Viability of olfactory ensheathing cells after hypoxia and serum deprivation: Implication for therapeutic transplantation

Olfactory ensheathing cells (OECs) represent glial cells supporting neuronal turnover in the olfactory system. In vitro, OECs promote axonal growth as a source of neurotrophic growth factors; in vivo, they produce myelin, promoting remyelination of damaged axons. Consequently, OEC transplantation appears to be a promising treatment for spinal cord injury, although the functional recovery is limited. This might be ascribed to the microenvironment at the lesion site, lacking growth factors (GFs), nutrients, and oxygen. To mimic this condition, we used an in vitro approach by growing primary neonatal mouse OECs under hypoxic conditions and/or serum deprivation. In addition, we compared OECs survival/proliferation with that of primary cultures of Schwann cells (SCs) and astrocytes under the same experimental conditions. Cultures were analyzed by immunocytochemistry, and cell viability was evaluated by MTT assay. Different GFs, such as NGF, bFGF, and GDNF, and their combination were used to rescue cells from serum and/or oxygen deprivation. We show that the cell types were differently sensitive to the tested stress conditions and that OECs were the most sensitive among them. Moreover, OEC viability was rescued by bFGF under serum‐deprived or hypoxic condition but not under conditions of drastic serum deprivation and hypoxia. bFGF was effective also for the other cell types, whereas the effect of the other GFs was negligible. This model suggests that administration of bFGF might be considered useful to sustain cell survival/proliferation after transplantation of OECs either alone or in combination with other glial cell types. © 2014 Wiley Periodicals, Inc.     

Dental pulp stem cells stimulate neuronal differentiation of PC12 cells

Dental pulp stem cells (DPSCs) secrete neurotrophic factors which may play an important therapeutic role in neural development, maintenance and repair. To test this hypothesis, DPSCs-conditioned medium (DPSCs-CM) was collected from 72 hours serum-free DPSCs cultures. The impact of DPSCs-derived factors on PC12 survival, growth, migration and differentiation was investigated. PC12 cells were treated with nerve growth factor (NGF), DPSCs-CM or co-cultured with DPSCs using Transwell inserts for 8 days. The number of surviving cells with neurite outgrowths and the length of neurites were measured by image analysis. Immunocytochemical staining was used to evaluate the expression of neuronal markers NeuN, microtubule associated protein 2 (MAP-2) and cytoskeletal marker βIII-tubulin. Gene expression levels of axonal growth-associated protein 43 and synaptic protein Synapsin-I, NeuN, MAP-2 and βIII-tubulin were analysed by quantitative polymerase chain reaction (qRT-PCR). DPSCs-CM was analysed for the neurotrophic factors (NGF, brain-derived neurotrophic factor [BDNF], neurotrophin-3, and glial cell-derived neurotrophic factor [GDNF]) by specific ELISAs. Specific neutralizing antibodies against the detected neurotrophic factors were used to study their exact role on PC12 neuronal survival and neurite outgrowth extension. DPSCs-CM significantly promoted cell survival and induced the neurite outgrowth confirmed by NeuN, MAP-2 and βIII-tubulin immunostaining. Furthermore, DPSCs-CM was significantly more effective in stimulating PC12 neurite outgrowths than live DPSCs/PC12 co-cultures over the time studied. The morphology of induced PC12 cells in DPSCs-CM was similar to NGF positive controls; however, DPSCs-CM stimulation of cell survival was significantly higher than what was seen in NGF-treated cultures. The number of surviving PC12 cells treated with DPSCs-CM was markedly reduced by the addition of anti-GDNF, whilst PC12 neurite outgrowth was significantly attenuated by anti-NGF, anti-GDNF and anti-BDNF antibodies. These findings demonstrated that DPSCs were able to promote PC12 survival and differentiation. DPSCs-derived NGF, BDNF and GDNF were involved in the stimulatory action on neurite outgrowth, whereas GDNF also had a significant role in promoting PC12 survival. DPSCs-derived factors may be harnessed as a cell-free therapy for peripheral nerve repair. All experiments were conducted on dead animals that were not sacrificed for the purpose of the study. All the methods were carried out in accordance with Birmingham University guidelines and regulations and the ethical approval is not needed.

