Amphibian sympathetic ganglia as a model system for investigating regeneration in the vertebrate peripheral nervous system

1. The paravertebral sympathetic ganglion of the bullfrog serves as an excellent experimental system in which to study the response of vertebrate neurones to axotomy and the mechanisms associated with regeneration. 2. Various types of lesions to the axons (axotomy) of these neurones promote distinct and reproducible changes in the electrophysiological properties of the cell bodies which are not a consequence of changes in cell body morphology. 3. The axotomy-induced increase in spike width and decrease in the amplitude of the action potential after-hyperpolarization may allow an increase in Ca2+ influx and thereby promote regrowth. 4. The axotomy-induced decrease in after-hyperpolarization duration may reflect the disconnection of the neurone with its target and the loss of available nerve growth factor (NGF) from the target. 5. Experiments with NGF antibodies provide evidence that an NGF-like substances serves to maintain the normal electrophysiological characteristics of amphibian sympathetic neurones.     

Detection of brain-derived neurotrophic factor-like activity in fibroblasts and Schwann cells: inhibition by antibodies to NGF

mRNA coding for brain-derived neurotrophic factor (BDNF) has been detected in cultured L929 fibroblasts, rat dermal fibroblasts, and sciatic nerve Schwann cells, as well as in rat skin. Medium conditioned by cultured fibroblasts and Schwann cells also stimulates neurite growth from retinal explants and promotes the survival in culture of BDNF-responsive sensory neurons; biological activity is abolished by antibodies raised against NGF. These results suggest that molecules with BDNF-like activity may be produced by cells in the peripheral nervous system and that the BDNF-like activity in fibroblasts and Schwann cells is derived from molecules immunologically related to NGF. In support of this concept, antibodies against NGF have been found to reduce the biological activity of recombinant BDNF in culture and to cross-react with BDNF on Western blots.     

Tooth pulp tissue promotes neurite outgrowth from rat trigeminal ganglia in vitro

The mammalian tooth pulp becomes innervated by nociceptive and sympathetic axons relatively late during development, when part of the root has formed. In the adult, regenerating axons from an injured tooth nerve or sprouting axons from uninjured nerves in the vicinity rapidly reinnervate denervated tooth pulps. These observations indicate that tooth pulp tissue can use molecular factors to attract pulpal axons from local nerve trunks. The present study examines the hypothesis that these factors include nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and glial cell line derived neurotrophic factor (GDNF). Explants of trigeminal ganglia from neonatal rat pups showed a distinct neurite outgrowth when co-cultured with pulpal explants collected from molar teeth of 12-day old pups, or after application of a pulpal extract. Control cultures, containing single ganglionic explants, or explants co-cultured with heat-treated pulpal tissue, exhibited a sparse neurite outgrowth. Exogenous NGF and/or GDNF, but not exogenous BDNF, stimulated neurite outgrowth from ganglionic explants. Unexpectedly, application of antibodies against NGF, BDNF and/or GDNF to co-cultures of ganglionic and pulpal explants did not inhibit neuritogenesis. Control experiments showed that IgG molecules readily penetrate the gel used for culture and that even very high concentrations of NGF and GDNF antibodies in combination failed to block neurite growth. On the basis of these data we suggest that other as yet unknown neurite-promoting factors might be present and active in TG/pulpal co-cultures.     

