Reduction by reserpine of the accumulation of retrogradely transported [125I]nerve growth factor in sympathetic neurons.

Experiments were carried out to determine if stimuli which augment preganglionic nerve activity to sympathetic neurons, and thereby cause trans-synaptic induction, increase the retrograde transport of nerve growth factor (NGF). It was found that nerve activity had no effect on retrograde transport of [125I]NGF. It was found, however, that reserpine decreased retrograde transport of [125I]NGF and this inhibition was characterized. Reserpine decreased the maximal accumulation of intravenously administered [125I]NGF in superior cervical ganglia (SCG) by about 60%. It also caused a distinct shift in the time course of accumulation so that maximal accumulation was seen 12 h after [125I]NGF injection rather than at 9 h as in control animals. Reserpine had no effect on retrograde transport in sensory neurons. Dose--response curves showed that maximal inhibition occurred with doses of reserpine of 2.5 mg/kg i.p. and that reserpine was not able to completely block transport at any dose. The maximal inhibition of retrograde transport was achieved within 30 min of reserpine administration and inhibitory activity was unchanged for 36 h. The ability of sympathetic neurons to transport [125I]NGF subsequently recovered and was normal 96 h after reserpine administration. The inhibitory effect of reserpine appears to be due to an action at or very near to the nerve terminal since it was effective at reducing NGF transport at very low doses (0.33 microgram) when co-administered directly into the eye with [125I]NGF. An action of reserpine at the nerve terminal was further suggested by the inability of reserpine to affect transport if the drug was given 4 h after [125I]NGF administration. Based upon these data, it is suggested that there may be two pools of retrogradely transported NGF and that only more rapidly turning over pool is reserpine-sensitive. This pool may represent the retrogradely moving synaptic vesicles or some derivative of the vesicles.     

Demonstration of retrogradely transported endogenous nerve growth factor in axons of sympathetic neurons

The presence of retrogradely transported endogenous nerve growth factor (NGF) in sympathetic nerves of the guinea pig was demonstrated directly by fluorescent and peroxidase immunohistochemistry in the ligated superior postganglionic nerve of the superior cervical ganglion. Fixed, frozen sections of previously ligated nerve were incubated with either rabbit antiserum against guinea pig NGF (gpNGF), rabbit antibodies against gpNGF purified on a mouse NGF (mNGF) affinity column, the portion of rabbit antiserum against gpNGF that did not bind to the mNGF affinity column, or nonimmune rabbit serum. Positive staining on the peripheral side of the ligation was obtained only with unfractionated antiserum against gpNGF and with purified antibodies against gpNGF. The staining properties of the various antiserum preparations correlated with their ability to block gpNGF- and mNGF-induced neurite outgrowth in the embryonic chick dorsal root ganglion bioassay and in the PC12 bioassay. Homogenates of superior postganglionic nerve supported growth of embryonic chick dorsal root ganglia and differentiation of PC12 cells. This support was blocked by the specific antisera against NGF used in the immunohistochemistry experiments. These experiments demonstrate that endogenous NGF, presumably released by peripheral target tissues, is retrogradely transported in vivo.     

Retrogradely transported nerve growth factor increases ornithine decar☐ylase activity in rat superior cervical ganglia

Nerve growth factor (NGF) transported intraaxonally from the adrenergic nerve terminals in the iris to the cell bodies in the superior cervical ganglion resulted in an increase in the activity of the enzyme ornithine decarboxylase in the ganglion. This occurred in both six-day-old and adult rats with the relative effect of the retrogradely transported NGF being greater in adult animals. These results suggest that the induction of ornithine decarboxylase by NGF may be due to the stimulation of intracellular receptors.     