Chinese Library Classification No. R459.9; R364; R622     

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.     

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.     

Acidic and basic fibroblast growth factors enhance neurite outgrowth in cultured rat spinal cord neurons

Abstract

We have studied neurotrophic effects of acidic fibroblast growth factor (aFCF) and basic fibroblast growth factor (bFGF) on explanted ventral and dorsal spinal cord cultures from 13- and 14-day-old rat embryos. Cultures treated with aFCF and bFGF significantly enhanced neurite outgrowth with cultures of ventral spinal cord, but not with cultures of dorsal spinal cord. Our data suggest that aFCF and bFGF are potent neurotrophic factors on rat ventral spinal cord neurons in vitro. [Neurol Res 1995; 17: 70-72]     

Fibroblast Growth Factor Treatment Produces Differential Effects on Survival and Neurite Outgrowth from Identified Bulbospinal Neurons in Vitro

The in vivo application of appropriate trophic factors may enhance regeneration of bulbospinal projections after spinal cord injury. Currently, little is known about the sensitivities of specific bulbospinal neuron populations to the many identified trophic factors. We devised novel in vitro assays to study trophic effects on the survival and neurite outgrowth of identified bulbospinal neurons. Carbocyanine dye crystals implanted into the cervical spinal cord of embryonic day (E)5 chick embryos retrogradely labeled developing bulbospinal neurons. On E8, dissociated cultures containing labeled bulbospinal neurons were prepared. Fibroblast growth factor (FGF)-2 (but not FGF-1) promoted the survival of bulbospinal neurons. FGF receptor expression was widespread in the E8 brainstem, but not detected in young bulbospinal neurons, suggesting that nonneuronal cells mediated the FGF-stimulated survival response. Astrocytes synthesize a variety of trophic factors, and astrocyte-conditioned medium (ACM) also promoted the survival of bulbospinal neurons. As might be expected, FGF-2 function blocking antibodies did not suppress ACM-promoted survival, nor did an ELISA detect FGF-2 in ACM. This suggests that nonneuronal cells synthesize other factors in response to exogenous FGF-2 which promote the survival of bulbospinal neurons. Focusing on vestibulospinal neurons, dissociated (survival assay) or explant (neurite outgrowth assay) cultures were prepared. FGF-2 promoted both survival and neurite outgrowth of identified vestibulospinal neurons. Interestingly, FGF-1 promoted neurite outgrowth but not survival; the converse was true of FGF-9. Thus, differential effects of specific growth factors on survival or neurite outgrowth of bulbospinal neurons were distinguished.     

Cellular GDNF delivery promotes growth of motor and dorsal column sensory axons after partial and complete spinal cord transections and induces remyelination

Glial cell line-derived neurotrophic factor (GDNF) is the prototypical member of a growth factor family that signals via the cognate receptors ret and GDNF-receptor alpha-1. The latter receptors are expressed on a variety of neurons that project into the spinal cord, including supraspinal neurons, dorsal root ganglia, and local neurons. Although effects of GDNF on neuronal survival in the brain have previously been reported, GDNF effects on injured axons of the adult spinal cord have not been investigated. Using an ex vivo gene delivery approach that provides both trophic support and a cellular substrate for axonal growth, we implanted primary fibroblasts genetically modified to secrete GDNF into complete and partial mid-thoracic spinal cord transection sites. Compared to recipients of control grafts expressing a reporter gene, GDNF-expressing grafts promoted significant regeneration of several spinal systems, including dorsal column sensory, regionally projecting propriospinal, and local motor axons. Local GDNF expression also induced Schwann cell migration to the lesion site, leading to remyelination of regenerating axons. Thus, GDNF exerts tropic effects on adult spinal axons and Schwann cells that contribute to axon growth after injury.     

