Laminin and growth factor receptor activation stimulates differential growth responses in subpopulations of adult DRG neurons

Neurons in the adult rat dorsal root ganglion (DRG) can be classified into at least three separate subpopulations based on morphologic and phenotypic differences. In this study we have focused on the growth response of these specific subpopulations in vitro with respect to laminin (LN) and growth factor receptor activation. Using a cell selection approach we show that LN-induced neurite growth occurs in the absence of added trophic factors only in heavy-chain neurofilament-positive and calcitonin gene-related peptide-positive DRG neurons [nerve growth factor (NGF)-responsive population]. In contrast, LN alone is not sufficient to stimulate significant neurite growth from lectin Griffonia simplicifolia IB4-positive neurons (IB4+ve), although it is still required to elicit a growth response from these cells in the presence of glial-derived neurotrophic factor (GDNF, e.g. neurite growth occurred only when cells were plated on LN in the presence of GDNF). By using chemical inhibitors we demonstrate that only the phosphatidylinositol 3 kinase (PI 3-K)/Akt pathway is required for neurite growth from the NGF-responsive cell population. However, both the PI 3-K/Akt and MEK/mitogen-activated protein kinase signaling pathways are required for neurite growth from the IB4+ve cell population. Thus, we have identified specific signaling events and environmental requirements associated with neurite growth for different subpopulations of adult DRG neurons, pointing to potential therapeutic targets while identifying an inability for any one treatment alone to repair peripheral nerve damage.     

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.     

Expression of soluble Nogo-66 receptor and brain-derived neurotrophic factor in transduced rat bone marrow stromal cells

Bone marrow stromal cells (BMSCs) are promising candidates for cell transplantation in the central nervous system. When grafted into injury sites, they may be able to form ‘guiding strands’ for host axonal growth, and secrete nerve growth factor and brain-derived neurotrophic factor (BDNF) to support injured neurons and axons.However, they have no effect on the inhibitory molecules secreted locally following neuronal injury. The Nogo-66 receptor (NgR) plays a key role in inhibiting axon regeneration in the central nervous system. Exogenous soluble NgR can competitively bind to inhibitors and improve locomotor function recovery. In this study, a gene encoding soluble NgR was cloned and transduced into rat BMSCs using a lentiviral vector. Expression of soluble NgR was detected in the rat BMSCs. NgR-expressing BMSCs also secreted BDNF during culture in vitro. These results indicate that transduced BMSCs not only antagonize the effects of molecules inhibiting axon growth but also express neurotrophic factors, and thus have the potential to promote axon regeneration via more than one mechanism.     

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.     

Factors contributing to neurotrophin-independent survival of adult sensory neurons

Dorsal root ganglion (DRG) sensory neurons become less dependent upon neurotrophins for their survival as they mature. DRG neurons from young adult rats were dissociated and cultured in vitro in serum-free defined medium. We show that adult DRG sensory neurons are able to survive for at least 2 weeks in culture in the absence of nerve growth factor (NGF). We then investigated potential mechanisms contributing to this apparent neurotrophin-independent survival in these neurons through the use of inhibitors of cellular signaling pathways. The phosphoinositide kinase-3 (PI 3-K) inhibitor LY294002, and a protein kinase C (PKC) inhibitor, chelerythrine resulted in significant decreases in neuronal survival. Neither the mitogen activated protein kinase kinase (MEK) inhibitor U0126 nor two other PKC inhibitors (bisindolylmaleimide and rottlerin) had any significant effect on survival. Our results point to the importance of PI 3-K and PKC signaling in the neurotrophin-independent survival of adult DRG neurons.     

Alterations in tyrosine kinase receptor (Trk) expression induced by insulin-like growth factor-1 in cultured dorsal root ganglion neurons

