A glial cell line-derived neurotrophic factor-secreting clone of the Schwann cell line SCTM41 enhances survival and fiber outgrowth from embryonic nigral neurons grafted to the striatum and to the lesioned substantia nigra.

We have developed a novel Schwann cell line, SCTM41, derived from postnatal sciatic nerve cultures and have stably transfected a clone with a rat glial cell line-derived neurotrophic factor (GDNF) construct. Coculture with this GDNF-secreting clone enhances in vitro survival and fiber growth of embryonic dopaminergic neurons. In the rat unilateral 6-OHDA lesion model of Parkinson's disease, we have therefore made cografts of these cells with embryonic day 14 ventral mesencephalic grafts and assayed for effects on dopaminergic cell survival and process outgrowth. We show that cografts of GDNF-secreting Schwann cell lines improve the survival of intrastriatal embryonic dopaminergic neuronal grafts and improve neurite outgrowth into the host neuropil but have no additional effect on amphetamine-induced rotation. We next looked to see whether bridge grafts of GDNF-secreting SCTM41 cells would promote the growth of axons to their striatal targets from dopaminergic neurons implanted orthotopically into the 6-OHDA-lesioned substantia nigra. We show that such bridge grafts increase the survival of implanted embryonic dopaminergic neurons and promote the growth of axons through the grafts to the striatum.     

Novel pluripotential neural progenitor lines exhibiting rapid controlled differentiation to neurotransmitter receptor‐expressing neurons and glia

The immortalization of progenitor cells from embryonic murine hippocampus using oncogene‐carrying retroviral vectors is described. Use of a vector encoding the oncogene v‐myc results in lines of nestin‐positive progenitor cells. Limited differentiation ensues if the cells are cultured in the presence of dibutyryl cyclic adenosine monophosphate. In contrast, use of a vector in which the extracellular portion of the epidermal growth factor (EGF) receptor is fused to the neu tyrosine kinase generates lines of pluripotential nestin‐positive progenitor cells, which differentiate upon withdrawal of EGF into neurons and glia. Differentiated neurons expressing action potentials and neurotransmitter receptors make up a high proportion of the cells. These cell lines are useful tools to investigate the characteristics of differentiating neurons and glia, as well as to screen neuroactive drugs. This work has been reported in preliminary form as an abstract (1996 Society for Neuroscience Abstract, #606.20, p. 1537).     

In vitro and in vivo characterisation of glial cells immortalised with a temperature sensitive SV40 T antigen-containing retrovirus

An oncogene-carrying repiication-defective retrovirus was used to establish immortalised lines of murine glial cells. Primary cultures of early postnatal cerebellar cells were infected with a retrovirus based on the Murine Moloney Leukemia Virus containing a temperature-sensitive mutant of the Simian Virus 40 large T antigen (SV40 T) oncogene and a gene coding for resistance to the antibiotic G418. Infected cells were selected in G418 and after several in vitro passages cells expressing the O4 antigen were established as a cell line. At a later time point O4-positive single-cell clones were established. Two different types of clones were obtained: (1) “plastic” clones consisting of cells which initially had a morphological and antigenic phenotype of young glial precursor cells but which gradually lost these features, and (2) “stable” cell clones including a clone with the immunological and electrophysiological characteristics of Schwann cells. Culture of the latter cells in the presence of 1 mM dibutyryl cyclic adenosine monophosphate for a period of at least 10 days induced a change in shape and a shift in antigen expression towards a more “diferentiated” maturation stage. When the SV40 T O4-positive immortalised cell line isolated on the cell sorter was transplanted into demyelinated lesions in adult rats, cells were observed ensheathing axons and forming limited amounts of PNS-type myelin. Glial cells immortalised with a temperature-sensitive mutant of the SV40 T oncogene thus retain many physiological properties of their primary culture counterparts and can be induced to undergo limited differentiation in vitro and in vivo. These cell lines, which represent immature CNS glia or Schwann cells, are providing useful tools for investigating the role of cell surface antigens involved in neuron-glial interactions. © 1994 Wiley-Liss, Inc.     

In vitro expansion of a multipotent population of human neural progenitor cells.

