GPI-1046 stimulates chicken dorsal root ganglion neurite outgrowth in the presence of nerve growth factor at low concentration in vitro.
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The purpose of this investigation was to re-evaluate the neurotrophic effect of GPI-1046 on neurite outgrowth in vitro. GPI-1046 was synthesized and identified with mass spectrometry, nuclear magnetic resonance and elemental analysis. Chicken dorsal root ganglions (DRGs) were removed and divided into three groups: (1) The DRGs were cultured in DMEM containing different concentrations of GPI-1046; (2) The DRGs were cultured in DMEM containing nerve growth factor (NGF) alone at 0.8 and 8 ng/mL, respectively; (3) The DRGs were cultured in DMEM containing both different concentrations of GPI-1046 and NGF at 0.8 ng/mL. The results showed that GPI-1046 alone could not stimulate chicken DRG neurite outgrowth; however, GPI-1046 stimulated DRG neurite outgrowth only in the presence of NGF at low concentration in the culture medium.Keywords:
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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.
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Most neurotrophic factors are members of one of three families: the neurotrophins, the glial cell-line derived neurotrophic factor family ligands (GFLs) and the neuropoietic cytokines. Each family activates distinct but overlapping cellular pathways. Several studies have shown additive or synergistic interactions between neurotrophic factors from different families, though generally only a single combination has been studied. Because of possible interactions between the neurotrophic factors, the optimum concentration of a factor in a mixture may differ from the optimum when applied individually. Additionally, the effect of combinations of neurotrophic factors from each of the three families on neurite extension is unclear. This study examines the effects of several combinations of the neurotrophin nerve growth factor (NGF), the GFL glial cell-line derived neurotrophic factor (GDNF) and the neuropoietic cytokine ciliary neurotrophic factor (CNTF) on neurite outgrowth from young rat dorsal root ganglion (DRG) explants. The combination of 50 ng ml−1 NGF and 10 ng ml−1 of each GDNF and CNTF induced the highest level of neurite outgrowth at a 752 ± 53% increase over untreated DRGs and increased the longest neurite length to 2031 ± 97 µm compared to 916 ± 64 µm for untreated DRGs. The optimum concentrations of the three factors applied in combination corresponded to the optimum concentration of each factor when applied individually. These results indicate that the efficacy of future therapies for nerve repair would be enhanced by the controlled release of a combination of neurotrophins, GFLs and neuropoietic cytokines at higher concentrations than used in previous conduit designs.
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ABSTRACT Introduction There is a clinical need to improve the outcomes of peripheral nerve regeneration and repair after injury. In addition to its immunosuppressive effects, FK506 (tacrolimus) has been shown to have neuroregenerative properties. To determine biologically relevant local FK506 and growth factor concentrations, we performed an in vitro bioassay using dorsal root ganglion (DRG) from chicken embryos. Methods Neurite elongation and neurite branching were analyzed microscopically after addition of FK506, glial cell line‐derived neurotrophic factor (GDNF), and nerve growth factor (NGF), each alone and in combination. Results FK506 induced modest neurite elongation (∼500–800 µm) without improving neurite branching significantly. The combination of FK506 with NGF, GDNF, or both, exerted a potentiating or competitive effect on neurite elongation (∼700–1100 µm) based on dosage and competitive effect on neurite branching (∼0.2–0.4). Conclusions These results strongly suggest that the interaction of FK506 with GDNF and NGF mediates distinct enhancement of neurite growth. Muscle Nerve 55 : 570–581, 2017
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Abstract Fifteen‐day embryonic rat dorsal root ganglion (DRG) neurons were exposed to 1 to 200 ng/ml nerve growth factor (NFG). Maximal neurite outgrowth was obtained with 10 to 20 ng/ml. Neurite outgrowth was reduced to 89% of maximal by increasing NGF to 50 ng/ml, to 66% by 100 ng/ml, and to 18% by 200 ng/ml NGF. Identical effects were seen with mouse 2.5S NGF and recombinant human NGF. Neuron cell counts demonstrated that significant cell death did not occur. In time course experiments, significant inhibition, compared with control, began within 1 hour of adding 200 ng/ml and 3 hours of adding 50 ng/ml NGF. The inhibitory effect of NGF on neurite outgrowth was reversed within 3 hours when DRG were incubated with 5 ng/ml NGF after treatment with 50 or 200 ng/ml NGF medium for 12 hours. The inhibition demonstrated for neurons did not occur in PC12 cells; axonal growth was not inhibited by up to 1,000 ng/ml NGF. Excess brain‐derived neurotrophic factor or neurotrophin‐3 did not inhibit neurite outgrowth. We conclude that high concentrations of NGF produces specific and reversible arrest of neurite outgrowth from sensory neurons. This observation has important clinical implications, because these inhibitory concentrations have been exceeded when NGF has been administered into the central nervous system of humans and animals.
