Stimulation of growth of new axonal sprouts from lesioned monoamine neurones in adult rat brain by nerve growth factor

A strong stimulating effect of intraventricularly administered NGF proteins has been demonstrated on the growth of new axonal sprouts from lesioned central catecholamine and indolamine neurones in the adult rat. The effect was observed as an increase in the growth of the lesioned ascending noradrenaline, dopamine, and indolamine neurone systems into a denervated iris transplanted to the caudal diencephalon. The response was similar with high molecular (7S) and low molecular (2.5S and the β-subunit of 7S NGF) NGF preparations, and it was dose-dependent. The results suggest that NGF, given as a single injection at the time of transplantation and axonal lesioning, did not alter the time sequence of events in the growth process (i.e. the time necessary for the initiation of sprouting) but rather that it accelerated or potentiated the outgrowth of the new axonal sprouts during the time when it normally occurred. It is suggested that the NGF sensitivity of regenerating central noradrenaline, dopamine, and indolamine neurones might reflect a general dependence of these neurones on endogenous NGF during ontogenesis and regeneration.     

Effects of brain-derived neurotrophic factor and nerve growth factor on remaining neurons in the lesioned nucleus basalis magnocellularis

Rats received a unilateral lesion of the nucleus basalis magnocellularis (NBM) by infusion of ibotenic acid. Starting 2 weeks after the lesion, the animals were treated with nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) by intraparenchymal infusion of 3 μg per day for 4 weeks. Lesioned control animals received a similar amount of cytochromec. The activity of cholone acethyltransferase (ChAT) in the frontal neocortex was signigicantly reduced by the lesion (−39%). However, the intraparenchymal treatment with NGF or BDNF did not affect cortical ChAT activity. The number of p75 NGF receptor-immunoreactive neurons in the NBM was significantly decreased (−49%) by the lesion and was not affected by NGF or BDNF. The size of the remaining neurons was significantly increased by NGF (+32%), but not by BDNF (+12%). Similarly, in situ hybridization showed enhanced expression of the p75 NGF receptor following treatment with NGF, but not with BDNF. These results suggest that although BDNF occurs in the target area of cholinergic NBM neurons, its effects on these neurons are less pronounced than those of NGF.     

Inhibition of the regenerative growth of central noradrenergic neurons by intracerebrally administered anti-NGF serum

Serial coronal or sagittal sections were stained for myelin and examined in 6 inbred, 4 hybrid, and 2 outbred mouse strains. Absent corpus callosum was seen only in BALB/cJ as reported by Wimer, but a wide range of the size of corpus callosum was also noted. The action of a major gene was not evident in backcross or F2 generations; polygenic and perhaps epistatic inheritance was indicated. In A/J, and to a lesser extent A/HeJ and BALB/cJ, the columns of fornix frequently collided with the anterior commissure and either passed around it to make a normal termination or deflected dorsally to make an abnormal termination in lateral septum. In some BALB/cJ brains the anterior commissure instead was displaced and passed behind or through the columns of fornix. Backcross and F2 data suggested inheritance was polygenic and that genetic variation affected the spatio-temporal coordination of ontogeny of the two tracts. Finally, unusual longitudinal bundles were detected in the septal region of BALB/cJ. Results of crosses were consistent with the hypothesis that a single, incompletely dominant gene was acting, but further study of both the anatomy and heredity of the defect was deemed necessary.     

Effect of nerve growth factor on the lesioned septohippocampal cholinergic system of aged rats

Nerve growth factor (NGF) was injected intraventricularly into aged (24 months) rats with unilateral lesions of the lateral fimbria. The activity of choline acetyltransferase (ChAT) was determined in the septum and hippocampus from the normal unlesioned rats, lesioned and cytochrome c-treated rats (controls), and lesioned and NGF-treated rats at different times after the lesion. NGF-injection for 15 days after the lesion resulted in an increase of the ChAT activity in both the contralateral hippocampus and the entire septum, to about 130% of that in the normal animals, but resulted in a slight increase in the ipsilateral lesioned hippocampus, when compared to the activity in the ipsilateral side of the cytochrome c-treated controls. NGF-injection for 30 days after the lesion resulted in a 48% increase of the ChAT activity in the ipsilateral hippocampus as compared to cytochrome c-treated controls, but failed to result in a significant increase in the contralateral hippocampus. These findings indicate that atrophic cholinergic neurons in aged animals are similarly responsive to NGF treatment, like these in the young animals. Moreover, these findings suggest that the responses of basal forebrain cholinergic neurons to NGF treatment varies with time after the lesion and imply that the NGF administration can promote the collateral sprouting from spared cholinergic fibers after the lesion in the aged forebrain.     