Ciliary neurotrophic factor stimulates neurite outgrowth from spinal cord neurons

Ciliary neurotrophic factor (CNTF) has been shown to promote the survival of motoneurons, but its effects on axonal outgrowth have not been examined in detail. Since nerve growth factor (NGF) promotes the outgrowth of neurites within the same populations of neurons that depend on NGF for survival, we investigated whether CNTF would stimulate neurite outgrowth from motoneurons in addition to enhancing their survival. We found that CNTF is a powerful promoter of neurite outgrowth from cultured chick embryo ventral spinal cord neurons. An effect of CNTF on neurite outgrowth was detectable within 7 hours, and at a concentration of 10 ng/ml, CNTF enhanced neurite length by about 3- to 4-fold within 48 hours. The neurite growth-promoting effect of CNTF does not appear to be a consequence of its survival-promoting effect. To determine whether the effect of CNTF on spinal cord neurons was specific for motoneurons, we analyzed cell survival and neurite outgrowth for motoneurons labeled with diI, as well as for neurons taken from the dorsal half of the spinal cord, which lacks motoneurons. We found that the effect of CNTF was about the same for motoneurons as it was for neurons from the dorsal spinal cord. The responsiveness of a variety of spinal cord neurons to CNTF may broaden the appeal of CNTF as a candidate for the treatment of spinal cord injury or disease. © 1996 Wiley-Liss, Inc.     

Retinoic acid responsive gene product,midkine, has neurotrophic functions for mouse spinal cord and dorsal root ganglion neurons in culture

Midkine (MK) is the product of a retinoic acid responsive gene and is a member of a new family of heparin-binding growth factors. Neurotrophic effects of MK were examined using cultured spinal cord and dorsal root ganglion (DRG) neurons derived from fetal mouse. MK, which was added to the culture medium at concentrations of 1–100 ng/ml, promoted survival of both types of neurons approximately 5-fold after 7 days in culture. For spinal cord neurons, the increased survival was reflected in an increase of choline acetyltransferase activity. MK also promoted neurite extension in spinal cord (2-fold) and DRG (1.7-fold) neurons. The survival-promoting activity of MK to these neurons was comparable to that of basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF). In spite of its significant effects on fetal neurons, MK was ineffective in sustaining survival of DRG neurons derived from postnatal mice. From these results, we conclude that MK is a neurotrophic factor to embryonic spinal cord and DRG neurons, and we propose that MK plays a significant role in embryogenesis of the nervous system. © 1993 Wiley-Liss, Inc.     

Neuronal differentiation elicited by glial cell line-derived neurotrophic factor and ciliary neurotrophic factor in adrenal chromaffin cell line tsAM5D immortalized with temperature-sensitive SV40 T-antigen

To understand the characteristics of tsAM5D cells immortalized with the temperature-sensitive simian virus 40 large T-antigen, we first examined the responsiveness of the cells to ligands of the glial cell line-derived neurotrophic factor (GDNF) family. tsAM5D cells proliferated at the permissive temperature of 33 degrees C in response to either GDNF or neurturin, but not persephin or artemin. At the nonpermissive temperature of 39 degrees C, GDNF or neurturin caused tsAM5D cells to differentiate into neuron-like cells; however, the differentiated cells died in a time-dependent manner. Interestingly, ciliary neurotrophic factor (CNTF) did not affect the GDNF-mediated cell proliferation at 33 degrees C but promoted the survival and differentiation of GDNF-treated cells at 39 degrees C. In the presence of GDNF plus CNTF, the morphological change induced by the temperature shift was associated with up-regulated expression of various neuronal marker genes, indicating that the cells had undergone neuronal differentiation. In addition, tsAM5D cells caused to differentiate by GDNF plus CNTF at 39 degrees C became dependent solely on nerve growth factor (NGF) for their survival and neurite outgrowth. Moreover, upon treatment with GDNF plus CNTF, the dopaminergic phenotype was suppressed by the temperature shift. Thus, we demonstrated that tsAM5D cells had the capacity to differentiate terminally into neuron-like cells in response to GDNF plus CNTF when the oncogene was inactivated by the temperature shift. This cell line provides a useful model system for studying the role of a variety of signaling molecules for GDNF/CNTF-induced neuronal differentiation.     