Insulin-like growth factor-1 (IGF-1) is a neurotrophic factor expressed in small dorsal root ganglion (DRG) neurons. IGF-1 promotes neuronal survival by activating its receptor (IGF-1R). Whether IGF-1 and its signaling pathways influence the expression of tyrosine kinase receptors TrkA, TrkB and TrkC in DRG neurons remains unknown. In the present study, primary cultured DRG neurons were used to determine the effects of IGF-1 on TrkA, TrkB and TrkC expression. The involvement of extracellular signal-regulated protein kinase (ERK1/2) and the effects of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways on IGF-1 were also evaluated. DRG neurons were cultured for 48 h and then exposed to IGF-1, PD98059 plus IGF-1, LY294002 plus IGF-1, and PD98059 plus LY294002 plus IGF-1 for an additional 24 h. The DRG neurons were continuously exposed to culture medium as a control. All cultures were then processed for detection of mRNA levels of TrkA, TrkB and TrkC using real-time PCR analysis. Protein levels of TrkA, TrkB and TrkC were detected using a Western blot assay. The expression of TrkA, TrkB and TrkC in situ was determined by a fluorescent labeling technique. The levels of phosphorylated ERK1/2 (pERK1/2) and phosphorylated Akt (pAkt) were detected using a Western blot assay. The results indicated that in primary cultured DRG neurons, IGF-1 increased the expression of TrkA and TrkB and their mRNAs but not TrkC or its mRNA. Neither the ERK1/2 inhibitor PD98059 nor the PI3K inhibitor LY294002 alone blocked the effect of IGF-1, but the use of both inhibitors together was effective. IGF-1 may play an important role in regulating the expression of different Trk receptors in DRG neurons through the ERK1/2 and PI3K/Akt signaling pathways. These results suggest that IGF-1 signaling might be a potential target on modifying distinct Trk receptor-mediated biological effects.     

Prostaglandin E2 contributes to the synthesis of brain-derived neurotrophic factor in primary sensory neuron in ganglion explant cultures and in a neuropathic pain model

Brain-derived neurotrophic factor (BDNF) exists in small to medium size neurons in adult rat dorsal root ganglion (DRG) and serves as a modulator at the first synapse of the pain transmission pathway in the spinal dorsal horn. Peripheral nerve injury increases BDNF expression in DRG neurons, an event involved in the genesis of neuropathic pain. In the present study, we tested the hypothesis that prostaglandin E2 (PGE2) over-produced in injured nerves contributes to the up-regulation of BDNF in DRG neurons. Two weeks after partial sciatic nerve ligation (PSNL), BDNF levels in the ipsilateral L4–L6 DRG of injured rats were significantly increased compared to the contralateral side. Perineural injection of a selective cyclooxygenase (COX2) inhibitor or a PGE2 EP4 receptor antagonist not only dose-dependently relieved PSNL elicited mechanical hypersensitivity, but also suppressed the increased BDNF levels in DRG neurons. PSNL shifted BDNF expression in the ipsilateral DRG from small to medium and larger size injured neurons. BDNF is mainly co-expressed with the EP1 and EP4 while moderately with the EP2 and EP3 receptor subtypes in naïve and PSNL rats. PSNL also shifted the expression of EP1–4 receptors to a larger size population of DRG neurons. In DRG explant cultures, a stabilized PGE2 analog 16,16 dimethyl PGE2 (dmPGE2) or the agonists of EP1 and EP4 receptors significantly increased BDNF levels and the phosphorylated protein kinase A (PKA), extracellular signal-regulated kinase (ERK)/mitogen activated protein kinase (MAPK) and cAMP response element binding protein (CREB). The EP1 and EP4 antagonists, a sequester of nerve growth factor (NGF), the inhibitors of PKA and MEK as well as CREB small interfering RNA suppressed dmPGE2-induced BDNF. Taken together, EP1 and EP4 receptor subtypes, PKA, ERK/MAPK and CREB signaling pathways as well as NGF are involved in PGE2-induced BDNF synthesis in DRG neurons. Injured nerve derived-PGE2 contributes to BDNF up-regulation in DRG neurons following nerve injury. Facilitating the synthesis of BDNF in primary sensory neurons is a novel mechanism underlying the role of PGE2 in the genesis of neuropathic pain.     