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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.
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The purpose of this investigation was to re-evaluate the neurotrophic effect of GPI-1046 on neurite outgrowth in vitro. GPI-1046 was synthesized and identified with mass spectrometry, nuclear magnetic resonance and elemental analysis. Chicken dorsal root ganglions (DRGs) were removed and divided into three groups: (1) The DRGs were cultured in DMEM containing different concentrations of GPI-1046; (2) The DRGs were cultured in DMEM containing nerve growth factor (NGF) alone at 0.8 and 8 ng/mL, respectively; (3) The DRGs were cultured in DMEM containing both different concentrations of GPI-1046 and NGF at 0.8 ng/mL. The results showed that GPI-1046 alone could not stimulate chicken DRG neurite outgrowth; however, GPI-1046 stimulated DRG neurite outgrowth only in the presence of NGF at low concentration in the culture medium.
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Neurite outgrowth from dorsal root ganglion (DRG) explants is a method of evaluating neurotrophic activity of growth factors. When complete medium containing collagen was supplemented with nerve growth factor (NGF) DRG outgrowth was observed after 18 h. In the absence of NGF and in the presence of collagen, the DRG outgrowth took place after 72 h. In wells not supplemented with collagen gel in substratum, no DRG outgrowth was observed. Partially, DRG differentiation was observed in the presence of NGF. In the absence of NGF and collagen, there was no DRG outgrowth detected. It seems that, in some circumstances, cells degenerated by DRG may be an indication of an apoptosis phenomenon. Therefore, we suggested that collagen as a substratum is more effective than
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We used compartmented cultures to study the regulation of adult sensory neurite growth by neurotrophins. We examined the effects of the neurotrophins nerve growth factor (NGF), neurotrophin-3 (NT3), and BDNF on distal neurite elongation from adult rat dorsal root ganglion (DRG) neurons. Neurons were plated in the center compartments of three-chambered dishes in the absence of neurotrophin, and neurite extension into the distal (side) compartments containing NGF, BDNF, or NT3 was quantitated. Initial proximal neurite growth did not require any of the neurotrophins, while subsequent elongation into distal compartments required NGF. After neurites had extended into NGF-containing distal compartments, removal of NGF by treatment with anti-NGF resulted in the cessation of growth with minimal neurite retraction. In contrast to the effects of NGF, no distal neurite elongation was observed into compartments with BDNF or NT3. To examine possible additive influences, neurite extension into compartments containing BDNF plus NGF or NT3 plus NGF was quantitated. There was no increased neurite extension into NGF plus NT3 compartments, while the combination of BDNF plus NGF resulted in an inhibition of neurite extension compared with NGF alone. We then investigated whether the regrowth of neurites that had originally grown into NGF subsequent to in vitro axotomy still required NGF. The results demonstrated that unlike adult sensory nerve regeneration in vivo, the in vitro regrowth did require NGF, and neither BDNF nor NT3 was able to substitute for NGF. Since the initial growth from neurons after dissociation (which is also a regenerative response) did not require NGF, it would appear that neuritic growth and regrowth of adult DRG neurons in vitro includes both NGF-independent and NGF-dependent components. The compartmented culture system provides a unique model to further study aspects of this differential regulation of neurite growth. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 395–410, 1997
