GDNF to the rescue: GDNF delivery effects on motor neurons and nerves,and muscle re-innervation after peripheral nerve injuries

Peripheral nerve injuries commonly occur due to trauma, like a traffic accident. Peripheral nerves get severed, causing motor neuron death and potential muscle atrophy. The current golden standard to treat peripheral nerve lesions, especially lesions with large(≥ 3 cm) nerve gaps, is the use of a nerve autograft or reimplantation in cases where nerve root avulsions occur. If not tended early, degeneration of motor neurons and loss of axon regeneration can occur, leading to loss of function. Alth...     

Characterization of a Schwann cell neurite-promoting activity that directs motoneuron axon outgrowth

Schwann cells support and facilitate axonal growth during development and successful regeneration in the peripheral nerve. In the regenerating rat sciaticnerve, Schwann cells provide a trophic milieu for primary sensory, sympathetic, and motoneurons. We have characterized a neurotrophic activity produced by adult rat sciatic nerve Schwann cells and a spontaneously immortal Schwann cell clone (iSC). This activity elicits neurite outgrowth from chick embryo explants of both CNS and PNS. The iSC activity has been concentrated by cation-exchange chromatography and compared to known neurotrophins in bioassay. Pooled bound fractions elicit neurite outgrowth from sympathetic, ciliary and motoneurons. In collagen matrix cocultures of iSC and E4 ventral horn(before motor axon extension to muscle targets), the iSC activity can direct the initial axonal extension from motoneurons. The data presented suggest that Schwann cell-produced activity may mediate motoneuron axonal extension before contact with their peripheral source of neurotrophin. © 1994 Wiley-Liss, Inc.     

Role of neurotrophic factors in enhancing linear axonal growth of ganglionic sensory neurons in vitro

Neurotrophins play a major role in the regulation of neuronal growth such as neurite sprouting or regeneration in response to nerve injuries. The role of nerve growth factor, neurotrophin-3, and brain-derived neurotrophic factor in maintaining the survival of peripheral neurons remains poorly understood. In regenerative medicine, different modalities have been investigated for the delivery of growth factors to the injured neurons, in search of a suitable system for clinical applications. This study was to investigate the influence of nerve growth factor, neurotrophin-3 and brain-derived neurotrophic factor on the growth of neurites using two in vitro models of dorsal root ganglia explants and dorsal root ganglia-derived primary cell dissociated cultures. Quantitative data showed that the total neurite length and tortuosity were differently influenced by trophic factors. Nerve growth factor and, indirectly, brain-derived neurotrophic factor stimulate the tortuous growth of sensory fibers and the formation of cell clusters. Neurotrophin-3, however, enhances neurite growth in terms of length and linearity allowing for a more organized and directed axonal elongation towards a peripheral target compared to the other growth factors. These findings could be of considerable importance for any clinical application of neurotrophic factors in peripheral nerve regeneration. Ethical approval was obtained from the Regione Piemonte Animal Ethics Committee ASLTO1(file # 864/2016-PR) on September 14, 2016.     

Roles of neural stem cells in the repair of peripheral nerve injury

Currently, researchers are using neural stem cell transplantation to promote regeneration after peripheral nerve injury, as neural stem cells play an important role in peripheral nerve injury repair. This article reviews recent research progress of the role of neural stem cells in the repair of peripheral nerve injury. Neural stem cells can not only differentiate into neurons, astrocytes and oligodendrocytes, but can also differentiate into Schwann-like cells, which promote neurite outgrowth around the injury. Transplanted neural stem cells can differentiate into motor neurons that innervate muscles and promote the recovery of neurological function. To promote the repair of peripheral nerve injury, neural stem cells secrete various neurotrophic factors, including brain-derived neurotrophic factor, fibroblast growth factor, nerve growth factor, insulin-like growth factor and hepatocyte growth factor. In addition, neural stem cells also promote regeneration of the axonal myelin sheath, angiogenesis, and immune regulation. It can be concluded that neural stem cells promote the repair of peripheral nerve injury through a variety of ways.     