Sympathetic neurons synthesize and secrete pro‐nerve growth factor protein

Postmitotic sympathetic neuronal survival is dependent upon nerve growth factor (NGF) provided by peripheral targets, and this dependency serves as a central tenet of the neurotrophic hypothesis. In some other systems, NGF has been shown to play an autocrine role, although the pervasiveness and significance of this phenomenon within the nervous system remain unclear. We show here that rat sympathetic neurons synthesize and secrete NGF. NGF mRNA is expressed in nearly half of superior cervical ganglion sympathetic neurons at embryonic day 17, rising to over 90% in the early postnatal period, and declining in the adult. Neuronal immunoreactivity is reduced when retrograde transport is interrupted by axotomy, but persists in a subpopulation of neurons despite diminished mRNA expression, suggesting that intrinsic protein synthesis occurs. Cultured neonatal neurons express NGF mRNA, which is maintained even when they are undergoing apoptosis. To determine which NGF isoforms are secreted, we performed metabolic labeling and immunoprecipitation of NGF‐immunoreactive proteins synthesized by cultured NGF‐dependent and ‐independent neurons. Conditioned medium contained high molecular weight NGF precursor proteins, which varied depending upon the state of NGF dependence. Mature NGF was undetectable by these methods. High molecular weight NGF isoforms were also detected in ganglion homogenates, and persisted at diminished levels following axotomy. We conclude that sympathetic neurons express NGF mRNA, and synthesize and secrete pro‐NGF protein. These findings suggest that a potential NGF‐sympathetic neuron autocrine loop may exist in this prototypic target‐dependent system, but that the secreted forms of this neurotrophin apparently do not support neuronal survival. © 2003 Wiley Periodicals, Inc. J Neurobiol 38–53, 2003     

Sympathetic neurons synthesize and secrete pro-nerve growth factor protein

Postmitotic sympathetic neuronal survival is dependent upon nerve growth factor (NGF) provided by peripheral targets, and this dependency serves as a central tenet of the neurotrophic hypothesis. In some other systems, NGF has been shown to play an autocrine role, although the pervasiveness and significance of this phenomenon within the nervous system remain unclear. We show here that rat sympathetic neurons synthesize and secrete NGF. NGF mRNA is expressed in nearly half of superior cervical ganglion sympathetic neurons at embryonic day 17, rising to over 90% in the early postnatal period, and declining in the adult. Neuronal immunoreactivity is reduced when retrograde transport is interrupted by axotomy, but persists in a subpopulation of neurons despite diminished mRNA expression, suggesting that intrinsic protein synthesis occurs. Cultured neonatal neurons express NGF mRNA, which is maintained even when they are undergoing apoptosis. To determine which NGF isoforms are secreted, we performed metabolic labeling and immunoprecipitation of NGF-immunoreactive proteins synthesized by cultured NGF-dependent and -independent neurons. Conditioned medium contained high molecular weight NGF precursor proteins, which varied depending upon the state of NGF dependence. Mature NGF was undetectable by these methods. High molecular weight NGF isoforms were also detected in ganglion homogenates, and persisted at diminished levels following axotomy. We conclude that sympathetic neurons express NGF mRNA, and synthesize and secrete pro-NGF protein. These findings suggest that a potential NGF-sympathetic neuron autocrine loop may exist in this prototypic target-dependent system, but that the secreted forms of this neurotrophin apparently do not support neuronal survival.     

Regeneration restores some of the altered electrical properties of axotomized bullfrog B-cells

In bullfrog B-type sympathetic neurones axon injury produces substantial changes in somal membrane properties. These include a shortening of action potential afterhyperpolarization (AHP) and an increase in action potential (AP) duration. In the present experiments we compared two injury situations: nerve crush, which was followed by regeneration, and nerve cut, after which regeneration to the original target was prevented, to investigate whether these electrophysiological changes were related to axon regeneration. Both crush and cut injuries produced a similar maximum decrease in AHP duration (to 33 and 30%) by 14 days after axotomy. After nerve crush, AHP duration recovered to within control values by 42 days, while after cut it remained depressed. AHP amplitude decreased to the same extent after nerve crush or cut (to 62 and 58%), but the rate of decrease was slower following crush when compared with cut, and following both types of injury it still remained depressed at 42 and 49 days. Changes in AP duration also took longer to occur following nerve crush, reaching maximal values at 35-42 days, at which time AHP duration had returned to within the normal range. The early reduction in AHP duration and its rapid recovery in regenerating neurones suggests that the current underlying this membrane property is regulated by events associated with axon outgrowth and peripheral reconnection. In contrast, changes in AHP amplitude and AP repolarization appeared to be independent of the occurrence of axon regeneration and remained abnormal at 49 days despite the recovery of AHP duration. These results imply that the electrophysiological changes seen in B-cells following injury are differentially regulated during subsequent regeneration.     

