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.     

神经生长因子对培养的大鼠背根神经节神经元P物质释放的调节作用

目的观察神经生长因子（nerve growth factor, NGF）对原代培养的背根神经节（dorsal root ganglion, DRG）神经元中P物质（substance P, SP）的基础释放量和辣椒素诱发释放量的调节效应。方法将15 天胚龄的Wistar大鼠DRG神经元培养于含有不同浓度NGF的DMEM/F12培养液中,不含NGF的培养液培养的神经元作为对照。72小时后,用RT-PCR检测神经元中SP mRNA和辣椒素受体（vanilloid receptor 1, VR1）mRNA的表达,用放射免疫分析（radioimmunoassay,RIA）法检测SP的基础释放量和辣椒素（100 nmol/L）刺激10 min后的诱发释放量。结果SPmRNA和VR1 mRNA在NGF孵育的标本中表达增加,并与孵育液中NGF的浓度呈剂量依赖关系。SP的基础释放量和辣椒素诱发释放量在NGF孵育的标本中均增加,而且诱发释放量与NGF的浓度呈剂量依赖关系。结论NGF使DRG神经元SP的基础释放量和诱发释放量增加,表明NGF能增加初级传入神经元感受伤害刺激的敏感性,该效应可能与SP和VR1的mRNA表达增加有关。     

神经生长因子对培养的大鼠背根神经节神经元P物质释放的调节作用(英文)

目的观察神经生长因子(nerve growth factor, NGF)对原代培养的背根神经节(dorsal root ganglion, DRG)神经元中P物质(substance P, SP)的基础释放量和辣椒素诱发释放量的调节效应。方法将15 天胚龄的Wistar大鼠DRG神经元培养于含有不同浓度NGF的DMEM/F12培养液中,不含NGF的培养液培养的神经元作为对照。72小时后,用RT-PCR检测神经元中SP mRNA和辣椒素受体(vanilloid receptor 1, VR1)mRNA的表达,用放射免疫分析(radioimmunoassay,RIA)法检测SP的基础释放量和辣椒素(100 nmol/L)刺激10 min后的诱发释放量。结果SPmRNA和VR1 mRNA在NGF孵育的标本中表达增加,并与孵育液中NGF的浓度呈剂量依赖关系。SP的基础释放量和辣椒素诱发释放量在NGF孵育的标本中均增加,而且诱发释放量与NGF的浓度呈剂量依赖关系。结论NGF使DRG神经元SP的基础释放量和诱发释放量增加,表明NGF能增加初级传入神经元感受伤害刺激的敏感性,该效应可能与SP和VR1的mRNA表达增加有关。     

Effects of autoimmune NGF deprivation in the adult rabbit and offspring

An experimental autoimmune approach to the production of nerve growth factor deprivation, which we have previously described in the rat and guinea pig, has been applied to the rabbit. This species was chosen for study because of several potential advantages. The rabbit produces large litters and has a relatively short gestation period. More importantly, rabbits generate high titers of antibody against mouse NGF and large amounts of maternal antibody are passively transferred to the developing rabbit fetus compared to most other species, particularly the rat. The sympathetic nervous system of adult rabbit immunized against mouse NGF underwent degeneration with up to an 85% decrease in neuronal numbers in the superior cervical ganglion after 10 months of immunization, thus providing further evidence that NGF is required for the survival of mature sympathetic neurons. Despite the fact that newborn rabbits born to anti-NGF producing mothers had much higher titers of anti-NGF than did rats, the effects on the developing sympathetic and sensory nervous systems were not found to be any greater than in rats. Reductions in norepinephrine levels in the heart and spleen of adult rabbits born to anti-NGF producing mothers were greater than in small intestine. Prenatal exposure to maternal anti-NGF caused reductions (up to 70%) in the number of neurons in the dorsal root ganglia. Substance-P immunoreactivity was reduced in the substantia gelatinosa of the spinal cord of rabbit exposed to maternal anti-NGF. These changes, however, were not greater than seen in the rat. We conclide that although the rabbits offers some advantage in the study of the effects of NGF deprivation in the adult animal, it appears less well suited than the rat or guinea pig to the study of the effects of NGF deprivation on development.     