Repair of peripheral nerve transections with fibrin sealant containing neurotrophic factors

Peripheral nerve injury is often followed by incomplete recovery of function and sometimes associated with neuropathic pain. There is, therefore, need for therapies which improve the speed of recovery and the final functional outcome after peripheral nerve injuries. In addition, neuropathic pain is not easily dealt with clinically and should preferably be eliminated. Neurotrophic factors have well-documented abilities to support neuron survival and stimulate neurite outgrowth, making them excellent candidates for use in repairing injured nerves. We investigated the possible beneficial effects of repairing the transected rat sciatic nerve by local application of a fibrin sealant containing nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), or acidic fibroblast growth factor (aFGF). Fibrin sealant was used in conjunction with sutures. Evaluation of motor and sensory function, autotomy, and histological parameters was carried out from 1 to 12 weeks after injury. We demonstrate that NGF cotreatment decreased the occurance of autotomy, suggesting a reduction of neuropathic pain, and improved the performance in motor and sensory tests. In addition, the number of regenerating motoneurons was significantly increased after NGF administration. GDNF increased the speed of sensory recovery, but also markedly increased autotomy, indicating an increased degree of neuropathic pain. aFGF did not alter the outcome of the motor or sensory tests. Fibrin sealant could easily be used in conjunction with sutures to deliver neurotrophic substances locally to the damaged nerve and to enhance recovery of nerve function.     

Neurite outgrowth in normal and injured primary sensory neurons reveals different regulation by nerve growth factor (NGF) and artemin

Neurotrophic factors have been intensively studied as potential therapeutic agents for promoting neural regeneration and functional recovery after nerve injury. Artemin is a member of the glial cell line-derived neurotrophic factor (GDNF) family of ligands (GFLs) that forms a signalling complex with GFRα3 and the tyrosine kinase Ret. Systemic administration of artemin in rodents is reported to facilitate regeneration of primary sensory neurons following axotomy, improve recovery of sensory function, and reduce sensory hypersensitivity that is a cause of pain. However, the biological mechanisms that underlie these effects are mostly unknown. This study has investigated the biological significance of the colocalisation of GFRα3 with TrkA (neurotrophin receptor for nerve growth factor [NGF]) in the peptidergic type of unmyelinated (C-fibre) sensory neurons in rat dorsal root ganglia (DRG). In vitro neurite outgrowth assays were used to study the effects of artemin and NGF by comparing DRG neurons that were previously uninjured, or were axotomised in vivo by transecting a visceral or somatic peripheral nerve. We found that artemin could facilitate neurite initiation but in comparison to NGF had low efficacy for facilitating neurite elongation and branching. This low efficacy was not increased when a preconditioning in vivo nerve injury was used to induce a pro-regenerative state. Neurite initiation was unaffected by artemin when PI3 kinase and Src family kinase signalling were blocked, but NGF had a reduced effect.     

体外低频电刺激促进背根神经元突起的生长：上调钙离子介导的脑源性神经营养因子表达可为其途径

摘要 背景：短时低频电刺激已被证明可显著促进周围神经系统损伤后轴突的再生,目前对电刺激是如何促进其突起生长还有待证实。 目的：体外培养背根神经元,观察短时低频电刺激对神经元突起生长的影响,探讨电刺激发挥作用可能的细胞信号分子。 设计、时间及地点：体外培养背根神经元及离体电刺激处理,于2007-05/2008-10在上海交通大学医学院完成。 材料：新生48h Sprague-Dawley大鼠20只(中科院上海生命科学研究所动科所)。 方法：体外培养背根神经元,随机分为两组,正常对照组(n = 6)及电刺激组(n = 8)。电刺激组施予离体电刺激(20Hz, 100μs, 3V),持续作用1h。为探讨电刺激发挥作用经由的细胞信号分子,在施予电刺激前预先加入钙离子通道阻滞剂Nifedipine孵育4小时,再给予电刺激,再次检测各组神经元突起的生长情况。 主要观察指标：β-tubulin染神经元,测量各组神经元突起的长度。RT-PCR、 western blot和ELISA分别检测神经元BDNF的表达和分泌。 结果：短时低频电刺激促进神经元突起的生长,增强其表达和分泌BDNF (P < 0.05)。Nifedipine的使用削弱了电刺激对神经元突起生长及BDNF合成的促进作用 (P < 0.05)。 结论：短时低频电刺激促进体外培养的背根神经元突起的生长及BDNF的合成,初步认为电刺激对神经元突起生长的促进作用,至少通过促发钙内流所致BDNF表达和分泌增多所致。 关键词：电刺激;背根神经元;突起生长;BDNF;Ca2+     

Bone mesenchymal stromal cells stimulate neurite outgrowth of spinal neurons by secreting neurotrophic factors