Reduction in nerve growth factor availability leads to a conditioning lesion‐like effect in sympathetic neurons

Axotomized peripheral neurons are capable of regeneration, and the rate of regeneration can be enhanced by a conditioning lesion (i.e., a lesion prior to the lesion after which neurite outgrowth is measured). A possible signal that could trigger the conditioning lesion effect is the reduction in availability of a target‐derived factor resulting from the disconnection of a neuron from its target tissue. We tested this hypothesis with respect to nerve growth factor (NGF) and sympathetic neurons by administering an antiserum to NGF to adult mice for 7 days prior to explantation or dissociation of the superior cervical ganglion (SCG) and subsequently measuring neurite outgrowth. The antiserum treatment dramatically lowered the concentration of NGF in the SCG and increased the rate of neurite outgrowth in both explants and cell cultures. The increase in neurite outgrowth was similar in magnitude to that seen after a conditioning lesion. To determine if exogenous NGF could block the effect of a conditioning lesion, mice were injected with NGF or cytochrome C immediately prior to unilateral axotomy of the SCG, and for 7 days thereafter. A conditioning lesion effect of similar magnitude was seen in NGF‐treated and control animals. While NGF treatment increased NGF levels in the contralateral control ganglion, it did not significantly elevate levels in the axotomized ganglion. The results suggest that the decreased availability of NGF after axotomy is a sufficient stimulus to induce the conditioning lesion effect in sympathetic neurons. While NGF administration did not prevent the conditioning lesion effect, this may be due to the markedly decreased ability of sympathetic neurons to accumulate the growth factor after axotomy. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Reduction in nerve growth factor availability leads to a conditioning lesion-like effect in sympathetic neurons

Axotomized peripheral neurons are capable of regeneration, and the rate of regeneration can be enhanced by a conditioning lesion (i.e., a lesion prior to the lesion after which neurite outgrowth is measured). A possible signal that could trigger the conditioning lesion effect is the reduction in availability of a target-derived factor resulting from the disconnection of a neuron from its target tissue. We tested this hypothesis with respect to nerve growth factor (NGF) and sympathetic neurons by administering an antiserum to NGF to adult mice for 7 days prior to explantation or dissociation of the superior cervical ganglion (SCG) and subsequently measuring neurite outgrowth. The antiserum treatment dramatically lowered the concentration of NGF in the SCG and increased the rate of neurite outgrowth in both explants and cell cultures. The increase in neurite outgrowth was similar in magnitude to that seen after a conditioning lesion. To determine if exogenous NGF could block the effect of a conditioning lesion, mice were injected with NGF or cytochrome C immediately prior to unilateral axotomy of the SCG, and for 7 days thereafter. A conditioning lesion effect of similar magnitude was seen in NGF-treated and control animals. While NGF treatment increased NGF levels in the contralateral control ganglion, it did not significantly elevate levels in the axotomized ganglion. The results suggest that the decreased availability of NGF after axotomy is a sufficient stimulus to induce the conditioning lesion effect in sympathetic neurons. While NGF administration did not prevent the conditioning lesion effect, this may be due to the markedly decreased ability of sympathetic neurons to accumulate the growth factor after axotomy.     

Differential time course of the response to axotomy induced by cut or crush in the leech AP cell

The time course of the reaction to axotomy in the leech AP cell was determined by measuring the duration of the spontaneous spikes at different times after the operation. The axotomy performed by section of the segmental roots containing the AP axon induced an increase of the spike duration, which persisted over 30 days. A different time course was found when the axotomy was performed by nerve crush: the changes in duration of the spontaneous spikes, which occurred during the early 2 weeks, were significantly reduced afterwards. Dye staining of some cells axotomized by crushing revealed that the reversion of the changes, which had been set up by axotomy, was in some cases concomitant with the reconnection between proximal and distal axon stumps. The section of a single axonal branch was never sufficient to affect the membrane properties of the AP cells. It is concluded that the changes observed in axotomized AP cells are not produced by simple axonal injury and that the maintainance of normal properties in the somatic membrane requires the presence of at least part of the distal axon arborization.     