Responsiveness of cultured septal and hippocampal neurons to ethanol and neurotrophic substances

Dissociated septal and hippocampal neurons from E18 fetal rats were cultured with varying concentrations of ethanol (0.6–2.4 g/dl) and in cultures containing ethanol plus nerve growth factor (NGF) or basic fibroblast growth factor (bFGF). These substances have been shown to provide neurotrophic support for these populations and to afford neuroprotection against certain toxic substances or conditions applied to some neuronal populations. Both the septal and hippocampal neurons responded to ethanol in a dosedependent manner. Survival of septal neurons was generally unaffected by initial ethanol concentrations of 0.6 and 1.2 g/dl but was considerably impaired by higher concentrations (1.8 and 2.4 g/dl), while neurite outgrowth was compromised by all ethanol concentrations except the lowest one applied. The hippocampal neurons survived ethanol concentrations up to 2.4 g/dl, although process extension was decreased in concentrations of 1.2 g/dl and higher. NGF or bFGF in the culture medium (in cultures without ethanol) did not affect neuronal survival or process outgrowth in either population, probably owing to the relatively high plating densities of the cultures. NGF did tend to have a moderate ameliorative effect on the ethanol neurotoxicity in the septal cultures, however, and was slightly effective in this regard in hippocampal cultures at intermediate ethanol concentrations (1.8 g/dl). High concentrations of ethanol (2.4 g/dl) reduced the proportion of cholinergic cells in the septal preparations by approximately 50%. This neuronal loss could be reversed by inclusion of high concentrations of NGF in the culture medium (100 ng/ml) but not by a lower concentration (20 ng/ml). bFGF provided some protection against ethanol cytotoxicity with respect to both populations. The implications of these results for studies of fetal alcohol effects are discussed, as well as their relation to prior reports of trophic factor neuroprotection. © 1994 Wiley-Liss, Inc.     

IGF-1 and BDNF promote chick bulbospinal neurite outgrowth in vitro

Injured neurons in the CNS do not experience significant functional regeneration and so spinal cord insult often results in permanently compromised locomotor ability. The capability of a severed axon to re-grow is thought to depend on numerous factors, one of which is the decreased availability of neurotrophic factors. Application of trophic factors to axotomized neurons has been shown to enhance survival and neurite outgrowth. Although brainstem-spinal connections play a pivotal role in motor dysfunction after spinal cord injury, relatively little is known about the trophic sensitivity of these populations. This study explores the response of bulbospinal populations to various trophic factors. Several growth factors were initially examined for potential trophic effects on the projection neurons of the brainstem. Brain derived neurotrophic factor (BDNF) and insulin-like growth factor (IGF-1) significantly enhance mean process length in both the vestibulospinal neurons and spinal projection neurons from the raphe nuclei. Nerve growth factor (NGF), neurotrophin-4 (NT-4) and glial derived neurotrophic factor (GDNF) did not effect process outgrowth in vestibulospinal neurons. At the developmental stages used in this study, it was determined that receptors for BDNF and IGF-1 were present both on bulbospinal neurons and on surrounding cells with a non-neuronal morphology.     

Changing responsiveness of developing midbrain dopaminergic neurons for extracellular growth factors