Effects of nerve growth factor on TTX- and capsaicin-sensitivity in adult rat sensory neurons

We have investigated the effects of nerve growth factor (NGF, 2.5 ng/ml for 1–2 weeks) on enriched adult rat dorsal root ganglion (DRG) neurons maintained in cell culture in defined media. Whole-cell recordings in cells cultured in the absence and presence of NGF revealed no significant difference in resting membrane potential and input resistance. However, the threshold for spike generation was significantly lower in untreated cells than in treated cells; −25 ± 1.1mV vs−19 ± 2.2mV, respectively. The sensitivity of the Na⁺ spike to tetrodotoxin (TTX, 1 μM) was different in cells cultured in the absence or presence of NGF. For example, spikes were abolished by TTX in 100% of untreated cells, while in NGF-treated cells the spike was abolished in only 41% of the neurons. Chemosensitivity of DRG neurons was also different in the absence and presence of NGF. For example, the percent of neurons in which a current activated by 8-methyl-N-vanillyl-6-nonenamide (capsaicin, 500 nM) was detected, increased from 18% in untreated cells to 55% in NGF-treated cells. NGF did not influence the number of cells surviving. The results indicate that NGF can regulate TTX and capsaicin sensitivity in these adult rat sensory neurons. Our experimental protocol indicates that this effect is not mediated by a factor in the serum or released from non-neuronal cells.     

Effects of cyclooxygenase 2 inhibitor on growth-associated protein 43 and nerve growth factor expression in dorsal root ganglion during neuropathic pain development

BACKGROUND： Inflammatory responses in injured nerves have been recognized as important factors for initially sensitizing nociceptive neurons. Cyclooxygenase （COX） is the rate-limiting enzyme in prostaglandin synthesis, and COX-2 inhibitor is involved in mechanisms of analgesia and anti-inflammation. OBJECTIVE： To investigate the effects of COX-2 inhibitor on thermal and mechanical hyperalgesia, as well as expression of growth associated protein 43 （GAP-43） and nerve growth factor （NGF） in dorsal root ganglion, in a rat model of neuropathic pain due to chronic constriction injury. DESIGN, TIME AND SETTING： A randomized, controlled, comparison study that was performed at the Surgical Department and Pathological Laboratory, Second Affiliated Hospital of Shantou University Medical College from September 2006 to September 2007. MATERIALS： COX-2 inhibitor, Iornoxicam, was purchased from Nycomed Pharmaceutical （Austria）; rabbit anti-GAP-43, and rabbit anti-NGF polyclonal antibodies were purchased from Boster, Wuhan, China. METHODS： A total of 50 adult, Wistar rats were randomly assigned to four groups： normal control （n = 5）, model （n = 15）, normal saline control （n = 15）, and Iornoxicam treatment （n =15）. With exception of the control group, the sciatic nerve of all rats was loosely ligated to establish a model of chronic constriction injury. The model rats were divided into three subgroups according to varying post-operative survival periods： 3, 7 and 14 days （n = 5）, respectively. Rats in the Iornoxicam treatment group were intraperitoneally injected with 1.3 mg/kg lornoxicam every 12 hours throughout the entire experimental procedure. Rats in the normal saline control group were intraperitoneally injected with 1.3 mL/kg saline. MAIN OUTCOME MEASURES： Immunohistochemistry revealed expression of GAP-43 and NGF in the L5 dorsal root ganglions. Mechanical withdrawal threshold and thermal withdrawal latency were used to observe neurological behavioral changes in rats. RESULTS： The relative gray values of GAP-43- and NGF-positive neurons in the model group were remarkably increased compared with the normal control rats （P 〈 0.01）, while the relative gray values in the Iomoxicam treatment group were significantly less than the model and normal saline control groups （P 〈 0.01）. Mechanical withdrawal threshold and thermal withdrawal latency gradually decreased with increasing injury time in the model, normal saline control, and Iornoxicam treatment groups, and were significantly less than the normal control group （P 〈 0.05）. In addition, mechanical withdrawal threshold and thermal withdrawal latency were significantly greater in the Iornoxicam treatment group compared with the model and normal saline control groups （P 〈 0.05）. CONCLUSION： Intraperitoneal injection of the COX-2 inhibitor Iornoxicam attenuated mechanical and thermal hyperalgesia induced by sciatic nerve chronic constriction injury and inhibited the increased expression of GAP-43 and NGF.     

Purification of adult rat sciatic nerve ciliary neuronotrophic factor

The ciliary neuronotrophic factor (CNTF), a protein required for the survival of cultured avian embryonic parasympathetic ciliary ganglionic neurons, was recently purified from extracts of selected chick intraocular tissues. Here we report the purification of a mammalian CNTF activity from extracts of adult rat sciatic nerve using a fractionation procedure similar to that employed for isolating chick eye CNTF. About 2 micrograms of CNTF protein can be obtained from each 1.5 g batch of nerve tissue. Like the chick CNTF, the mammalian factor displays trophic activity for dorsal root and sympathetic as well as ciliary ganglionic neurons. The nerve CNTF activity differs from its chick counterpart in molecular weight and chromatographic behavior on ion-exchange columns. Unlike purified nerve growth factor (NGF), nerve CNTF activity is insensitive to anti-NGF antibodies and is unable to support the survival of 8-day chick embryo dorsal root ganglion neurons.     