It has been demonstrated that bone mesenchymal stromal cells (BMSCs) stimulate neurite outgrowth from dorsal root ganglion (DRG) neurons. The present in vitro study tested the hypothesis that BMSCs stimulate the neurite outgrowth from spinal neurons by secreting neurotrophic factors. Spinal neurons were cocultured with BMSCs, fibroblasts and control medium in a non-contact system. Neurite outgrowth of spinal neurons cocultured with BMSCs was significantly greater than the neurite outgrowth observed in neurons cultured with control medium or with fibroblasts. In addition, BMSC-conditioned medium increased the length of neurites from spinal neurons compared to those of neurons cultured in the control medium or in the fibroblasts-conditioned medium. BMSCs expressed brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). The concentrations of BDNF and GDNF in BMSC-conditioned medium were 132±12 and 70±6 pg ml(-1), respectively. The addition of anti-BDNF and anti-GDNF antibodies to BMSC-conditioned medium partially blocked the neurite-promoting effect of the BMSC-conditioned medium. In conclusion, our results demonstrate that BMSCs promote neurite outgrowth in spinal neurons by secreting soluble factors. The neurite-promoting effect of BMSCs is partially mediated by BDNF and GDNF.     

Enteric glia mediate neuronal outgrowth through release of neurotrophic factors

Previous studies have shown that transplanted enteric glia enhance axonal regeneration, reduce tissue damage, and promote functional recovery following spinal cord injury. However, the mechanisms by which enteric glia mediate these beneficial effects are unknown. Neurotrophic factors can promote neuronal differentiation, survival and neurite extension. We hypothesized that enteric glia may exert their protective effects against spinal cord injury partially through the secretion of neurotrophic factors. In the present study, we demonstrated that primary enteric glia cells release nerve growth factor, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor over time with their concentrations reaching approximately 250, 100 and 50 pg/mL of culture medium respectively after 48 hours. The biological relevance of this secretion was assessed by incubating dissociated dorsal root ganglion neuronal cultures in enteric glia-conditioned medium with and/or without neutralizing antibodies to each of these proteins and evaluating the differences in neurite growth. We discovered that conditioned medium enhances neurite outgrowth in dorsal root ganglion neurons. Even though there was no detectable amount of neurotrophin-3 secretion using ELISA analysis, the neurite outgrowth effect can be attenuated by the antibody-mediated neutralization of each of the aforementioned neurotrophic factors. Therefore, enteric glia secrete nerve growth factor, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor and neurotrophin-3 into their surrounding environment in concentrations that can cause a biological effect.     

Methyl 3,4-dihydroxybenzoate promotes neurite outgrowth of cortical neurons cultured in vitro

Cerebral cortical neurons from neonatal rats were cultured in the presence of methyl 3,4-dihydroxybenzoate (MDHB;2,4,and 8 μM).Results showed that MDHB significantly promoted neurite outgrowth and microtubule-associated protein 2 mRNA expression,and increased neuronal survival in a dose-dependent manner.Moreover,MDHB induced brain-derived neurotrophic factor expression.These findings suggest that MDHB has a neurotrophic effect,which may be due to its ability to increase brain-derived neurotrophic factor expression.     

Neurotrophic Actions of Bone Marrow Stromal Cells on Primary Culture of Dorsal Root Ganglion Tissues and Neurons

Application of adult bone marrow stromal cells (BMSCs) provides therapeutic benefits to the treatment of neurological insults. The aim of this study was to explore the potential of nonhematopoietic BMSCs to produce soluble factors and stimulate signaling pathways in neurons that mediate trophic effects. A combination of enzyme-linked immunosorbent assay and two-dimensional gel electrophoresis coupled with mass spectrometry showed that the BMSCs released into the culture medium an array of soluble factors such as nerve growth factor, brain-derived neurotrophic factor, basic fibroblast growth factor, and ciliary neurotrophic factor, which have been shown to exhibit potent neurotrophic effects on neural cells. Immunochemistry, cell viability assay, and quantitative real-time RT-PCR collectively showed that neurite outgrowth and neurogenesis in cultured rat dorsal root ganglion (DRG) explants and neurons were enhanced after they were cocultured with rat BMSCs. Western blot analysis revealed that BMSC-conditioned medium activated phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated protein kinase and/or phosphoinositide 3-kinase/serine/threonine kinase (PI3K/Akt) in primary culture of rat DRG neurons, which suggested that BMSCs trigger endogenous survival signaling pathways in neurons through their secreted soluble factors. Our data help to elucidate the mechanisms by which BMSCs function as a cell therapy agent in peripheral nerve regeneration.     