Retrograde transport of nerve growth factor in lesioned goldfish retinal ganglion cells

Previous experiments have shown that nerve growth factor (NGF) enhances regeneration of goldfish optic nerve after local application of NGF at the site of the lesion. However, the site and mechanism of action of NGF are not yet known. One possibility is that NGF is taken up at the site of the lesion and retrogradely transported to the cell bodies of the retinal ganglion cells and thereby exerts its trophic effects. The present work was carried out to assess the role of retrograde transport of NGF in this enhanced regeneration of goldfish retinal ganglion cells. In intact retinal ganglion cells of the goldfish, 125I-labeled NGF was found not to be retrogradely transported from the optic tectum to the retina, suggesting that retinal ganglion cells do not possess specific NGF receptors. However, if [125I]NGF was injected at the site of an optic nerve lesion at the time of lesion, [125I]NGF was retrogradely transported from the site of a lesion of the optic nerve to the cell body of retinal ganglion cells. The accumulated radioactivity was shown to be intact NGF by SDS-PAGE. The ability of NGF to decrease the time required for recovery of visual function was observed only when NGF was administered at the time of the injury. Likewise, no transport of [125I]NGF was observed when it was injected at the crush site 16 hr or longer after crush. Thus, there is a temporal correlation between the ability of intact [125I]NGF to be retrogradely transported from a lesion site to the retina and the regenerative effect of NGF. Autoradiography showed that the [125I]NGF accumulated only in retinal ganglion cells. The transport of NGF in the lesioned goldfish visual system was not specific for NGF in that other proteins (cytochrome c, bovine serum albumin) were transported equally well. Likewise, transport of [125I]NGF was not prevented by concomitant administration of excess unlabeled NGF. The retrograde transport of [125I]NGF therefore was not selective and did not appear to be mediated by specific NGF receptors in this system. This nonspecific transport of [125I]NGF did not occur in the axotomized spinal motor neurons in the neonatal or adult rat or in the newt. However, receptor-mediated transport is seen in lesioned sensory neurons in both species.(ABSTRACT TRUNCATED AT 400 WORDS)     

NGF促再生周围神经中血管生成及其机制研究

目的:探讨再生周围神经中NGF促血管生成及其有关机制。方法:用单通道的硅胶管硅胶45只Wistar大鼠1cm的坐骨神经缺损,在硅胶管内给药,并将它们随机分为四组:生理盐水组、几丁糖组(医用几丁糖简称几丁糖,此组15只,其中5只用作透射电镜观察),NGF+几丁糖组,NGF+几丁糖+抗VEGF组,每组10只,在术后14天时,用免疫组化的方法检测大鼠再生坐骨神经新生血管内皮细胞中CD34、vWf、VEGF、trkA的表达,并作半定量分析。同时用激光共聚焦扫描显微镜检测其表达情况。结果:生理盐水组、几丁糖组再生坐骨神经中的血管内皮细胞能表达TrkA、CD34、vWf、VEGF,两组间比较没有显著性差异,NGF+几丁糖组上述四抗原的表达比生理盐水组、几丁糖组有明显增加(P<0.01或P<0.05),NGF+几丁糖+抗VEGF组再生坐骨神经血管中的CD34和VEGF的表达完全被抑制,vWf、TrkA的表达与NGF+几丁糖组相比也显著减少(P<0.01)。激光共聚焦扫描发现,再生坐骨神经的血管中VEGF与CD34其表达明显而强烈,凡CD34表达阳性的,VEGF也表达阳性,VEGF的阳性表达比CD34要多。VEGF与vWf也呈明显其表达。结论:NGF能促进再生周围神经的血管生成;NGF促再生周围神经血管生成过程中,VEGF起着重要的作用,trkA、CD34也与之相关;NGF经trkA-VEGF-CD34通路来促进周围神经中毛细血管的生成;NGF促较大管径血管生成时,除经trkA-VEGF通路外,还可能存在其它途径。     