Neurotrophic and growth factor gene expression profiling of mouse bone marrow stromal cells induced by ischemic brain extracts

Treatment of rodents after stroke with bone marrow stromal cells (BMSCs) improves functional outcome. However, the mechanisms underlying this benefit have not been ascertained. This study focused on the contribution of neurotrophic and growth factors produced by BMSCs to therapeutic benefit. Rats were subjected to middle cerebral artery occlusion and the ischemic brain extract supernatant was collected to prepare the conditioned medium. The counterpart normal brain extract from non‐ischemic rats was employed as the experimental control. Using microarray assay, we measured the changes of the neurotrophin associated gene expression profile in BMSCs cultured in different media. Furthermore, real‐time RT‐PCR and fluorescent immunocytochemistry were utilized to validate the gene changes. The morphology of BMSCs, cultured in the ischemic brain‐conditioned medium for 12 h, was dramatically altered from a polygonal and flat appearance to a fibroblast‐like long and thin cell appearance, compared to those in the normal brain‐conditioned medium and the serum replacement medium. Forty‐four neurotrophin‐associated genes in BMSCs were identified by microarray assay under all three culture media. Twelve out of the 44 genes (7 neurotrophic and growth factor genes, 5 receptor genes) increased in BMSCs cultured in the ischemic brain‐conditioned medium compared to the normal brain‐conditioned medium. Real time RT‐PCR and immunocytochemistry validated that the ischemic brain‐conditioned medium significantly increased 6/7 neurotrophic and growth factor genes, compared with the normal brain‐conditioned medium. These six genes consisted of fibroblast growth factor 2, insulin‐like growth factor 1, vascular endothelial growth factor A, nerve growth factor beta, brain‐derived neurotrophic factor and epidermal growth factor. Our results indicate that transplanted BMSCs may work as ‘small molecular factories’ by secreting neurotrophins, growth factors and other supportive substances after stroke, which may produce therapeutic benefits in the ischemic brain.     

Nerve growth factors (NGF, BDNF) enhance axonal regeneration but are not required for survival of adult sensory neurons

Largely on the basis of studies with nerve growth factor (NGF), it is now widely accepted that development of the peripheral nervous system of vertebrates is dependent in part on the interaction of immature sensory and autonomic neurons with specific survival factors that are derived from peripheral target fields. I have found, in marked contrast to an absolute requirement for NGF during development, that adult rat dorsal root ganglion sensory neurons are not dependent on NGF or other survival factors for long-term (3-4 weeks) maintenance in vitro. When dissociated and enriched, at least 70-80% of adult DRG neurons survived and extended long processes either in the absence of exogenously added NGF or upon the removal of any possible source of endogenous NGF or other neurotrophic activity (i.e., nonneuronal cells, in chemically defined culture medium, in the presence of an excess of anti-NGF antibodies, or when cultured as single neurons in microwells). Although not required for survival or expression of a range of complex morphologies, both NGF and brain-derived neurotrophic factor (BDNF) were found to stimulate the regeneration of axons from adult DRG neurons.     

Nerve growth factor regulates galanin and c-jun overexpression occurring in dorsal root ganglion cells after intravesical resiniferatoxin application

Galanin and c-jun expression after a single bladder instillation of resiniferatoxin was studied by immunocytochemistry in L6 dorsal root ganglia (DRG) neurons of the rat. The role of nerve growth factor depletion in causing that effect was also investigated. Three days after instillation of a 100 nM resiniferatoxin solution a marked increase in the number of galanin and c-Jun immunoreactive DRG cells was evident bilaterally. The increments were still present at 8 days and disappeared 1 month after treatment. Systemic administration of nerve growth factor, 100 microg/kg, prevented both overexpressions. Results suggest that the changes induced in bladder sensory neurons by intravesical resiniferatoxin are due, at least in part, to the temporary deprivation of bladder-derived neurotrophic factors, namely nerve growth factor, in those neurons.     

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。     

Differential activation of MAPK in injured and uninjured DRG neurons following chronic constriction injury of the sciatic nerve in rats