Peripheral nerve induces macrophage neurotrophic activities: regulation of neuronal process outgrowth, intracellular signaling and synaptic function

Rat cortical neurons cultured in conditioned media from human monocyte-derived macrophages (MDM) show increased neuronal protein synthesis, neurite outgrowth, mitogen-activating protein kinase activity, and synaptic function. Neurotrophic properties of human MDM-conditioned media are significantly enhanced by human peripheral nerve and to a more limited extent by CD40 ligand pre-stimulation. Such positive effects of MDM secretions on neuronal function parallel the secretion of brain-derived neurotrophic factor (BDNF). MDM activation cues may serve to balance toxic activities produced during neurodegenerative diseases and thus, under certain circumstances, mitigate neuronal degeneration.     

Bone mesenchymal stromal cells stimulate neurite outgrowth of spinal neurons by secreting neurotrophic factors

Abstract

It has been demonstrated that bone mesenchymal stromal cells (BMSCs) stimulate neurite outgrowth from dorsal root ganglion (DRG) neurons. The present in vitro study tested the hypothesis that BMSCs stimulate the neurite outgrowth from spinal neurons by secreting neurotrophic factors. Spinal neurons were cocultured with BMSCs, fibroblasts and control medium in a non-contact system. Neurite outgrowth of spinal neurons cocultured with BMSCs was significantly greater than the neurite outgrowth observed in neurons cultured with control medium or with fibroblasts. In addition, BMSC-conditioned medium increased the length of neurites from spinal neurons compared to those of neurons cultured in the control medium or in the fibroblasts-conditioned medium. BMSCs expressed brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). The concentrations of BDNF and GDNF in BMSC-conditioned medium were 132±12 and 70±6 pg ml^?1, respectively. The addition of anti-BDNF and anti-GDNF antibodies to BMSC-conditioned medium partially blocked the neurite-promoting effect of the BMSC-conditioned medium. In conclusion, our results demonstrate that BMSCs promote neurite outgrowth in spinal neurons by secreting soluble factors. The neurite-promoting effect of BMSCs is partially mediated by BDNF and GDNF.     

Extracellular matrix from human umbilical cord-derived mesenchymal stem cells as a scaffold for peripheral nerve regeneration

The extracellular matrix, which includes collagens, laminin, or fibronectin, plays an important role in peripheral nerve regeneration. Recently, a Schwann cell-derived extracellular matrix with classical biomaterial was used to mimic the neural niche. However, extensive clinical use of Schwann cells remains limited because of the limited origin, loss of an autologous nerve, and extended in vitro culture times. In the present study, human umbilical cord-derived mesenchymal stem cells (hUCMSCs), which are easily accessible and more proliferative than Schwann cells, were used to prepare an extracellular matrix. We identiifed the morphology and function of hUCMSCs and investi-gated their effect on peripheral nerve regeneration. Compared with a non-coated dish tissue culture, the hUCMSC-derived extracellular matrix enhanced Schwann cell proliferation, upregulated gene and protein expression levels of brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, and vascular endothelial growth factor in Schwann cells, and enhanced neurite outgrowth from dorsal root ganglion neurons. These ifndings suggest that the hUCMSC-derived extracellular matrix promotes peripheral nerve repair and can be used as a basis for the rational design of engineered neural niches.     

Human umbilical cord mesenchymal stem cells promote peripheral nerve repair via paracrine mechanisms

Human umbilical cord-derived mesenchymal stem cells(h UCMSCs) represent a promising young-state stem cell source for cell-based therapy. h UCMSC transplantation into the transected sciatic nerve promotes axonal regeneration and functional recovery. To further clarify the paracrine effects of h UCMSCs on nerve regeneration, we performed human cytokine antibody array analysis, which revealed that h UCMSCs express 14 important neurotrophic factors. Enzyme-linked immunosorbent assay and immunohistochemistry showed that brain-derived neurotrophic factor, glial-derived neurotrophic factor, hepatocyte growth factor, neurotrophin-3, basic fibroblast growth factor, type I collagen, fibronectin and laminin were highly expressed. Treatment with h UCMSC-conditioned medium enhanced Schwann cell viability and proliferation, increased nerve growth factor and brain-derived neurotrophic factor expression in Schwann cells, and enhanced neurite growth from dorsal root ganglion explants. These findings suggest that paracrine action may be a key mechanism underlying the effects of h UCMSCs in peripheral nerve repair.     