Different effects of nerve growth factor on cultured sympathetic and sensory neurons

Long-term cultures of dissociated nodose ganglion (NG) and superior cervical ganglion (SCG) neurons from newborn rabbits were used to compare their response to nerve growth factor (7S NGF). SCG neurons required added NGF for their survival and a concentration of 1 μg/ml was found to be optimal. NG neurons, on the other hand, survived well for a long term without addition of NGF, but its application (1 μg/ml) was found to be effective in accelerating the growth of fibers (neurites) and neuronal somata. It is concluded that unlike SCG, NG neurons do not depend on exogenous NGF but may require an intrinsic trophic-like factor which may be contained in the serum of the medium, emanating from glial cells or by metabolic cooperation between neurons.     

Epidermal growth factor influences the neurotrophic/differentiating action of nerve growth factor

Exposure of naive PC12 cells, sympathetic neurons from rat superior cervical ganglia, and brain-derived septal neurons to epidermal and nerve growth factors simultaneously resulted in some alteration of cellular events induced by nerve growth factor alone. A more pronounced decline of catecholamine content, no additional change in acetylcholinesterase activity, and additive stimulation of RNA and protein syntheses were found in PC12 cells. Earlier elevation of the enzyme activity was observed in sympathetic but not in septal neurons. Epidermal growth factor appeared to support independently the same level of acetylcholinesterase activity in septal neurons as revealed for nerve growth factor during the first week and cell survival throughout 2 weeks of observation. The data obtained indicate that epidermal growth factor augments temporarily some effects of nerve growth factor, thus supporting the idea of an important role of mitogenic growth factors in neural development as complementary and/or substitutive regulators of nerve cell differentiation and survival.     

Time and dose dependencies of effects of nerve growth factor on sympathetic and sensory neurons in neonatal rats

Nerve growth factor (NGF) plays a role in the development of several components of the sympathetic and sensory nervous systems. The objectives of this study were to examine the time and dose dependencies of some of the well known effects of NGF on sympathetic ganglia and to examine qualitatively and quantitatively the recently described effects on sensory ganglia of neonatal rats. Single doses of NGF as low as 0.1 mg/kg produce increases in tyrosine hydroxylase (TOH) activity in superior cervical ganglia (SCG), and doses of 3 mg/kg produce maximal effects. Larger doses and longer treatments are required to see increases in protein content of the SCG. Larger doses are also required to affect TOH activity in the adrenal gland. Increases in TOH activity in SCG can be observed within 18 h of injection. Chronic NGF treatment for three weeks produces no change in blood pressure or heart rate in neonatal rats. Chronic administration of NGF (1 or 3 mg/kg/day) results in dose-related increases in the protein content of dorsal root ganglia (DRG). The increase in protein content of the DRG was associated with an increase in the diameter of smaller neurons (those<30 μm in diameter), but NGF caused no change in the number of neurons.     

Effect of injury on nerve growth factor uptake by sensory ganglia

The level of the nerve growth factor protein, NGF, in vivo has a profound influence on axonal sprouting by sensory neurons of vertebrate dorsal root ganglia. There is evidence also that NGF may play similar roles in cholinergic central structures in brain. In both instances, retrograde transport of NGF has been demonstrated. Here we examined uptake of NGF by DRG neurons in response to contusion injury of the spinal cord. Under these conditions there was uptake and transport of NGF into large DRG neurons via central processes but no uptake by non-DRG central neurons. Thus, any effects of NGF on spinal neurons or their processes would be secondary to the direct effects of NGF on DRG neurons.     

Substrate-bound nerve growth factor promotes neurite growth in peripheral nerve

Nerve growth factor (NGF), in addition to its well-known effects as a soluble neurite growth-promoting factor, also appears to promote the elongation of neurites when it is adsorbed to tissue culture substrates. Peripheral nerve Schwann cells appear to possess a receptor for NGF on their surfaces which is induced substantially after axotomy. We have found that the adsorption of NGF onto cryostat sections of the distal stump of previously severed sciatic nerve enhances neurite growth over this tissue. This finding, coupled with the two previous observations, suggests that Schwann cell surface NGF receptors serve to bind to NGF-like growth factors so as to provide favorable surfaces for regenerating peripheral nerve axons.     