The isolation and expansion of human neural progenitor cells have important potential clinical applications, because these cells may be used as graft material in cell therapies to regenerate tissue and/or function in patients with central nervous system (CNS) disorders. This paper describes a continuously dividing multipotent population of progenitor cells in the human embryonic forebrain that can be propagated in vitro. These cells can be maintained and expanded using a serum-free defined medium containing basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), and epidermal growth factor (EGF). Using these three factors, the cell cultures expand and remain multipotent for at least 1 year in vitro. This period of expansion results in a 10(7)-fold increase of this heterogeneous population of cells. Upon differentiation, they form neurons, astrocytes, and oligodendrocytes, the three main phenotypes in the CNS. Moreover, GABA-immunoreactive and tyrosine hydroxylase-immunoreactive neurons can be identified. These results demonstrate the feasibility of long-term in vitro expansion of human neural progenitor cells. The advantages of such a population of neural precursors for allogeneic transplantation include the ability to provide an expandable, well-characterized, defined cell source which can form specific neuronal or glial subtypes.     

In vitro differentiation of neural stem cells derived from human olfactory bulb into dopaminergic‐like neurons

This study describes a new accessible source of neuronal stem cells that can be used in Parkinson's disease cell transplant. The human olfactory bulb contains neural stem cells (NSCs) that are responsible for neurogenesis in the brain and the replacement of damaged cellular components throughout life. NSCs are capable of differentiating into neuronal and glial cells. We isolated NSCs from the olfactory bulb of brain‐death donors and differentiated them into dopaminergic neurons. The olfactory bulb tissues obtained were cultured in Dulbecco's modified Eagle's medium/nutrient mixture F12, B27 supplemented with basic fibroblast growth factor, epidermal growth factor and leukemia inhibitory factor. The NSCs and proliferation markers were assessed. The multipotentiality of olfactory bulb NSCs was demonstrated by their capacity to differentiate into neurons, oligodendrocytes and astrocytes. To generate dopaminergic neurons, olfactory bulb NSCs were differentiated in neurobasal medium, supplemented with B27, and treated with sonic hedgehog, fibroblast growth factor 8 and glial cell‐derived neurotrophic factor from the 7th to the 21st day, followed by detection of dopaminergic neuronal markers including tyrosine hydroxylase and aromatic l ‐amino acid decarboxylase. The cells were expanded, established in continuous cell lines and differentiated into the two classical neuronal phenotypes. The percentage of co‐positive cells (microtubule‐associated protein 2 and tyrosine hydroxylase; aromatic l‐amino acid decarboxylase and tyrosine hydroxylase) in the treated cells was significantly higher than in the untreated cells. These results illustrate the existence of multipotent NSCs in the adult human olfactory bulb that are capable of differentiating toward putative dopaminergic neurons in the presence of trophic factors. Taken together, our data encourage further investigations of the possible use of olfactory bulb NSCs as a promising cell‐based therapeutic strategy for Parkinson's disease.     

Differentiation of the O-2A progenitor cell line CG-4 into oligodendrocytes and astrocytes following transplantation into glia-deficient areas of CNS white matter

The in vitro properties of the CG4 cell line have led to its increasing use as a cell line with which to study the behaviour of the O-2A progenitor cell. In this study we have examined the in vivo behaviour of the CG4 cell line following transplantation into areas of adult rat spinal cord white matter which have been permanently depleted of glial cells by the combination of local X-irradiation and direct injection of 0.1% ethidium bromide. Twenty-one days after transplantation, both myelin-forming oligodendrocytes and glial fibrillary acidic protein-positive astrocytes were identified within the lesion, indicating that the CG4 cell line has bipotential differentiation properties when introduced into a pathological environment consisting of demyelinated axons but devoid of oligodendrocytes or astrocytes. In this respect, the CG4 cell line resembles other glial progenitor cell lines that have been transplanted into similar lesions. In some areas of the lesion, remyelination was observed that was similar in extent to that achieved by growth factor-expanded populations of O-2A progenitor cells. The transplant origin of the cell types within the lesion was confirmed by retroviral incorporation of the lacZ marker gene, the expression of which allowed their identification by histochemistry. In conclusion, the in vivo properties of the CG4 cell line make it a highly suitable line with which to study the behaviour of O-2A progenitors following transplantation into normal and damaged CNS. © 1995 Wiley-Liss, Inc.     