Neuroprotective effects of ultrasound-guided nerve growth factor injections after sciatic nerve injury

Nerve growth factor (NGF) plays an important role in promoting neuroregeneration after peripheral nerve injury. However, its effects are limited by its short half-life; it is therefore important to identify an effective mode of administration. High-frequency ultrasound (HFU) is increasingly used in the clinic for high-resolution visualization of tissues, and has been proposed as a method for identifying and evaluating peripheral nerve damage after injury. In addition, HFU is widely used for guiding needle placement when administering drugs to a specific site. We hypothesized that HFU guiding would optimize the neuroprotective effects of NGF on sciatic nerve injury in the rabbit. We performed behavioral, ultrasound, electrophysiological, histological, and immunohistochemical evaluation of HFU-guided NGF injections administered immediately after injury, or 14 days later, and compared this mode of administration with intramuscular NGF injections. Across all assessments, HFU-guided NGF injections gave consistently better outcomes than intramuscular NGF injections administered immediately or 14 days after injury, with immediate treatment also yielding better structural and functional results than when the treatment was delayed by 14 days. Our findings indicate that NGF should be administered as early as possible after peripheral nerve injury, and highlight the striking neuroprotective effects of HFU-guided NGF injections on peripheral nerve injury compared with intramuscular administration.     

Intrahippocampal injections of antiserum to nerve growth factor inhibit sympathohippocampal sprouting

Following lesions of the fimbria/fornix system in the rat, noradrenergic sympathetic fibers grow into the hippocampal formation. It has been postulated that these fibers collateralize in response to the presence of a neurotrophic substance similar to Nerve Growth Factor (NGF). We tested this by injecting into the rat hippocampus antibody to NGF, or control serum (contralateral control), immediately prior to a bilateral fimbria/fornix transection. In fluorescent histochemical preparations at four to five weeks following surgery, there are fewer large, brightly fluorescent fibers around the injection site on the experimental side when compared with the contralateral control side. These results support the hypothesis that NGF, or an NGF-like substance, plays an important role in the sprouting of sympathetic fibers into the denervated hippocampal formation.     

The response of adrenergic neurones to axotomy and nerve growth factor.

Division of the axons of adrenergic neurones by crushing the postganglionic nerve trunks of rat superior cervical ganglia (SCG) at 6 days of age resulted in a permanent atrophy of the SCG reflected by a persistent decrease in the total protein content and in the activities of the enzymes tyrosine hydroxylase and DOPA decarboxylase. Administration of nerve growth factor (NGF) to rats with unilateral axotomy at a dose of 10 mug/g/day for the period 7-21 days of age resulted in hypertrophy of both normal and axotomised SCG. There was a progressive rise in the total protein content and in the activities of the two enzymes till the end of the treatment period in both SCG. After treatment ceased there was a progressive fall in the total protein content and activities of the two enzymes reaching a stable level after 4 weeks. The level reached for treated unoperated SCG remained elevated when compared to untreated control SCG. Axotomised treated SCG had approximately the same biochemical parameters as untreated control SCG and very much elevated over untreated axotomised SCG. These final levels persisted for at least 56 days after treatment had ceased. Animals showed a persistent ptosis after axotomy at 6 days of age but treatment with NGF resulted in a functional recovery by 11 weeks of age. It is suggested that there is normally a retrograde transfer of a factor durind development from the target cell to the perikarya of the neurone permitting survival if the appropriate connections are made. Failure to make such a contact results in cedd death. The cell death occurring normally, and the cell death resulting from axotomy, can both be prevented by NGF treatment leading to an hypertrophy of both SCG. This consistent with the hypothesis than NGF is the retrograde trophic agent for the sympathetic nervous system in the developing animal.     

Evaluation of the effects of nerve growth factor and anti-nerve growth factor on the development of central catecholamine-containing neurons

Intracisternal NGF or anti-NGF has been found to produce no long-term major alterations in central norepinephrine (NE) or dopamine levels when administered to neonatal rats. While NGF and anti-NGF were found to produce significant changes in brain NE content within one week of treatment, changes in central NE were no longer detectable at 30 days of age. Modification of the growth response of the central adrenergic neurons following 6-OHDA treatment was also not affected by NGF or anti-NGF when evaluated 3 weeks after treatment. However, centrally administered anti-NGF did induce a loss of peripheral NE terminals, which was attributed to leakage of the anti-NGF from the central injection site.     