NGF receptor-mediated reduction in axonal NGF uptake and retrograde transport following sciatic nerve injury and during regeneration.

Injury to the rat sciatic nerve leads to the induction of nerve growth factor (NGF) receptors on the denervated Schwann cells and their disappearance on the regenerating axons of the axotomized, normally NGF-sensitive sensory and sympathetic neurons. This disappearance in the axonal expression and retrograde transport of NGF receptors is associated with a similarly dramatic reduction in the axonal uptake and retrograde transport of NGF following axotomy and during regeneration. In view of the massive NGF synthesis occurring in the injured nerve, these results suggest that, while sensory and sympathetic neurons are the primary targets of NGF in the normal peripheral nervous system, the denervated Schwann cells may become its primary target in the aftermath of nerve injury.     

Effects of monoclonal antibodies to bovine nerve growth factor on NGF-induced cell differentiation in culture

Five Hybridoma clones producing monoclonal antibodies (MAT) to bovine nerve growth factor (NGF) were developed. The biological effects of antibodies were studied: the influence of MAT on neurit outgrowth induced by NGF in rat pheochromocytoma PC12 or spinal chicken ganglia was investigated. MAT fell into two groups. Two of them inhibited neurit induction by NGF, three others stimulated this process. The stimulation of the neurit outgrowth by MAT was observed at low concentration of NGF (3 ng/ml of culture medium). Mechanisms of antibodies effects are discussed.     

Expression of nerve growth factor receptor during human peripheral nerve development

The expression of NGF receptors on human Schwann cells during development and myelination and in culture was analyzed using a murine monoclonal antibody to human NGF receptor. Nonmyelinated femoral nerves from 13- to 14-week fetuses stained strongly for NGF receptor, whereas tissues from later stages of development showed a decrease in the staining intensity. These changes correlated with the initiation of myelination (17-19 weeks), as observed by phase-contrast and electron microscopy, and the reactivity with monoclonal antibody 4C5, a marker of mature Schwann cells. In adult nerves, only the perineurium and few endoneurial cells were stained with anti-NGF receptor antibody. Cultured human fetal Schwann cells were positive for NGF receptor by immunofluorescence irregardless of donor age or length of time in culture. The decreased staining of NGF receptor with nerve maturation may reflect a dependence of antigen expression on Schwann cell differentiation and/or neuron-Schwann cell interaction.     

Chick embryo nerve growth factor : Fractionation and biological activity

A nerve growth factor (NGF)-like factor initiating nerve fibre outgrowth from sympathetic ganglia in culture was partially purified from chick embryo extract by cation-exchange chromatography followed by hydrophobic interaction chromatography on octylsulfide agarose. The NGF-like factor was markedly activated upon gel filtration in the presence of 6 M urea. Further analysis of the activated chick NGF by immunoblotting following SDS-PAGE, and by inhibition of bioassay response using antibodies to mouse beta NGF demonstrated a distinct antigenic cross-reactivity. The size of the chick embryo NGF was also indistinguishable from that of the mouse beta NGF with a molecular weight (MW) of about 14,000. The findings demonstrate directly the presence of biologically active NGF protein in the developing 18-day chick embryo.     

Development of slow Ca2+-Na+ channels during organ culture of young embryonic chick hearts

Young (3-days-old) embryonic chick hearts have slowly-rising spontaneous action potentials, dependent on tetrodotoxin-insensitive slow Na+ channels. When the hearts were placed into organ culture for 5-11 days, action potential duration was markedly increased by 260-370%, and a notch appeared between the initial spike phase and the plateau phase in some hearts. The spike amplitude was mainly dependent on [Na]0, whereas the plateau amplitude was dependent on [Ca]0. Thus, the young embryonic hearts develop slow Ca2+-Na+ channels (while retaining the slow Na+ channels) during organ culture, and the spike phase and the plateau phase of the slow action potentials are mainly dependent on currents through slow Na+ channels and through slow Ca2+-Na+ channels, respectively. The effects of Mn2+ (a specific blocker of slow Ca2+-Na+ channels) and verapamil (a blocker of slow Na+ channels as well as of slow Ca2+-Na+ channels) on the spike phase and the plateau phase were examined. Mn2+ (0.5 mM) and verapamil (5 microM) depressed the plateau duration and overshoot. Verapamil did not decrease the maximum rate of rise (Vmax), but Mn++ produced a small, but significant, decrease. High concentrations (10/30 microM) of verapamil depressed the action potential amplitude and Vmax, and abolished the spontaneous action potentials. These results indicate that slow Ca2+-Na+ channels appear de novo during organ culture of young embryonic hearts.     