The use of cultured autologous Schwann cells to remyelinate areas of persistent demyelination in the central nervous system

Areas of persistent demyelination were created in the dorsal columns of the cat spinal cord by injecting ethidium bromide into white matter which had previously been exposed to 40 Grays of X-irradiation. In the centre of such lesions demyelinated axons occurred in a glial-free area while axons next to normal tissue were separated by astrocyte processes. No remyelination occurs in such lesions (Blakemore 1984). Autologous Schwann cells and fibroblasts cultured from a peripheral nerve biopsy were injected into such lesions and the extent of Schwann cell remyelination examined. Only lesions injected with viable cells showed remyelination by Schwann cells; in no lesion were all the demyelinated axons remyelinated. Three forms of association of Schwann cell with axons were detected. In the centre of the lesions Schwann cells either remyelinated axons around or near to blood vessels, or lay next to demyelinated axons and did not form myelin. Schwann cell remyelination was also detected in the astrocyte-containing areas around the edges of some lesions. It was concluded that the extent of Schwann cell remyelination was influenced by the mode of entry of the cells into the lesion and by the architecture of the lesion. The presence or absence of stable extracellular matrix is believed to be the prime factor which influenced Schwann cell remyelination. The relevance of these observations to artificial repair of the lesions of multiple sclerosis is discussed.     

多细胞因子诱导成年鼠骨髓基质细胞向神经细胞分化的实验研究

目的观察大鼠骨髓基质细胞(rBMSCs)的生长特点及诱导条件下分化成神经细胞的能力,并对其机制进行初步探讨。方法以密度梯度离心分离骨髓基质细胞,在神经干细胞培养液中培养,采用四唑盐(MTT)法观察在培养液中添加碱性成纤维细胞生长因子(bFGF)、表皮生长因子(EGF)对BMSCs增殖的影响;观察添加脑源性神经生长因子(BDNF)、神经生长因子(NGF)和维甲酸(RA)对rBMSCs的诱导分化情况;采用免疫组织化学法(ABC)检测诱导后的细胞表达神经元特异性烯醇化酶(NSE)、神经元核蛋白(NeuN)和胶质原性纤维酸性蛋白抗体(GFAP)等特异性标志物的情况;以流式细胞分选确定神经元的比例。结果bFGF和EGF能在体外促进rBMSCs增殖,BDNF、NGF和RA能诱导rBMSCs来源的神经干细胞(NSCs)表达NSE、GFAP等特异性标志物。结论EGF、bFGF、BDNF、NGF、RA及适宜的培养液可使rBMSCs定向转化为NSCs,获得足够的目的细胞,进而分化为神经元样和神经胶质样细胞。     

In vitro differentiation of quail neural crest cells into sensory-like neuroblasts

This study shows that quail neural crest cells can differentiate in vitro into sensory-like neuroblasts. The putative sensory neuroblasts were large and spherical, possessing large diameter, bipolar or pseudo-unipolar, long processes that lacked multiple varicosities characteristic of autonomic neurons. They bound HNK-1, a monoclonal antibody against a cell surface epitope expressed by early neural crest cells but not by young neural tube-derived cells. Many of the sensory-like neuroblasts had substance P (SP)-like immunoreactivity. Some exhibited histochemical carbonic anhydrase activity; carbonic anhydrase is shown in this study to stain a subpopulation of spinal sensory neurons in adult quail and embryos 9 days and older, whereas ventral root axons and neurons in sympathetic ganglia are non-reactive at all ages. Double staining indicated that unlike the multipolar neuroblasts developing in the same cultures, SP-like immunoreactive neuroblasts do not contain detectable levels of tyrosine hydroxylase or dopamine-beta-hydroxylase. Finally, the neuronal nature of the cultured sensory-like neuroblasts was further documented by double labeling for antibodies against the 68 kDa neurofilament polypeptide and substance P.     

Establishment and characterization of a multipotential neural cell line that can conditionally generate neurons,astrocytes, and oligodendrocytes in vitro

In the mammalian central nervous system (CNS), multipotential neural stem cells in the neuroepithelium generate the three major types of neural cells, namely, neurons, astrocytes, and oligodendrocytes. To explore the molecular mechanisms underlying proliferation and differentiation of these neural stem cells, we established a cell line named MNS-57 from the embryonic day 12 rat neuroepithelium by introducing the mycer fusion gene, in which c-myc can be conditionally activated by adding oestrogen to the culture medium. MNS-57 cells expressed nestin, vimentin, and the RC1 antigen, which are potential markers for neural stem cells. We show that under particular culture conditions, MNS-57 cells can conditionally generate neurons, astrocytes, and oligodendrocytes in vitro, indicating that they are likely to originate from multipotential neural stem cells. Incubating MNS-57 cells with either oestrogen, which activates mycer, or growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) stimulated their growth, and the combination of oestrogen and bFGF (or EGF) had a synergistically stronger mitogenic effect than the single factors. Furthermore, both c-myc activation and bFGF appeared to be necessary for the differentiation of MNS-57 cells, and only when stimulated by both signals simultaneously, the cells committed to generating multiple neural cell types. Thus, the property of the cell line is unique in that its differentiation into neurons and glia can be conditionally manipulated invitro in an exogenous signal-dependent manner. We propose that the cell line described here will provide an useful in vitro model to understand genetic and environmental mechanisms that control the generation of neural cell diversity in the CNS. © 1995 Wiley-Liss, Inc.     