Numerous purified growth factors as well as yet-unidentified neurotrophic activities within mesencephalic glia support the survival of dopaminergic neurons. To further characterize the functional role of these multiple growth factor influences in dopaminergic cell development, various purified growth factors as well as mesencephalic glial-conditioned medium (CM) were screened for effects on dopaminergic cell survival and glial numbers in serum-free low density cultures of the dissociated embryonic day (E) 15 and E17 rat mesencephalon. In E15 mesencephalic cultures, dopaminergic cell survival increased with brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), transforming growth factor α (TGFα), insulin-like growth factor-1 (IGF-1), platelet-derived growth factor-BB (PDGF-BB), and interleukin-6 (IL-6). bFGF, TGFα, PDGF, and IL-6 also stimulated glial proliferation as demonstrated by autoradiographic labeling for ³H-thymidine. Moreover, CM derived from the mesencephalic glial cell line Mes42 completely prevented the death of E15 dopaminergic neurons within the initial days of cultivation. In E17 mesencephalic cultures, survival-promoting effects on dopaminergic neurons were present with BDNF, GDNF, and bFGF. TGFα, IGF-1, PDGF-BB, and IL-6 stimulated glial proliferation but did not affect dopaminergic cell survival. Similarly, mesencephalic glial-CM completely failed to support the survival of E17 dopaminergic neurons. These observations demonstrate that during embryonic development, dopaminergic cell survival sequentially depends on distinct sets of growth factors. The concomitant loss of sensitivity of developing dopaminergic neurons for mesencephalic glial-CM as well as TGFα, IGF-1, PDGF-BB, and IL-6 further provides evidence that these growth factors indirectly affect early dopaminergic neurons through glial-mediated processes and suggests a crucial role of glia during the initial stages of neuronal development. J. Neurosci. Res. 51:508–516, 1998. © 1998 Wiley-Liss, Inc.     

Establishment of pure neuronal cultures from fetal rat spinal cord and proliferation of the neuronal precursor cells in the presence of fibroblast growth factor.

A primary culture system of nearly pure neuronal cells from 14-day-old fetal rat spinal cord has been developed by combining a preplating step, the use of a chemically defined serum-free medium, and borated polylysine-coated dishes that prevented the formation of cell aggregates. About 98% of the cells were found to be immunostained with neuron-specific enolase antibodies, confirming their neuronal nature. The cultures are composed essentially of a population of non-motoneurons and contain few motoneurons, characterized by their large size and multipolar aspect, the presence of acetylcholinesterase (AChE), and the intense immunoreaction for growth-associated protein GAP-43. Neuronal precursor cells are also present in these cultures and proliferate during the first 3 days. The addition of bovine brain basic fibroblast growth factor (bFGF) stimulates their proliferation over a period of 2 days, as determined by measurement of [125I]iododeoxyuridine incorporation and by immunocytochemical reaction after bromodeoxyuridine incorporation into nuclei. The proliferating cells were characterized as neurons by immunostaining against neuron-specific enolase. Recombinant human bFGF and bovine brain acidic FGF (aFGF) exerted similar effects. Other growth factors, including epidermal growth factor (EGF), transforming growth factor beta 1 (TGF-beta 1), and thrombin, were without effect on the proliferative activity of these neuronal cells. bFGF has no effect on the survival of motoneurons and on the fiber outgrowth of the whole neuronal population. However, bFGF affects the development of bipolar AChE-positive neurons, probably belonging to the non-motoneuron population. The data indicate that bFGF and aFGF are mitogens for neuroblasts from rat spinal cord in culture and that bFGF influences the development of a subpopulation of spinal neurons that are AChE-positive.     

Differential effects of lentiviral vector-mediated overexpression of nerve growth factor and glial cell line-derived neurotrophic factor on regenerating sensory and motor axons in the transected peripheral nerve

Even after reconstructive surgery, major functional impairments remain in the majority of patients with peripheral nerve injuries. The application of novel emerging therapeutic strategies, such as lentiviral (LV) vectors, may help to stimulate peripheral nerve regeneration at a molecular level. In the experiments described here, we examined the effect of LV vector-mediated overexpression of nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) on regeneration of the rat peripheral nerve in a transection/repair model in vivo. We showed that LV vectors can be used to locally elevate levels of NGF and GDNF in the injured rat peripheral nerve and this has profound and differential effects on regenerating sensory and motor neurons. For sensory neurons, increased levels of NGF and GDNF do not affect the number of regenerated neurons 1 cm distal to a lesion at 4 weeks post-lesion but do cause changes in the expression of markers for different populations of nociceptive neurons. These changes are accompanied by significant alterations in the recovery of nociceptive function. For motoneurons, overexpression of GDNF causes trapping of regenerating axons, impairing both long-distance axonal outgrowth and reinnervation of target muscles, whereas NGF has no effect on these parameters. These observations show the feasibility of combining surgical repair of the transected nerve with the application of viral vectors. Furthermore, they show a difference between the regenerative responses of motor and sensory neurons to locally increased levels of NGF and GDNF.     

