脑溢安对脑出血大鼠脑内GDNF蛋白及mRNA表达的影响

目的观察脑出血模型大鼠脑内胶质细胞源性神经营养因子（GDNF）蛋白和mRNA的分布及中药脑溢安对其影响。方法通过微量注射器向苍白球注入Ⅶ型胶原酶0．4u建立脑出血模型。用免疫组织化学方法和原住杂交法分别观察脑出血后2h，6h，1d，4d，7d，14d共6个时间点GDNF蛋白及mRNA的表达变化，以阳性细胞计数作为观察指标。结果脑出血后2hGDNF蛋白主要表达于血肿周围的星型胶质细胞，6h表达开始增高，1d达高峰，以后逐渐降低，GDNF蛋白到第7天基本恢复到正常水平，14d后GDNF阳性细胞消失；在1d，4d两个时间点上，两组阳性细胞计数比较差异有显著性（P〈0．05）；而GDNF mRNA主要表达于神经元，1d后达高峰，7d后两者的表达水平继续下降但仍高于正常，14d后降至基础水平，在6h，1d，4d三个时间点上，两组阳性细胞计数比较差异有显著性（P〈0．01）。结论脑溢安可促进脑出血后GDNF蛋白及mRNA的表达。     

Recombinant adeno-associated virus vector expressing glial cell line-derived neurotrophic factor reduces ischemia-induced damage

To explore the potential of using the recombinant adeno-associated viral (rAAV) vector, expressing glial cell line-derived neurotrophic factor (GDNF) as the gene therapy for stroke, we injected rAAV vectors expressing GDNF (rAAV-GDNF) into the cortex of rats which had been experiencing transient bilateral common carotid artery ligation and right middle cerebral artery ligation for 90 min. GDNF levels in cortical tissues of rAAV-GDNF-injected animals were significantly higher than in the control animals injected with rAAV-expressing lacZ (rAAV-lacZ), indicating that rAAV can deliver and express the GDNF gene in cortical tissues. Triphenyltetrazolium chloride tissue stain analysis revealed that the rAAV-delivered GDNF gene could rescue the brain tissues from ischemia-induced injury. Cortical tissues which received rAAV-GDNF injections had both significantly smaller total volumes of infarction and smaller areas of infarction on each brain slice than those which were injected with rAAV-lacZ. An in situ labeling analysis demonstrated significantly less apoptotic cells in cortical tissues rescued by rAAV-GDNF, indicating prevention of apoptosis as the mechanism of cortical cell protection. Moreover, immunohistochemistry staining of Neu-N indicated that the rescued brain tissues contained the same number of Neu-N-positive neuronal cells as contralateral undamaged brain tissues. This provides strong evidence that cortical neuronal cells can be rescued by GDNF gene therapy. Indeed, these findings show that the rAAV is a potential delivery vector of GDNF gene for the therapy of stroke.     

维甲酸和联合应用神经营养因子对新生大鼠神经干细胞分化的影响

目的研究全反式维甲酸（ATRA）及联合应用神经营养因子（BDNF，GDNF）对体外培养的神经干细胞（NSCs）分化的影响。方法取新生SD大鼠的前脑室下带（SVZ）区，按NSCs的常规培养方法分离、培养。用免疫细胞化学法鉴定巢蛋白（nestin）、微管相关蛋白-2（MAP-2）、胶质原纤维酸性蛋白（GFAP）的表达，以此来观察ATRA、BDNF、GDNF单独或联合应用对次代神经球细胞分化的作用。结果原代及次代神经球均显示nestin阳性，并可分化为MAP-2阳性神经元样细胞及GFAP阳性胶质细胞样细胞。1μmol／LATRA可促进NSCs分化为MAP-2阳性细胞的比例达（29．14±5．00）％，显著高于对照组的（7．19±1．21）％，差异有高度统计学意义（P〈0．001）。ATRA联合应用10ng／ml的BDNF或GDNF，与单独使用ATRA比较，并不显著提高NSCs分化为MAP-2阳性细胞的比例。结论ATRA可促进神经干细胞向神经元方向分化，ATRA联合应用BDNF或GDNF无明显的协同作用。     