Growth responses of different subpopulations of adult sensory neurons to neurotrophic factors in vitro

Different subpopulations of adult primary sensory neurons in the dorsal root ganglia express receptors for different trophic factors, and are therefore potentially responsive to distinct trophic signals. We have compared the effect of the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and NT-3, and of glial cell line-derived neurotrophic factor (GDNF) on neurite outgrowth in dissociated cultures of sensory neurons from the lumbar ganglia of young adult rats, and attempted to establish subset-specific effects of these trophic factors. We analysed three parameters of neurite growth (percentage of process-bearing neurons, length of longest neurite and total neurite length), which may correlate with particular types of axon growth in vivo, and may therefore respond differently to trophic factor presence. Our results showed that percentage of process-bearing neurons and total neurite length were influenced by trophic factors, whilst the length of the longest neurite was trophic factor independent. Only NGF and GDNF were found to enhance significantly the proportion of process-bearing neurons in vitro. GDNF was more effective than NGF on small, IB4- neurons, which are known to develop GDNF responsiveness early in postnatal development. NGF, and to a much lesser extent GDNF, enhanced the total length of the neurites produced by neurons in culture. BDNF exerted an inhibitory effect on growth, and both BDNF and NT-3 could partially block some of the growth-promoting effects of NGF on specific neuronal subpopulations.     

Bone marrow stromal cells promote neurite outgrowth of spinal motor neurons by means of neurotrophic factors in vitro

Transplantation of bone marrow stromal cells (BMSCs) into spinal cord injury models has shown significant neural function recovery; however, the underlying mechanisms have not been fully understood. In the present study we examined the effect of BMSCs on neurite outgrowth of spinal motor neuron using an in vitro co-culture system. The ventral horn of the spinal grey matter was harvested from neonatal Sprague–Dawley rats, cultured with BMSCs, and immunostained for neurofilament-200 (NF-200). Neurite outgrowth of spinal motor neurons was measured using Image J software. ELISA was used to quantify neurotrophic factors such as brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) in culture media, and antibodies or exogenous neurotrophic factors were used to block or mimic the effect of BMSCs on neurite outgrowth, respectively. The results showed that neurite outgrowth significantly increased in spinal motor neurons after co-cultured with BMSCs, while the secretion level of BDNF, GDNF and NGF was dramatically elevated in co-culture. However, the neurite outgrowth-promoting effect of BMSCs was found to significantly reduced using antibodies to BDNF, GDNF and NGF. In addition, a fraction of BMSCs was found to exhibit NF-200 immunoreactivity. These results indicated that BMSCs could promote neurite outgrowth of motor neurons by means of neurotrophic factors. The findings of the present study provided new cues for the treatment of spinal cord injury.     

Regulation of Semaphorin III/Collapsin-1 Gene Expression during Peripheral Nerve Regeneration

The competence of neurons to regenerate depends on their ability to initiate a program of gene expression supporting growth and on the growth-permissive properties of glial cells in the distal stump of the injured nerve. Most studies on intrinsic molecular mechanisms governing peripheral nerve regeneration have focussed on the lesion-induced expression of proteins promoting growth cone motility, neurite extension, and adhesion. However, little is known about the expression of intrinsic chemorepulsive proteins and their receptors, after peripheral nerve injury and during nerve regeneration. Here we report the effect of peripheral nerve injury on the expression of the genes encoding sema III/coll-1 and its receptor neuropilin-1, which are known to be expressed in adult sensory and/or motor neurons. We have shown that peripheral nerve crush or transection results in a decline in sema III/coll-1 mRNA expression in injured spinal and facial motor neurons. This decline was paralleled by an induction in the expression of the growth-associated protein B-50/GAP-43. As sema III/coll-1 returned to normal levels following nerve crush, B-50/GAP-43 returned to precrush levels. Thus, the decline in sema III/coll-1 mRNA coincided with sensory and motor neuron regeneration. A sustained decline in sema III/coll-1 mRNA expression was found when regeneration was blocked by nerve transection and ligation. No changes were observed in neuropilin-1 mRNA levels after injury to sensory and motor neurons, suggesting that regenerating peripheral neurons continue to be sensitive to sema III/coll-1. Therefore we propose that a decreased expression of sema III/coll-1, one of the major ligands for neuropilin-1, during peripheral nerve regeneration is an important molecular event that is part of the adaptive response related to the success of regenerative neurite outgrowth occurring following peripheral nerve injury.