Staurosporine is a potent activator of neuronal,glial, and "CNS stem cell-like" neurosphere differentiation in murine embryonic stem cells

Staurosporine (STS), a broad spectrum protein kinase inhibitor, was previously shown to induce neurite outgrowth in several neuroblastoma cell lines. However, data on the neurotrophic potential of this alkaloid in embryonic stem cell systems were not available. Therefore, three mouse ES cell lines, IB10, RW4, and Bruce 4, were induced to enter neurogenesis in culture at low concentrations of STS. These cells differentiated into epidermal growth factor-responsive neural precursor cells, formed neurospheres, and further developed to neurons and astrocytes. The clonally derived neurospheres consisted of multipotent cells which exhibited some of the classical characteristics of early CNS stem cells and could be propagated in vitro. STS was antagonistic in several ways to retinoic acid (RA), a vitamin A metabolite, which promotes neuritogenesis. Results from RT-PCR experiments and inhibition studies with RA provided evidence that staurosporine exerted its neurotrophic effects through the induction of very late levels of the nerve growth factor and protein kinase C neurogenesis pathways.     

骨髓基质细胞分化为神经元样细胞后与皮质神经元间突触的建立

目的 观察与大脑皮质神经元共培养的骨髓基质细胞(BMSCs)经诱导分化成神经元样细胞后,与脑皮质神经元之间形成功能性突触的情况.方法 无菌条件下取绿色荧光蛋白(GFP)转基因小鼠骨髓,用贴壁筛选法体外培养获得GFP转基因小鼠BMSCs(GFP-GM-BMSCs),在体外培养、扩增、纯化.取第3代GFP-GM-BMSCs,种植到源于小鼠大脑的原代皮质神经元和胶质细胞中,培养介质为加有20 ng/mL表皮生长因子(EGF)、20 ng/mL碱性成纤维细胞生长因子(bFGF)的无血清培养基(Neurobasal-A+2%B27),体外模拟建立细胞移植的共培养体系.共培养第10天,利用FM1-43荧光染料染色活动突触小泡的特性,通过荧光显微镜观察共培养的两种细胞之间形成的突触.结果 与神经元共培养的GFP-GM-BMSCs在含有EGF、bFGF的无血清培养基中7 d后分化为神经元样细胞.共培养10 d后,FM1-43染色阳性的突触囊泡明显增加,主要位于神经元样细胞胞体、突起及其末端结构上.结论 在体外模拟细胞移植共培养体系中,分化自GFP-GM-BMSCs的神经元样细胞能与神经元之间形成突触样连接.     

Green-fluorescent-protein-expressing mice as models for the study of axonal growth and regeneration in vitro

The culture of hippocampal-entorhinal brain slices is a widely used model for studying neuronal differentiation, axon growth and pathfinding in vitro. The application of tracers (e.g. biocytin) is a well-established method for studying single or multiple neurons and their extensions in this model. For quantifying the growth of high numbers of axons after lesion, however, genetically expressed enhanced green fluorescent protein (EGFP) has proven particularly useful for labeling living axons in vivo and in vitro. Here, we introduce several EGFP-expressing mouse lines which improve the organotypic brain slice model. The questions addressed determine which mouse line to use: beta-actin-EGFP mice for labeling all cells and their extensions; Tau-EGFP mice for labeling the axoplasma; or Thy-1.2-EGFP mice for labeling the axonal membrane. Cocultures of EGFP-positive entorhinal cortex explants with EGFP-negative hippocampal explants allow the monitoring of fluorescent axons growing into the hippocampus in an easily quantifiable manner.     