人GDNF基因的克隆

目的：克隆可表达人胶质细胞源性神经营养因子的基因。方法：从脑胶质细胞瘤患者术后的脑瘤组织中提取总ＲＮＡ，以ＲＴＰＣＲ方法扩增出了一段约５５８ｂｐ的ｃＤＮＡ片段，将这一片段克隆于ｐＵＣ１８载体中，进行全序列分析。结果：证实该研究所克隆的ｃＤＮＡ是编码正确的人ＧＤＮＦ基因。与发表于ＧｅｎｅＢａｎｋ上的序列相比，发现该研究克隆的基因有一段７８ｂｐ的核苷酸序列缺失，但不影响密码子的阅读框。结论：克隆到了编码正确的人ＧＤＮＦ的ｃＤＮＡ全序列，对帕金森病基因治疗的基础研究及临床应用具有重要意义。     

Development of pontine noradrenergic A5 neurons requires brain-derived neurotrophic factor

Pontine noradrenergic A5 neurons play a pivotal role in maturation and regulation of the brainstem respiratory rhythm-generating network. Analysis of newborn brain-derived neurotrophic factor (BDNF)-null mice revealed a marked loss of tyrosine hydroxylase-positive A5 neurons compared to wildtype controls that was rescued by null mutation of the proapoptotic gene Bax. In cultures of the A5 region from E12.5 rat embryos, BDNF significantly increased the number and branching of tyrosine hydroxylase-positive neurons. Immunoneutralization of endogenous glial cell line-derived neurotrophic factor partially inhibited the BDNF-dependent increase in the number of tyrosine hydroxylase-positive cells without affecting neurite number. The A5 nucleus is the first brainstem cell group identified at which BDNF is required in vivo for development of neurons critical for cardiorespiratory control.     

Intrathecal injection of GDNF and BDNF induces immediate early gene expression in rat spinal dorsal horn

Glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) are potent trophic factors for dorsal root ganglion cells. In addition, these factors are produced in subsets of dorsal root ganglion cells and transported anterogradely to their terminals in the superficial dorsal horn of the spinal cord, where they constitute the only source of GDNF and BDNF. We investigated the effect of 10 mug GDNF and BDNF injected by lumbar puncture on the expression of the immediate early gene (IEG) products c-Fos, c-Jun, and Krox-24 in the adult rat dorsal horn. In the dorsal horn of S1 spinal segments, GDNF and BDNF induced a strong increase in IEG expression, which was most pronounced in laminae I and II (2.9- to 4.5-fold). More distal from the injection site, in the dorsal horn of L1/L2 spinal segments, the increase in IEG expression was less pronounced, suggesting a concentration-dependent effect. In order to explain the effects of intrathecally injected GDNF, we investigated whether lumbo-sacral dorsal horn neurons expressed RET protein, the signal-transducing element of the receptor complex for GDNF. It was found that several of these neurons contained RET immunoreactivity and that some of the RET-labeled neurons had the appearance of nociceptive-specific cells, confirming their presumed role in pain transmission. Additionally, using double-labeling immunofluorescence combined with confocal microscopy, it was found that after intrathecal GDNF injection 35% of c-Fos-labeled cells were also labeled for RET. These results demonstrate that intrathecally administered GDNF and BDNF induce IEG expression in dorsal horn neurons in the adult rat, supposedly by way of their cognate receptors, which are present on these neurons. We further suggest that the endogenous release of GDNF and BDNF, triggered by nociceptive stimuli, is involved in the induction of changes in spinal nociceptive transmission as in various pain states.     