Sural nerve immunoreactivity for nerve growth factor receptor in a case of localized hypertrophic neuropathy

Summary Immunoreactivity for nerve growth factor receptor (NGFR) was examined using a monoclonal antibody against human NGFR in the sural nerve of a 24-year-old woman, affected by localized hypertrophic neuropathy (LHN). NGFR expression was correlated with electron microscopy and with immunoreactivity for S-100 protein, laminin, HLA-DR, HNK-1, P0 glycoprotein and neurofilament peptides. Our results indicate that in LHN most of whorl-forming cells are NGFR positive and S-100 protein or HLA-DR negative. These data along with the ultrastructural features suggest their origin from perineurium.Supported by funds from Dino Ferrari Center for Neuromuscular and Neurodegenerative Diseases, University of Milan and Italian MS Association     

Subpopulations of sympathetic neurones differ in their sensitivity to nerve growth factor antiserum

Sympathetic postganglionic nerve fibres supplying mesenteric arteries and intrinsic ileal neurones differ in their characteristics of regeneration. Since the latter population of neurones occurs predominantly in prevertebral ganglia, which have been reported to be spared to some extent after treatment with antiserum to nerve growth factor (anti-NGF), we have investigated whether the two populations were differentially sensitive to anti-NGF. Newborn rats were treated daily for the first postnatal week with either anti-NGF or 154 mM NaCl solution. At 4 and 8 weeks of age, the presence of a functional sympathetic innervation to the mesenteric arteries and the gut was determined and correlated with the fluorescence histochemical demonstration of noradrenergic fibres. At both ages, stimulation of extrinsic sympathetic fibres caused an inhibition of gut motility, while the mesenteric arteries completely lacked a sympathetic innervation. Retrograde labelling of nerve cell bodies in control and antiserum treated rats confirmed that the sympathetic neurones supplying the ileal neurones were located in the prevertebral, superior mesenteric and coeliac ganglia and in the splanchnic ganglia lying along the greater splanchnic nerves. By interference from retrograde labelling in control animals, sympathetic neurones supplying the mesenteric arteries were present in all these ganglia, as well as in the thoracic and lumbar paravertebral sympathetic chains. The results suggest that two functionally distinct populations of sympathetic neurones, which overlap considerably in their distributions, are differentially sensitive to the immunological postnatal removal of NGF.     

The effect of the retrograde axonal transport of nerve growth factor on the morphology of adrenergic neurones.

An injection of nerve growth factor (NGF) into one eye of neonatal rats results in an increase in the tyrosine hydroxylase activity of the ipsilateral superior cervical ganglion. This effect was seen maximally after the intraocular injection of a depot preparation of NGF linked to cellulose. The sympathetic neurones that innervate the eye can be identified by autoradiography after the retrograde axonal transport of either NGF or tetanus toxin labelled with [125I]iodine. It was only those cells having their terminals in the vicinity of the depot preparation. This demonstrates that NGF transported from the periphery to the cell bodies is effectively retained within the transporting cell and is not released to act on extracellular receptors within the ganglion. It is suggested that this specificity of action for NGF reaching the ganglion in this fashion is important during normal development in determining the survival of adrenergic neurones.     

Rescue of lesioned septal cholinergic neurons by nerve growth factor: specificity and requirement for chronic treatment