Effects of nerve growth factor and heart cell conditioned medium on neurite regeneration of aged sympathetic neurons in culture

The effects of nerve growth factor (NGF) and heart-cell-conditioned medium (HCM) on the neurite regeneration of aged sympathetic neurons were investigated in culture. Investigation of HCM was carried out by two different methods: one was the use of whole HCM on collagen substratum, which reflected component(s) effective in solution (HCM-S); the other was the use of polyornithine (PORN)-binding component(s) (P-HCM). Superior cervical ganglion neurons prepared from male mice from 6 to 30 months of age were cultured in MEM-10% FCS on collagen or gelatin-PORN substratum for 3 days. The number of neurons with neurites and the length of neurites were quantified as neurite production and elongation, respectively. Neuronal survival was not affected by addition of NGF, HCM-S or P-HCM. Neurite production of early adult neurons was enhanced by NGF, HCM-S or P-HCM. In contrast, neurite production of aged neurons was enhanced by only HCM-S, but not NGF or P-HCM. HCM-S did not promote neurite elongation in neurons at any age. Neurite elongation of early adult neurons was enhanced by NGF or P-HCM. Neurite elongation of aged neurons was enhanced by P-HCM. However, responsiveness of NGF for neurite elongation varied according to substrata. No age-related difference was found in neurite production and elongation in the absence of NGF, HCM-S or P-HCM. These results indicate that responsiveness of aged sympathetic neurons is various in different growth factors.     

Immunohistochemical phenotype of sensory neurons associated with sympathetic plexuses in the trigeminal ganglia of adult nerve growth factor transgenic mice

Following peripheral nerve injury, postganglionic sympathetic axons sprout into the affected sensory ganglia and form perineuronal sympathetic plexuses with somata of sensory neurons. This sympathosensory coupling contributes to the onset and persistence of injury-induced chronic pain. We have documented the presence of similar sympathetic plexuses in the trigeminal ganglia of adult mice that ectopically overexpress nerve growth factor (NGF), in the absence of nerve injury. In this study, we sought to further define the phenotype(s) of these trigeminal sensory neurons having sympathetic plexuses in our transgenic mice. Using quantitative immunofluorescence staining analyses, we show that the invading sympathetic axons specifically target sensory somata immunopositive for several biomarkers: NGF high-affinity receptor tyrosine kinase A (trkA), calcitonin gene-related peptide (CGRP), neurofilament heavy chain (NFH), and P2X purinoceptor 3 (P2X3). Based on these phenotypic characteristics, the majority of the sensory somata surrounded by sympathetic plexuses are likely to be NGF-responsive nociceptors (i.e., trkA expressing) that are peptidergic (i.e., CGRP expressing), myelinated (i.e., NFH expressing), and ATP sensitive (i.e., P2X3 expressing). Our data also show that very few sympathetic plexuses surround sensory somata expressing other nociceptive (pain) biomarkers, including substance P and acid-sensing ion channel 3. No sympathetic plexuses are associated with sensory somata that display isolectin B4 binding. Though the cellular mechanisms that trigger the formation of sympathetic plexus (with and without nerve injury) remain unknown, our new observations yield an unexpected specificity with which invading sympathetic axons appear to target a precise subtype of nociceptors. This selectivity likely contributes to pain development and maintenance associated with sympathosensory coupling.     