To investigate the intracellular signal transduction pathways involved in the pathophysiological mechanisms of neuropathic pain after partial nerve injury, we examined the activation of extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) in the dorsal root ganglion (DRG) in the chronic constriction injury (CCI) model. The CCI induced an increase in the phosphorylation of ERK in predominantly injured medium-sized and large-sized DRG neurons and in satellite glial cells. Treatment with the MAPK kinase 1/2 inhibitor, U0126, suppressed CCI-induced mechanical allodynia and partially reversed the increase in neuropeptide Y (NPY) expression in damaged DRG neurons. In contrast, the CCI induced the activation of p38, mainly in uninjured small-to-medium-diameter DRG neurons and in satellite glial cells. The p38 inhibitor, SB203580, reversed the CCI-induced heat hyperalgesia and also the increase in brain-derived neurotrophic factor (BDNF) expression in intact DRG neurons. On the other hand, the nerve growth factor (NGF)-induced increase in BDNF expression in small-to-medium-diameter neurons was reversed by SB203580, whereas the anti-NGF-induced increase in NPY in medium-sized and large-sized neurons was partially blocked by U0126. Taken together, our results demonstrate that the activation of ERK and p38 and also the changes in NPY and BDNF expression may occur in different populations of DRG neurons after CCI, partially through alterations in the target-derived NGF. These changes in injured and intact primary afferents are likely to have a substantial role in pathological states, and MAPK pathways in nociceptors may be potential targets for the development of novel analgesics.     

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.     

Nerve Growth Factor Regulates Expression of the Nerve Growth Factor Receptor Gene in Adult Sensory Neurons

Sensory neurons of the adult rat dorsal root ganglion (DRG) can be maintained in culture in the absence of nerve growth factor (NGF). We have thus used dissociated cultures of these neurons to study effects of NGF on the regulation of expression of mRNA encoding the nerve growth factor receptor (NGF-R). In the absence of NGF, levels of NGF-R mRNA remained constant for 7 days in cultures of adult rat DRG neurons. In the presence of NGF, NGF-R mRNA levels rose two - three-fold after 2 days, reaching plateau levels (five - six-fold elevation) after 5 days. This NGF-induced up-regulation could be demonstrated even after prior NGF-deprivation for 3 - 4 days. NGF had no effect upon NGF-R mRNA levels in DRG non-neuronal cells. Epidermal growth factor (EGF), fibroblast growth factor (FGF) and ciliary neurotrophic factor (CNTF) were without effect on NGF-R mRNA levels, but 8-bromo-cAMP decreased NGF-R mRNA levels by 65% after 2 days. NGF also induced a rapid (30 min) rise in expression of c-fos in DRG neurons, but not in non-neuronal cells. Our results suggest that endogenous levels of NGF may regulate the expression of NGF-R in vivo.     

ALK (Anaplastic Lymphoma Kinase) expression in DRG neurons and its involvement in neuron–Schwann cells interaction

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) transiently expressed in specific regions of the central and peripheral nervous systems. In this study, we focused on the rat developing dorsal root ganglion (DRG). This ganglion is composed of heterogeneous sensory neurons characterized by the expression of RTK for neurotrophic factors, such as the nerve growth factor receptor TrkA or the glial-derived neurotrophic factor family receptor Ret, which are specifically detected in nociceptive neurons. In DRG, ALK expression reached a maximum around birth. We showed that ALK is specifically present in a subtype of neurons during DRG development, and that the majority of these neurons co-expressed TrkA and Ret. Interestingly, we identified only one form (220 kDa) of ALK in DRG neurons both in vivo and in vitro . On the opposite, in transfected cells as well as in brain extracts, ALK was identified as two forms (220 and 140 kDa). The DRG is composed of neurons and glial cells, principally satellite Schwann cells. Thus, we hypothesized that the presence of satellite Schwann cells was involved in the absence of truncated ALK. Using two different cell types, HEK293 cells stably expressing ALK, and MSC80 cells, a previously described Schwann cell line, we showed that a factor secreted by the Schwann cells is likely involved in the absence of ALK cleavage. All these data hence open new perspectives concerning the role of ALK in the specification of nociceptive DRG neurons and in the neurons–Schwann cells interaction.     

In vitro neuroprotective effects of ciliary neurotrophic factor on dorsal root ganglion neurons with glutamate-induced neurotoxicity