Neurotrophin and GDNF family ligands promote survival and alter excitotoxic vulnerability of neurons derived from murine embryonic stem cells

Embryonic stem (ES) cells are genetically manipulable pluripotential cells that can be differentiated in vitro into neurons, oligodendrocytes, and astrocytes. Given their potential utility as a source of replacement cells for the injured nervous system and the likelihood that transplantation interventions might include co-application of growth factors, we examined the effects of neurotrophin and GDNF family ligands on the survival and excitotoxic vulnerability of ES cell-derived neurons (ES neurons) grown in vitro. ES cells were differentiated down a neural lineage in vitro using the 4-/4+ protocol (Bain et al., Dev Biol 168:342-57, 1995). RT-PCR demonstrated expression of receptors for neurotrophins and GDNF family ligands in ES neural lineage cells. Neuronal expression of GFRalpha1, GFRalpha2, and ret was confirmed by immunocytochemistry. Exposure to 30-100 ng/ml GDNF or neurturin (NRTN) resulted in activation of ret. Addition of NT-3 and GDNF did not increase cell division but did increase the number of neurons in the cultures 7 days after plating. Pretreatment with NT-3 enhanced the vulnerability of ES neurons to NMDA-induced death (100 microM NMDA for 10 min) and enhanced the NMDA-induced increase in neuronal [Ca2+]i, but did not alter expression of NMDA receptor subunits NR2A or NR2B. In contrast, pretreatment with GDNF reduced the vulnerability of ES neurons to NMDA-induced death while modestly enhancing the NMDA-induced increase in neuronal [Ca2+]i. These findings demonstrate that the response of ES-derived neurons to neurotrophins and GDNF family ligands is largely similar to that of other cultured central neurons.     

Galectin-1 in regenerating motoneurons

The exogenous application of recombinant galectin-1 has recently been shown to promote the rate of peripheral nerve regeneration. Endogenous neuronal galectin-1 expression has recently been demonstrated to increase after axotomy. Here we demonstrate a significant increase in the endogenous neuronal expression of galectin-1 mRNA in facial motoneurons after either a nerve resection or crush injury in mice. This increase in galectin-1 expression was due in part to the loss of target-derived factor(s) as indicated by both the return of galectin-1 expression to control levels following target re-innervation and the increase in galectin-1 expression after blockade of axonal transport by an interneuronal colchicine injection. Furthermore, interneuronal injections of glial-derived neurotrophic factor into the uninjured nerve also increased galectin-1 mRNA expression within facial motoneurons suggesting that positive signals may also be involved in the regulation of galectin-1 expression. Galectin-1 null mutant mice showed an attenuated rate of functional recovery of whisking movement after a facial nerve crush.     

Overexpression of glial cell line-derived neurotrophic factor in the CNS rescues motoneurons from programmed cell death and promotes their long-term survival following axotomy

To study the role of one of the most potent motoneuron (MN) survival factors, glial cell line-derived neurotrophic factor (GDNF) derived from the CNS, we generated transgenic animals overexpressing GDNF under the control of an astrocyte-specific GFAP promoter. In situ hybridization revealed that GDNF was expressed at high levels in astrocytes throughout the brain and spinal cord. We analyzed the effects of CNS-derived GDNF on MN survival during the period of programmed cell death (PCD) and after nerve axotomy. In GFAP-GDNF mice at E15, E18, and P1, the survival of brachial MNs was increased on average by 30%, lumbar MNs by 20%, and thoracic MNs at P1 by 33%. GDNF also prevented MN PCD in several cranial motor nuclei. We demonstrated for the first time that the number of MNs in the mouse abducens nucleus was also increased by 40%, thus extending known MN populations that are responsive to GDNF. Next, we tested if GDNF could support complete and relatively long-term survival of MNs following neonatal facial nerve axotomy. We found that virtually all MNs (91%) in GFAP-GDNF mice survived for up to 18 weeks post-axotomy. This is the longest GDNF-mediated survival of neonatal MNs reported following axotomy. Most of surviving MNs were not atrophic, and MN-specific ChAT and neurofilament immunoreactivity (IR) were preserved. Furthermore, GDNF attenuated axotomy-induced astroglial activation. These data demonstrate that overexpression of GDNF in the CNS has very profound effects on MN survival both during the PCD period and after neuronal injury. GFAP-GDNF mice will be valuable to study the effects of CNS-derived GDNF in mouse models of MN degenerative diseases and axonal regeneration in vivo.