Long-term tripotent differentiation capacity of human neural stem (NS) cells in adherent culture

Stem cell lines that provide a renewable and scaleable supply of central nervous system cell types would constitute an invaluable resource for basic and applied neurobiology. Here we describe the generation and long-term expansion of multiple human foetal neural stem (NS) cell lines in monolayer culture without genetic immortalization. Adherent human NS cells are propagated in the presence of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2), under which conditions they stably express neural precursor markers and exhibit negligible differentiation into neurons or glia. However, they produce astrocytes, oligodendrocytes, and neurons upon exposure to appropriate differentiation factors. Single cell cloning demonstrates that human NS cells are tripotent. They retain a diploid karyotype and constant neurogenic capacity after over 100 generations. In contrast to human neurospheres, we observe no requirement for the cytokine leukaemia inhibitory factor (LIF) for continued expansion of adherent human NS cells. Human NS cells can be stably transfected to provide reporter lines and readily imaged in live monolayer cultures, creating the potential for high content genetic and chemical screens.     

细胞因子联合纹状体条件培养液定向诱导间充质干细胞体外分化为多巴胺能神经元

背景：在众多体外诱导间充质干细胞向多巴胺能神经元的诱导分化研究中，诱导阳性率仍不理想。 目的：实验应用碱性成纤维细胞生长因子、表皮生长因子和纹状体条件培养液定向诱导大鼠骨髓间充质干细胞分化为多巴胺能神经元，拟探讨提高诱导阳性率的方法。 设计、时间及地点：以细胞为对象的对照观察细胞学实验，于2006-07/2007-12在山东大学齐鲁儿童医院和山东大学第二医院血液实验室完成。 材料：健康成年Wistar大鼠用于骨髓间充质干细胞的分离，新生Wistar大鼠用于纹状体条件培养液的制备。 方法：采用贴壁法分离纯化健康成年Wistar大鼠骨髓间充质干细胞进行传代培养。取出生24 h内新生Wistar大鼠，完整剥离其大脑组织制备纹状体条件培养液。取体外培养的第5代间充质干细胞，用含碱性成纤维细胞生长因子和表皮生长因子的预诱导液进行预诱导，24 h后去除预诱导液，换用纹状体条件培养液进行诱导。 主要观察指标：倒置显微镜下观察细胞形态变化，并应用细胞免疫化学技术检测细胞内神经元特异烯醇化酶和酪氨酸羟化酶表达。 结果：大鼠骨髓间充质干细胞经碱性成纤维细胞生长因子、表皮生长因子和纹状体条件培养液诱导后细胞胞体逐渐回缩成团，形成梭形，部分细胞可见突起伸出，类似神经元。细胞免疫化学检测，诱导后细胞神经元特异烯醇化酶阳性表达率为( 72.70±14.81)%，酪氨酸羟化酶阳性表达率为(34.50±15.93)%。 结论：应用碱性成纤维细胞生长因子、表皮生长因子联合纹状体条件培养液诱导分化体系，获得了高比例的神经元，其中包括较多的多巴胺能神经元。     

Enhanced axonal regeneration following combined demyelination plus schwann cell transplantation therapy in the injured adult spinal cord.

We have treated spinal cord injured rats with demyelination plus Schwann cell transplantation and assessed neurite outgrowth in a quantifiable model of axonal regeneration. Axonal injuries of differing severity were induced in the dorsal funiculus of adult rats using a micromanipulator-controlled Scouten knife. Demyelinated regions were produced so as to overlap with the injury site by the injection of galactocerebroside antibodies plus complement one segment cranial to the axonal injury site. Schwann cells were isolated from the sciatic nerve, expanded in vitro, and transplanted into the injury site 1 day later. Animals were killed after an additional 7 days. Schwann cells were evenly distributed throughout the region of demyelination, which extended 6-7 mm cranial to the axonal injury site. The severity of axonal injury was quantified by counting degenerate axons in transverse resin sections. The degree of axonal regeneration was assessed by an electron microscopic analysis of growth cone frequency and distribution relative to the site of axonal injury. Quantification of growth cones at a distance from the site of axonal injury indicated a strong linear relationship (P < 0.001) between the number of growth cones and the number of severed axons; the ratio of growth cones to severed axons was increased by 26.5% in demyelinated plus transplanted animals compared to demyelinated animals without a transplant. Furthermore, only the demyelinated plus transplanted animals contained growth cones associated with myelin in white matter immediately outside of the region of complete demyelination. Growth cones were absent in transplanted-only animals at a distance from the site of axonal injury. These findings indicate that combined demyelination plus Schwann cell transplantation therapy enhances axonal regeneration following injury and suggests that growth cones are able to overcome myelin-associated inhibitors of neurite outgrowth in the presence of trophic support.     