Therapeutic Effect of Vinorine on Sciatic Nerve Injured Rat

Vinorine is a monoterpenoid indole alkaloid, a type of natural alkaloids. Growing reports exhibited the numerous pharmacology activities of vinorine such as anti-inflammation, anti-bacterial and anti-tumor. In this study, the effect of vinorine injection (7.5, 15 and 30 mg/kg) on motor function, sensation and nerve regeneration in sciatic nerve crush injury rat was investigated. The results of behavioral analysis, electrophysiological analysis and muscle histological analysis suggested that vinorine promoted the motor function recovery after sciatic nerve injury. The results of mechanical withdrawal thresholds assay and hot plate test demonstrated that vinorine improved the sensation recovery after sciatic nerve injury. The results of Fluoro-gold retrograde labeling, transmission electron microscope assay, toluidine blue and HE staining showed that vinorine attenuated the nerve damage caused by sciatic nerve injury and promoted the nerve regeneration. Furthermore, nerve growth factor (NGF) and its downstream extracellular signal-regulated kinase (ERK) signaling pathway participated in the neuro-recovery effect of vinorine after crush. In conclusion, vinorine treatment accelerated the sciatic nerve regeneration, motor function recovery and sensation recovery after crush injury via regulation of NGF and ERK activity. These results suggested that vinorine is a promising agent for never injury therapy.     

Three Distinct Types of Monoclonal Antibodies After Long-Term Immunization of Rats with Mouse Nerve Growth Factor

This article reports the results of a systematic investigation of the different types of antibodies produced in the course of a long-term immunization of rats with mouse nerve growth factor (NGF). We have characterized three types of monoclonal antibodies, namely: (1) antibodies that bind to NGF and inhibit its binding to target cells and its biological activity in culture (type A); (2) antibodies that bind to and precipitate NGF but do not inhibit its binding to target cells or its biological activity (type B); (3) antibodies that fail to recognize NGF itself, but inhibit nonetheless its binding to target cells (type C). These antibodies bind to an antigen present on NGF target cells and not on rat fibroblasts lacking NGF receptor. They appear thus to be antiidiotypic antibodies directed against the NGF receptor, developed as a consequence of the long-term immunization with NGF.     

Embryonic rat adrenal glands in organ culture: Effects of dexamethasone,nerve growth factor and its antibodies on pheochromoblast differentiation

Summary In the present study we sought to determine the developmental potentialities and restrictions of adrenal medullary cells (pheochromoblasts) by investigating their morphological and biochemical response to nerve growth factor (NGF), anti-NGF antibodies and dexamethasone (DEX) after explantation into culture at different embryonic stages. With the exception of explants taken at embryonic day 15 (E 15) cultures of embryonic adrenal glands showed neurite outgrowth, which was not influenced by the addition of NGF, anti-NGF antibodies or DEX to the culture medium during the 4-day-culture period.Pheochromoblasts in E 17+4 explants showed spontaneous ultramorphological and biochemical maturation in terms of an increase in the number of catecholamine storage vesicles (CSVs) per m² of cytoplasmic area, diameters of the cores of CSVs, percentages of electron-lucent cores of CSVs indicative of increased storage of adrenaline, overall catecholamine (CA) content and relative amount of adrenaline. NGF did not significantly affect this maturational process. Anti-NGF antibodies slightly decreased the proportion of adrenaline. The most pronounced maturation was seen in response to DEX and DEX plus NGF, although a maturational state equivalent to the E 21 stage was not achieved. E 21+4 explants showed neither spontaneous nor drug-induced biochemical maturation. Medullary cells in NGF-treated E 21 explants frequently retained the morphological features of pheochromoblasts. Treatment with anti-NGF antibodies significantly reduced the portion of adrenaline as compared to any other treatment. We conclude that under the culture conditions employed (1) a few pheochromoblasts spontaneously express a neuronal phenotype, (2) differentiation of pheochromoblasts towards chromaffin cells is enhanced by glucocorticoids but not by NGF, and (3) anti-NGF antibodies do not impair spontaneous neuritic growth and morphological maturation of pheochromoblasts, but cause a small reduction in the relative amount of adrenaline.     