Reduction of nerve growth factor receptor immunoreactivity in ischaemic gerbil hippocampal CA1 neurons after treatment with L-threo-3,4-dihydroxyphenylserine (DOPS)

Abstract

Nerve growth factor (NGF) synthesis in cultured mouse L-M fibroblast and astroglial cells can be increased after the treatment with L-threo-3,4-dihydroxyphenylserine (DOPS). Since the increase of NGF is not blocked by the treatment with decarboxylase inhibitor, DOPS may have direct effect to increase the NGF content. NGF and its receptor (NGFR) are suggested to play an important role in the neuronal survival and regeneration under pathologic conditions. In this study, we studied a possible protective effect of DOPS against the hippocampal CA1 cell death after transient forebrain ischaemia in gerbils in relation to the change of NGFR immunoreactivity. We found that treatment with DOPS (300 mg kg^–1) in combination with a decarboxylase inhibitor (benserazide, 10 mg kg^–1) protected ischaemic hippocampal CA1 cell against delayed neuronal death (neuronal density = 125 ± 24 mm^–1) as compared to the treatment with vehicle (49 ± 11 mm^–1) (p < 0.01). The immunoreactivity for NGFR was scarcely present in the sham-control CA1 area but was induced from 1 h and markedly expressed at 7 days after recirculation in the vehicle group. However; it was slightly and transiently induced from 8 h to 2 days in the DOPS plus benserazide treated group. These data suggest that the protective role of DOPS on the ischaemic hippocampal CA1 cells may act through the NGF and its receptor system. [Neurol Res 1994; 16: 201–204]     

Protective effect of nerve growth factor against glutamate-induced neurotoxicity in cultured cortical neurons

The effect of recombinant human nerve growth factor (hNGF) and mouse NGF on cultured rat cortical neurons was examined. The DNA fragment coding the human NGF gene was isolated and inserted downstream from the SV40 promoter in a plasmid containing the dihydrofolate reductase cDNA, and this plasmid was introduced into Chinese hamster ovary (CHO) cells to establish cells producing recombinant hNGF. The recombinant hNGF protein secreted by CHO cells was confirmed to be biologically active in an assay using PC12 cells. Brief exposure of cortical cells to glutamate followed by incubation with glutamate-free medium reduced cell viability by 60–70% when compared with the control culture. Simultaneous addition of recombinant hNGF or mouse NGF to rat cortical cultures with glutamate did not affect this reduction of cell viability. However, 24 h pretreatment of rat cortical cultures with recombinant hNGF or mouse NGF resulted in a significant reduction of glutamate-induced neuronal damage. Mouse NGF also protected cortical neurons against N-methyl-d-aspartate (NMDA)- and kainate-induced neuronal damage. These findings suggest that NGF can protect cortical neurons against glutamate-induced neurotoxicity.     

Expression of axonal protein degradation machinery in sympathetic neurons is regulated by nerve growth factor

Deficiencies in protein degradation and proteolytic function within neurons are linked to a number of neurodegenerative diseases and developmental disorders. Compartmentalized cultures of peripheral neurons were used to investigate the properties and relative abundance of the proteolytic machinery in the axons and cell bodies of sympathetic and sensory neurons. Immunoblotting of axonal proteins demonstrated that LAMP2, LC3, and PSMA2 were abundant in axons, suggesting that lysosomes, autophagosomes and proteasomes were located in axons. Interestingly, the expression of proteins associated with lysosomes and proteasomes were upregulated selectively in axons by NGF stimulation of the distal axons of sympathetic neurons, suggesting that axonal growth and maintenance requires local protein turnover. The regulation of the abundance of both proteasomes and lysosomes in axons by NGF provides a link between protein degradation and the trophic status of peripheral neurons. Inhibition of proteasomes located in axons resulted in an accumulation of ubiquitinated proteins in these axons. In contrast, lysosome inhibition in axons did not result in an accumulation of ubiquitinated proteins or the transferrin receptor, a transmembrane protein degraded by lysosomes. Interestingly, lysosomes were transported both retrogradely and anterogradely, so it is likely that ubiquitinated proteins that are normally destined for degradation by lysosomes in axons can be transported to the cell bodies for degradation. In summary, proteasomal degradation occurs locally, whereas proteins degraded by lysosomes can most likely either be degraded locally in axons or be transported to cell bodies for degradation.     