We earlier reported that chronic intraventricular injections of NGF into adult rats with partial transection of the fimbria prevent the lesion-induced disappearance of cholinergic neurons in the medial septal nucleus and the diagonal band of Broca (Hefti, 1986). The present study assessed the specificity and treatment requirements of this effect of NGF. Immunohistochemical visualization of NGF receptors (NGF-R) revealed that these molecules are selectively located in forebrain cholinergic neurons of unlesioned brains. Fimbrial transection resulted in transient accumulation of NGF-R in proximal stumps of lesioned axons but failed to induce the expression of NGF-R by other cells in the septal area or near the lesion. Two to three weeks after lesioning, the number of septal neurons expressing NGF-R was reduced by approximately 50% in parallel with the reduction of the number of neurons expressing cholinergic marker enzymes. Repeated intraventricular NGF injections during 4 weeks prevented the disappearance of these cells. Fimbrial transections also reduced the number of septal GABAergic neurons visualized by glutamate decarboxylase immunohistochemistry. The loss of GABAergic neurons was not prevented by NGF. These findings suggest that NGF prevents the lesion-induced degeneration of cholinergic neurons by directly acting on NGF-R expressed by cholinergic cells and that NGF does not affect any neuron with an axonal lesion. Delayed start of the NGF treatment failed to prevent the disappearance of lesioned cholinergic neurons, providing evidence that NGF treatment indeed promotes the survival of these cells rather than simply upregulating the expression of transmitter-specific enzymes. A single injection of NGF at the time of the lesion was not sufficient to prevent the lesion-induced degeneration of cholinergic neurons. Furthermore, termination of chronic NGF treatment after 4 weeks was followed by loss of septal cholinergic neurons after an additional 4 weeks. These findings suggest that the continuous presence of NGF during more than 4 weeks is required to prevent the degeneration of cholinergic cells. The data are discussed in the context of a possible physiological role of NGF in the function of adult forebrain cholinergic neurons.     

Presence and regulation of transforming growth factor beta mRNA and protein in the normal and lesioned rat sciatic nerve

The transforming growth factors beta (TGF-β), a family of regulatory polypeptides, are involved in numerous vital processes including inflammation and wound healing. Since repair of a peripheral nerve lesion includes a series of well-defined steps of cellular actions possibly controlled by TGF-βs, and since TGF-β mRNA and immunoreactivity have been found in the normal peripheral nerve, we have examined TGF-β mRNA regulation and protein expression in the lesioned peripheral nerve. Sciatic nerves of adult rats were either crushed (allowing axonal regenration) or transected (to prevent axonal regeneration and to induce Wallerian degeneration in the distal stump). After intervals of 6 hours, 2 and 6 days post-lesion, the rats were sacrificed and each nerve was cut into four segments, two proximal and two distal to the lesion site. TGF-β 1-3 mRNA were determined for each segment. We demonstrate that TGF-ß1 mRNA levels are higher than those of TGF-ß3; the amplitude of mRNA regulation depends on time, type of lesion and localization relative to the lesion site. TGF-ß2 mRNA could not be detected. For TGF-ß1-3 immunocytochemistry, animals were sacrificed 12, 24, 48, 72 hours and 7 and 14 days after surgery. TGF-β immunoreactivity (IR) was observed for all isoforms in lesioned and unlesioned nerves. In the segment directly adjacent to the lesion at its proximal side, an increase of TGF-β-IR became apparent as soon as 12 hours after surgery; it remained elevated during the whole period observed in both models. In the segment adjoining the distal side of the lesion, an increase of TGF-β-IR was observed after 48 hours, which was still present after 14 days. At day 7 after crush or transection, an increase of TGF-β-IR was detected in the most distal segments, which reached its highest levels at the end of our observation period. Our results suggest that the presence of axonal contact might induce an enhancement of TGF-β expression by Schwann cells in the distal stump of a lesioned and regenerating peripheral nerve. Since we demonstrate an increase of TGF-β mRNA and protein expression also in the distal stump of transected nerves where Schwann cells are not able to contact sprouting axons from the proximal part, other regulatory pathways must exist. The acquisition of a “reactive” Schwann cell phenotype after peripheral nerve lesion might involve an upregulation of TGF-β expression. © 1994 Wiley-Liss, Inc.     

Distinct influences of nerve growth factor and a central cholinergic trophic factor on medial septal explants

A central cholinergic trophic factor (C-CTF), previously reported in hippocampal extracts, enhances acetylcholine synthesis (ACh) and to a lesser extent choline acetyltransferase (ChAT) activity in cultured explants of the rat medial septal nucleus. Nerve growth factor (NGF) has been reported to enhance ChAT in several systems in vitro and in vivo, and clearly stimulates septal explants. At optimal concentrations of NGF and C-CTF, there is an additive effect on ChAT activity. The effects of NGF on ACh synthesis are minimal. Antibodies to NGF block effects of added NGF but have no effects on C-CTF activity. The ability of C-CTF to enhance ACh synthesis appears related to its ability to enhance the acetylation of the choline that has been taken up by a sodium dependent, high affinity transport system. Thus, actions of NGF and C-CTF appear qualitatively and quantitatively distinct, yet both can influence the cholinergic activity of the developing medial septal nucleus.