cord. The predominant ontogenic increase in spinal cord neurotrophic activity was seen to occur during the first week after hatching. Sensory neurons from both spinal and cranial nerve ganglia were sustained in culture by spinal cord extracts, whereas sympathetic neurons did not respond. Neurons from older sensory ganglia (12-16 day old embryos) were much more responsive than similar neurons from young embryos (8 day).(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the action of euxanthone, a plant-derived compound that stimulates neurite outgrowth

We have investigated the neurite growth-stimulating properties of euxanthone, a xanthone derivative isolated from the Chinese medicinal plant Polygala caudata. Euxanthone was shown to exert a marked stimulatory action on neurite outgrowth from chick embryo dorsal root ganglia explanted in collagen gels, in the absence of added neurotrophins. It was also shown to promote cell survival in explanted chick embryo ganglia, and to stimulate neurite outgrowth from isolated adult rat primary sensory neurons in vitro. The further finding that euxanthone stimulates neurite outgrowth from explants of chick embryo retina and ventral spinal cord suggests an action on signaling pathways downstream of neuronal receptors for specific neurotrophic factors. Consistent with this, euxanthone did not promote neurite outgrowth from non-transfected PC12 cells, or from PC12 cells transfected with TrkB or TrkC, under conditions in which these cells extended neurites in response to, respectively, the neurotrophins nerve growth factor, brain-derived neurotrophic factor and neurotrophin 3. Western blot analysis of euxanthone-stimulated dorsal root ganglion explants showed that expression of phospho-mitogen-activated protein (MAP) kinase was up-regulated after 1 h of euxanthone-treatment. Inhibition of the MAP kinase pathway using PD98059, a specific inhibitor of MAP kinase kinase, blocked all euxanthone-stimulated neurite outgrowth. However, analysis of phospho-Akt expression indicated that the phosphatidylinositol-3 kinase-Akt pathway, another major signaling pathway engaged by neurotrophins, is not significantly activated by euxanthone. These results suggest that euxanthone promotes neurite outgrowth by selectively activating the MAP kinase pathway.     

胶质细胞源性神经营养因子在成年大鼠三叉神经节及三叉神经核团中的分布

目的 观察胶质细胞源性神经营养因子(GDNF)在成年大鼠三叉神经节及三叉神经核团内的分布，探讨其对三叉神经感觉神经元及运动神经元的作用。方法 抗GDNF多克隆抗体免疫组织化学ABC法。结果 成年大鼠三叉神经运动核、三叉神经感觉核簇及三叉神经节中出现GDNF免疫反应阳性。结论 成年大鼠三叉神经运动核、三叉神经感觉核簇及三叉神经节中存在GDNF神经元。     

The IL-6/sIL-6R fusion protein hyper-IL-6 promotes neurite outgrowth and neuron survival in cultured enteric neurons.

The undisturbed development of the enteric nervous system depends on the supply of various neurotrophic factors during ontogenesis. Besides glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) take part in its development. CNTF and LIF belong to the interleukin-6 (IL-6) family of cytokines. The combination of IL-6 and the soluble IL-6 receptor accelerates peripheral nerve regeneration. In this study, we examined the effect of the fusion protein Hyper-IL-6, which consists of IL-6 and the soluble receptor sIL-6R, on neurite outgrowth and neuronal survival in vitro. Myenteric plexus of newborn rats was dissected and dissociated. Cells were grown in either serum-free chemically defined medium alone or medium supplemented with sIL-6R, IL-6, sIL-6+IL-6, Hyper-IL-6, CNTF, LIF, or GDNF. Average neurite outgrowth per neuron was highest in GDNF-treated and Hyper-IL-6-treated cultures. The number of neurite-bearing neurons was reduced in GDNF cultures compared with Hyper-IL-6-treated cells, so that the total neurite outgrowth was maximal after Hyper-IL-6 stimulation. Hyper-IL-6 furthermore stimulated neuronal survival and morphologic differentiation of the enteric glia.