Type 1 astrocytes fail to inhibit Schwann cell remyelination of CNS axons in the absence of cells of the O-2A lineage.

The extent to which Schwann cells are able to remyelinate demyelinated CNS axons is influenced by the presence of astrocytes. In order to study further the nature of astrocyte control of Schwann cell remyelination in the CNS, cultures containing type 1 astrocytes and a small proportion of Schwann cells, but depleted of O-2A lineage cells by exposure to cytosine arabinoside and complement-mediated immunocytolysis, were transplanted into glial-free lesions in adult rat spinal cord in which the host response to demyelinated axons was suppressed by X-irradiation. Following transplantation of these O-2A lineage-depleted cultures into X-irradiated, demyelinating lesions, there was extensive remyelination of demyelinated axons by Schwann cells, a result which contrasted with those obtained from earlier experiments in which O-2A lineage cells were present within the transplant, and/or recruited from host tissue. This experiment shows that the presence of O-2A lineage cells is required in order for transplanted type 1 astrocytes to organise in a manner which inhibits extensive Schwann cell remyelination of CNS axons.     

Schwann cell-like myelination following transplantation of an olfactory bulb-ensheathing cell line into areas of demyelination in the adult CNS

In this study we have transplanted a clonal olfactory bulb-ensheathing cell line into focal areas of the rat spinal cord which contain demyelinated axons but neither oligodendrocytes nor astrocytes. The cell line was created by retroviral incorporation of the temperature-sensitive Tag gene into FACS-sorted 04+ cells from 7-day-old rat pup olfactory bulb. The spinal cord lesions were obtained by injecting small volumes of ethidium bromide into the dorsal white matter of spinal cord previously exposed to 40 Grays of X-irradiation. Many of the axons were remyelinated by P0+ myelin sheaths 21 days after transplantation. Light and electron microscopy revealed cells engaging and myelinating axons in a manner highly reminiscent of Schwann cells within similar lesions. GFAP+ cells were also present within the lesion. This study provides the first in vivo evidence that olfactory bulb-ensheathing cells are able to produce peripheral-type myelin sheaths around axons of the appropriate diameter. © 1996 Wiley-Liss, Inc.     

Neuronal properties and trophic activities of immortalized hippocampal cells from embryonic and young adult mice

The hippocampal formation elaborates trophic factors such as nerve growth factor (NGF) to support the cholinergic innervation it receives from the septal region. To further study the trophic interactions of this pathway, hippocampal cells from embryonic day 18 and postnatal day 21 mice were immortalized via somatic cell fusion to N18TG2 neuroblastoma cells. The hippocampal cell lines exhibit morphological and cytoskeletal features which are typical of their neuronal parents but which are not expressed by the neuroblastoma parent. When differentiated with retinoic acid, the hippocampal cell lines exhibit electrophysiological features similar to cultured hippocampal neurons. Many of the lines constitutively express high levels of NGF, and at least one cell line exerts a non-NGF trophic effect on the expression of choline acetyltransferase by septal neurons in vitro. These cell lines are potentially useful for investigating the neurochemical and excitable properties of hippocampal neurons and identifying novel trophic activities that promote the development and maintenance of the septohippocampal pathway.     

GDNF selectively promotes regeneration of injury-primed sensory neurons in the lesioned spinal cord

Axonal regeneration within the CNS fails due to the growth inhibitory environment and the limited intrinsic growth capacity of injured neurons. Injury to DRG peripheral axons induces expression of growth associated genes including members of the glial-derived neurotrophic factor (GDNF) signaling pathway and "preconditions" the injured cells into an active growth state, enhancing growth of their centrally projecting axons. Here, we show that preconditioning DRG neurons prior to culturing increased neurite outgrowth, which was further enhanced by GDNF in a bell-shaped growth response curve. In vivo, GDNF delivered directly to DRG cell bodies facilitated the preconditioning effect, further enhancing axonal regeneration beyond spinal cord lesions. Consistent with the in vitro results, the in vivo effect was seen only at low GDNF concentrations. We conclude that peripheral nerve injury upregulates GDNF signaling pathway components and that exogenous GDNF treatment selectively promotes axonal growth of injury-primed sensory neurons in a concentration-dependent fashion.