Placodal sensory neurons in culture: nodose ganglion neurons are unresponsive to NGF, lack NGF receptors but are supported by a liver-derived neurotrophic factor

Explant and dissociated neuron-enriched cultures of nodose ganglia (inferior or distal sensory ganglion of the Xth cranial nerve) were established from chick embryos taken between embryonic Day 4 (E4) and Day 16 (E16). The response of each type of culture to nerve growth factor (NGF) was examined over this developmental range. At the earliest ages taken (E4-E6), NGF elicited modest neurite outgrowth from ganglion explants cultured in collagen gel for 24 hr, although the effect of NGF on ganglia taken from E4 chicks was only marginally greater than spontaneous neurite extension from control ganglia of the same developmental age. The response of nodose explants to NGF was maximal at E6-E7, but declined to a negligible level in ganglia taken from E9-E10 or older chick embryos. In dissociated neuron-enriched cultures, nodose ganglion neurons were unresponsive to NGF throughtout the entire developmental age range between E5 and E12. In contrast to the lack of effect of NGF, up to 50% of nodose ganglion neurons survived and produced extensive neurites in dissociated cultures, on either collagen- or polylysine-coated substrates, in the presence of extracts of late embryonic or early posthatched chick liver (E18-P7). Antiserum to mouse NGF did not block the neurotrophic activity of chick (or rat or bovine) liver extracts. Whether cultured with chick liver extract alone or with chick liver extract plus NGF, nodose ganglion neurons taken from E6-E12 chick embryos and maintained in culture for 2 days were devoid of NGF receptors, as assessed by autoradiography of cultures incubated with 125I-NGF. Under similar conditions 70-95% of spinal sensory neurons (dorsal root ganglion--DRG) were heavily labeled. 2+     

Nerve growth factor acutely potentiates synaptic transmission in vitro and induces dendritic growth in vivo on adult neurons in airway parasympathetic ganglia

Elevated levels of nerve growth factor (NGF) and NGF-mediated neural plasticity may have a role in airway diseases such as asthma and chronic obstructive pulmonary disease (COPD). Although NGF is known to affect sensory and sympathetic nerves, especially during development, little is known regarding its effect on parasympathetic nerves, especially on adult neurons. The purpose of this study was to analyze the acute and chronic effects of NGF on the electrophysiological and anatomical properties of neurons in airway parasympathetic ganglia from adult guinea pigs. Using single cell recording, direct application of NGF caused a lasting decrease in the cumulative action potential afterhyperpolarization (AHP) and increased the amplitude of vagus nerve-stimulated nicotinic fast excitatory postsynaptic potentials. Neuronal responsiveness to nicotinic receptor stimulation was increased by NGF, which was blocked by the tyrosine kinase inhibitor, K-252a, implicating neurotrophin-specific (Trk) receptors. Neurotrophin-3 and brain-derived neurotrophic factor had no effect on the synaptic potentials, AHP, or nicotinic response; inhibition of cyclooxygenase with indomethacin inhibited the effect of NGF on the cumulative AHP. Forty-eight hours after in vivo application of NGF to the trachealis muscle caused an increase in dendritic length on innervating neurons. These results are the first to demonstrate that NGF increases the excitability of lower airway parasympathetic neurons, primarily through enhanced synaptic efficacy and changes to intrinsic neuron properties. NGF also had dramatic effects on the growth of dendrites in vivo. Such effects may indicate a new role for NGF in the regulation of parasympathetic tone in the diseased or inflamed lower airways.