The effects of nerve growth factor (NGF) and antiserum to NGF on the development of embryonic sympathetic neurons in vivo

The role of nerve growth factor (NGF) in the development of embryonic sympathetic neurons was examined in vivo. Individual mouse embryos received transuterine injections of NGF or antiserum to NGF (anti-NGF), and the effects on the superior cervical ganglion (SCG) were studied. Treatment with NGF at any gestational stage, from the time of ganglion aggregation to birth, increased ganglion tyrosine hydroxylase (T-OH) activity. Both the number of catecholaminergic neurons and T-OH activity per neutron were increased. Choline acetyltransferase (ChAc) activity was increased by NGF at early gestational stages, but not at later stages. These observations suggest that perikarya containing ChAc are responsive to NGF, whereas preganglionic nerve terminals are not. Treatment with anti-NGF rapidly and permanently decreased ganglion T-OH activity. The effects of anti-NGF were more pronounced at later gestational stages, suggesting that ganglia become increasingly dependent on NGF during development. Alteration of maternal levels of NGF had no effect on development of the embryonic SCG, suggesting that local embryonic concentrations of NGF are responsible for modulating sympathetic ontogeny.     

Regulation of substance P by nerve growth factor: disruption by capsaicin

Capsaicin depleted substance P from guinea pig dorsal root ganglia and inhibited the retrograde axoplasmic transport of nerve growth factor (NGF). Doses of capsaicin which depleted substance P also inhibited the retrograde axoplasmic transport of NGF. Inhibition of the retrograde transport of NGF by capsaicin preceded substance P depletion. Supplementation of guinea pigs with mouse NGF completely prevented capsaicin-induced substance P depletion. It is concluded that capsaicin depletes substance P from primary afferent neurons of the adult guinea pig by altering the availability of NGF. The data support a role for NGF in the normal maintenance of neuropeptide levels in some sensory neurons in the adult animal.     

人神经生长因子对大鼠中枢胆碱能神经元损伤后作用的实验研究

目的　研究人神经生长因子（ Ｈ Ｎ Ｇ Ｆ） 对大鼠中枢胆碱能神经元轴突切断后的保护作用。方法　向双侧海马伞切断所致的痴呆大鼠脑室内注入 Ｈ Ｎ Ｇ Ｆ, 采用穿梭箱训练, 检测大鼠学习记忆功能恢复状况, 对脑内隔斜带复合体（ Ｓ Ｄ Ｂ） 中隔区（ Ｍ Ｓ） 胆碱能神经元采用胆碱乙酰基转移酶（ Ｃ Ｈ Ａ Ｔ） 免疫组化法观察其存活状态。结果　 Ｈ Ｎ Ｇ Ｆ 组大鼠术后两周内穿梭箱回避次数高于对照组（ Ｐ＜ ００１） , 回避潜伏期短于对照组（ Ｐ＜ ００１） 。免疫组化及图像分析显示, 其胆碱能神经元存活状态优于对照组。结论　 Ｈ Ｎ Ｇ Ｆ 对大鼠中枢胆碱能神经元损伤具有短期保护作用; 减缓轴突切断引起的退行性变, 对痴呆大鼠学习记忆功能恢复有促进作用。     

Nerve growth factor and injured peripheral nerve regeneration

Nerve growth factor （NGF） exhibits many biological activities, such as supply of nutrients, neuroprotection, and the generation and rehabilitation of injured nerves. The neuroprotective and neurotrophic qualities of NGF are generally recognized. NGF may enhance axonal regeneration and myelination of peripheral nerves, as well as cooperatively promote functional recovery of injured nerves and limbs. The clinical efficacy of NGF and its therapeutic potentials are reviewed here. This paper also reviews the latest NGF research developments for repairing injured peripheral nerve, thereby providing scientific evidence for the appropriate clinical application of NGF.