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The compartmented culture method was used to study the regulation of adult sensory neurite growth by neurotrophins. We examined the effects of the neurotrophins nerve growth factor (NGF), neurotrophin-3 (NT3), and brain derived neurotrophic factor (BDNF) on neurite elongation from adult rat dorsal root ganglion (DRG) neurons. Initial proximal neurite growth (within center compartments) did not require neurotrophins. Subsequent elongation into distal compartments resulted from NGF but not NT3 or BDNF treatment. After neurites had extended into distal compartments, treatment with anti-NGF resulted in the cessation of growth with no significant neurite retraction. After axotomy of growing neurites in distal compartments, NGF was necessary to support regrowth. In the same paradigm, both NT3 and BDNF did not result regrowth. The results demonstrated that unlike in vivo nerve regeneration, in vitro regrowth does require NGF. -- Given the above results, the next focus of study was to determine the individual contributions ofthe NGF receptors to the growth response. The requirement of both TrkA and the p75 neurotrophin receptor in neurite growth was examined using several experimental interventions. As expected, inhibition of TrkA activation using K252a totally blocked distal neurite extension into NGF containing compartments. Results showed using BDNF to interfere with NGF binding to p75, found that the addition of BDNF to NGF containing distal compartments reduced distal neurite extension. In contrast MC192 which alters the interaction of NGF with p75, completely inhibited NGF dependent neurite growth. Both compounds were found to partially attenuate NGF induced TrkA phosphorylation. However only MC192 appeared to activate p75 based on immunocytochemical evidence showing nuclear localization of NFk-B. -- These results demonstrate that both TrkA and p75 play a role in neurite growth response to NGF. Furthermore our evidence suggests that any alteration in optimal TrkA-p75 interactions, or direct activation of p75 at the expense of TrkA, results in an inhibition of NGF-dependent neurite growth. -- NGF/TrkA signaling has a number of common pathways in which to elicit various physiological responses. Many of the same pathways are used by other growth factors that also are capable of producing neurite growth in adult sensory neurons. To investigate the influence of other growth factors in addition to NGF, the effects of IGF-1 EGF and FGF on neurite growth from adult rat dorsal root ganglion (DRG) neurons were examined. As expected, NGF elicited robust neuritic growth in both the dissociated and compartmented cultures. The growth response to IGF-1 was similar. There was minimal neurite growth in response to EGF or FGF. In addition, IGF-1 (but neither FGF nor EGF), when applied to cell bodies in compartmented cultures, potentiated the distal neurite growth into NGF- containing side compartments. -- In order to determine the contribution of signaling intermediates downstream of receptor activation, we used pharmacological inhibitors and western blotting. The PI 3- kinase inhibitor, LY294002 attenuated neurite growth evoked by NGF, IGF and EGF in dissociated cultures, while the MEK inhibitor PD98059 only diminished growth in IGF treated cultures. Immunoprecipitation and western blotting results demonstrated differential activation of MAPK, PI 3-kinase, PLCyl and SNT by the different factors. Activation of PI 3-kinase and SNT by both NGF and IGF-1 correlated with their effects on neurite growth. These results support the hypothesis that the PI 3-kinase pathway, and the SNT protein play an important role in neurogenesis. -- In conclusion neurite growth of adult DRG neurons is mediated by NGF in vitro and required TrkA activation. In comparison these results indicate that inhibition of NGF binding to p75 partially inhibits growth while activation of the receptor abolishes the response. Further assessment of NGF-TrkA signaling pathways, using other growth factors for comparison (i.e. IGF-1 and EGF) indicate that PI 3-kinase and SNT signaling intermediates are important contributors to the growth process. These experiments are a part of a larger focus on NGF and its role in neurite growth in neurons (adult DRG neurons) that do not require this factor for survival. Furthermore these studies will hopefully contribute to elucidating the processes involved in neurite growth.
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