Ciliary neurotrophic factor has neuroprotective effects mediated through signal transducer and Janus kinase (JAK) 2/activator of transcrip-tion 3 (STAT3) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways. Whether ciliary neurotrophic factor is neuroprotective for glutamate-induced excitotoxicity of dorsal root ganglion neurons is poorly understood. In the present study, the in vitro neuroprotective effects of ciliary neurotrophic factor against glutamate-induced excitotoxicity were determined in a primary culture of dorsal root ganglion neurons from Wistar rat embryos at embryonic day 15. Whether the JAK2/STAT3 and PI3K/Akt signaling pathways were related to the protective effects of ciliary neurotrophic factor was also determined. Glutamate exposure inhibited neurite outgrowth, cell viability, and growth-associated protein 43 expression and promoted apoptotic neuronal cell death, all of which were reversed by the administration of exogenous ciliary neurotrophic factor. Additionally, preincubation with either JAK2 inhibitor AG490 or PI3K inhibitor LY294002 blocked the neuroprotective effect of ciliary neurotrophic factor. These data indicate that the two pathways JAK2/STAT3 and PI3K/Akt play major roles in mediating the in vitro neuroprotective effects of ciliary neurotrophic factor on dorsal root ganglion neurons with glutamate-in-duced neurotoxicity.     

Rat astrocytes and Schwann cells in culture synthesize nerve growth factor-like neurite-promoting factors

Neurite-promoting activity in feeding medium conditioned by rat astrocytes and Schwann cells in culture was examined. The conditioned medium (CM) from both types of glial cultures stimulated extensive neurite outgrowth from embryonic chick dorsal root ganglia (DRG) as well as pheochromocytoma (PC12) cells. Both the DRG and PC12 cells also produce neurite outgrowth in the presence of nerve growth factor (NGF). With the DRG, the neurite growth rates observed with the glial cell CM were identical to growth rates seen with NGF. Although anti-NGF antibody did not inhibit the neurite outgrowth produced by either of the glial CM, a nerve growth factor radioreceptor assay did detect an NGF-like molecule in both CM. Since the extensive neurite outgrowth stimulated by the glial CM was not mimicked by pure laminin alone, we conclude that the glial neurite promoting factors are distinct from laminin.     

Expression of glial cell line-derived neurotrophic factor family of growth factors in peripheral nerve injury in rats

The glial cell line-derived neurotrophic factor (GDNF) family of growth factors may be involved in the regenerative support of neurons in the peripheral nervous system. In order to study the role of these growth factors and their receptors following rat peripheral nerve injury we examined the changes in their mRNA levels in the spinal cord, the dorsal root ganglia and the peripheral nerve trunk. Following transaction of the sciatic nerve GDNF mRNA was up-regulated rapidly in the denervated nerve distal to the cut along with the mRNA for one of its receptors, GFRalpha-1. GFRalpha-1 mRNA was also increased in the DRG ipsilateral to the nerve injury suggesting that GDNF may be involved in the trophic support of DRG sensory neurons. In contrast there were no analogous changes in the mRNA levels of neurturin, persephin and artemin following injury.     

Effects of IL-1beta, IL-6 or LIF on rat sensory neurons co-cultured with fibroblast-like cells.

Inflammation may affect the local presence of sensory nerve fibers in situ and inflammatory mediators influence sensory neurons in vitro. In the present study we have investigated effects of the cytokines interleukin-1beta (IL-1beta, interleukin-6 (IL-6), and leukemia inhibitory factor (LIF) on survival of and neurite growth from neonatal rat sensory neurons co-cultured with fibroblast-like cells prepared from neonatal rat skin (sFLCs) or perichondrium (pFLCs). The results showed that both FLC types expressed receptors for all three cytokines. Five ng/ml of either cytokine, but not lower or higher concentrations, supported survival of DRG neurons co-cultured with sFLCs. Neuronal survival was also enhanced by addition of the soluble IL-6 receptor (rsIL-6R) with or without IL-6. In co-cultures with pFLCs neuronal survival was promoted by IL-6, increasing with cytokine concentration. Addition of rsIL-6R without IL-6 did also stimulate neuronal survival. The growth of neurites from DRG neurons co-cultured with sFLCs was stimulated by 0.5 ng/ml LIF, unaffected by 5 ng/ml LIF and inhibited by 50 ng/ml LIF. Considering DRG neurons co-cultured with pFLCs, 50 ng/ml of either of the three cytokines, as well as rsIL-6R conditioned medium, stimulated neurite outgrowth. Some of the cytokine effects observed were reduced by application of antibodies against nerve growth factor (NGF). We conclude that that the cytokines examined affect DRG neurons in terms of survival or neuritogenesis, that the effects are influenced by cytokine concentration and the origin of the FLCs and that some of the effects are indirect, probably being mediated by factors released from FLCs.