Effect of nerve growth factor on the elongation of neurites from axotomized rat embryonic septal-basal forebrain neurons: an in vitro analysis

The administration of nerve growth factor (NGF) into the brain of a fornix-fimbria lesioned rat can rescue many cholinergic, septal-basal forebrain (SBF) neurons from imminent cell death. Unfortunately, it is unclear if NGF can stimulate regenerative growth from axotomized, SBF neurons. In the present study, we used an in vitro model system to determine if NGF could affect neurite outgrowth from nonaxotomized and/or axotomized, embryonic SBF neurons. Axotomized neurons were obtained by severing the neuritic fields surrounding embryonic day (E) 15 SBF explants maintained in primary culture. Acetylcholinesterase (AChE) histochemistry was used to assess the effects of NGF on cholinergic neurites. We report that 1) neurite outgrowth on type I collagen from E15 SBF neurons in primary culture (nonaxotomized neurons) was not affected by NGF. 2) NGF enhanced the outgrowth (regeneration) of axotomized, SBF neurons on a collagen substratum; however, neurons had to be treated with NGF both before and after axotomy to stimulate regeneration effectively. Application of NGF either before or after axotomy did not enhance regenerative neurite outgrowth. 3) SBF neurons had to be axotomized for NGF to facilitate neurite outgrowth. This is supported by the observation that SBF explants, initially maintained in NGF-supplemented medium in suspension culture, did not demonstrate enhanced neurite outgrowth in the presence of NGF when plated onto a substratum. 4) The regenerative growth of AChE-negative, as well as AChE-positive, neurites was facilitated by NGF treatment. In addition to data concerning neurite outgrowth, we also found that the NGF receptor, as recognized by the antibody 192-IgG, expands its distribution as time in culture progresses; i.e., staining, originally confined to cell bodies and proximal processes within the explant, later included neurites that emanated from the explant. Thus, our results demonstrate that NGF can stimulate regenerative growth from axotomized, but not nonaxotomized, embryonic SBF neurons. We hypothesize that, given the appropriate substratum for axon elongation in vivo, NGF can stimulate the regeneration of SBF neurons in the injured adult brain.     

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.     

Basic Fibroblast Growth Factor in the Adrenal Gland

The importance of trophic agents for the development and maintenance of neurons and their presence in mesenchyme-derived neuronal target organs such as muscle is well exemplified by the protein nerve growth factor (NGF) and its synthesis in target areas of sympathetic and sensory nerves. Stringent conceptualization of target organ-regulated neuronal maintenance would imply that neurons were able to provide trophic support to their presynaptic counterparts. We present data suggesting that basic fibroblast growth factor (bFGF), a mitogen and trophic factor for several neuron populations in vitro, may be such a protein involved in retrograde trophic neuron - neuron interaction. Basic FGF or a closely related protein is present in the adrenal medulla and its sympathetic neuron-like chromaffin cells. A polyclonal antibody specific for bFGF recognizes an 18 kD band in Western blots of bFGF-enriched bovine adrenal medulla extracts and immunostains isolated bovine chromaffin cells. This antibody also blocks the bFGF-like activity present in adrenal medullary extracts and chromaffin granule extracts that both promote in vitro survival of embryonic chick ciliary ganglionic neurons. Furthermore, like bFGF, the soluble proteins of bovine chromaffin granules are mitogenic for cultured bovine aorta endothelial cells. Electrothermal unilateral destruction of the adrenal medulla causes the disappearance of 25% of Nissl-stained neurons in the ipsilateral intermediolateral column (IML) of the spinal cord between levels Th7 and L1, which contains the preganglionic neurons projecting to the adrenal medulla. Substitution of the adrenal medulla by gel foams soaked with bFGF prevents neuron losses in the IML. The effects are specific in that NGF and cytochrome C are ineffective. Our results suggest that bFGF is located in chromaffin cells and maintains target-deprived autonomic spinal cord neurons, thus possibly acting as an interneuronal trophic messenger in vivo.