Transganglionic regulation of central terminals of dorsal root ganglion cells by nerve growth factor (NGF)

Blockade of axonal transport or transection of the rat sciatic nerve results in transganglionic degenerative atrophy (TDA) of nerve terminals containing fluoride-resistant acid phosphatase (FRAP) in the Rolando substance of the spinal cord. Application of vinblastine (9 micrograms) in a cuff around the sciatic nerve of adult rats blocked the retrograde transport of [125I]NGF in sensory fibers; this amount of vinblastine is identical to the threshold amount that induces TDA. Conversely, application of NGF to the proximal stump of the transected sciatic nerve prevented or delayed the occurrence of TDA as reflected by the maintenance of FRAP in the upper dorsal horn, that otherwise would inevitably disappear following the peripheral nerve lesion. These results suggest that endogenous NGF transported retrogradely in peripheral sensory fibers of the adult rat under normal conditions may be responsible for the regulation of the structural and functional integrity of the central terminals of these FRAP-containing primary sensory neurons and that TDA may be the consequence of the failure of NGF to reach the perikarya of these neurons.     

Apoptosis of spinal interneurons induced by sciatic nerve axotomy in the neonatal rat is counteracted by nerve growth factor and ciliary neurotrophic factor

We have previously shown that not only motoneurons and dorsal root ganglion cells but also small neurons, presumably interneurons in the spinal cord, may undergo apoptotic cell death as a result of neonatal peripheral nerve transection in the rat. With the aid of electron microscopy, we have here demonstrated that apoptosis in the spinal cord is confined to neurons and does not involve glial cells at the survival time studied (24 hours). To define the relative importance of the loss of a potential target (motoneuron) and a potential afferent input (dorsal root ganglion cell) for the induction of apoptosis in interneurons in this situation, we have compared the distributions and time courses for TUNEL labeling, which detects apoptotic cell nuclei, in the L5 segment of the spinal cord and the L5 dorsal root ganglion after sciatic nerve transection in the neonatal (P2) rat. In additional experiments, we studied the effects on TUNEL labeling of interneurons after treatment of the cut sciatic nerve with either ciliary neurotrophic factor (CNTF) to rescue motoneurons or nerve growth factor (NGF) to rescue dorsal root ganglion cells. The time courses of the TUNEL labeling in motoneurons and interneurons induced by the lesion show great similarities (peak at 8-48 hours postoperatively), whereas the labeling in dorsal root ganglion cells occurs later (24-72 hours). Both CNTF and NGF decrease the number of TUNEL-labeled interneurons, but there is a regional difference, in that CNTF preferentially saves interneurons in deep dorsal and ventral parts of the spinal cord, whereas the rescuing effects of NGF are seen mainly in the superficial dorsal horn. The results are interpreted as signs of a trophic dependence on both the target and the afferent input for the survival of interneurons neonatally.     

Viability of mature superior cervical ganglia transplants in peripheral nerve of adult rat

Allografts of mature rat superior cervical ganglia (SCG) survived for up to 120 days following transplantation to regenerating and degenerating peripheral nerves of adult rats. Transplants contained the constituents of normal superior cervical ganglia and there was evidence of outgrowth of catecholamine containing fibers from the transplants. However, fibers did not attain the length necessary to enter peripheral muscles and no muscle response could be elicited by electrical stimulation of the implanted nerve where the transplanted mature SCG was the only source of nerve fibers.     

Variation in number of small intensely fluorescent cells (SIF cells) in rat superior cervical ganglia

Changes in the relative synthesis rates of a number of specific proteins from rat dorsal root ganglia (DRG) were observed in response to transection of the sciatic nerve. The pattern of these changes was compared to that in rat sympathetic ganglia after transection of the postganglionic nerves. Although the overall pattern of proteins synthesized, and the pattern of changes seen after nerve transection, were quite similar in both ganglia, some specific differences were observed.     

Endogenous nerve growth factor regulates the sensitivity of nociceptors in the adult rat

Nerve growth factor (NGF) has a well characterized role in the development of the nervous system and there is evidence that it interacts with nociceptive primary afferent fibres. Here we applied a synthetic tyrosine kinase A IgG (trkA-IgG) fusion molecule for 10–12 days to the innervation territory of the purely cutaneous saphenous nerve in order to bind, and thereby neutralize endogenous NGF in adult rats. Using neurophysiological analysis of 152 nociceptors we now show that sequestration of NGF results in specific changes of their receptive field properties. The percentage of nociceptors responding to heat dropped significantly from a normal 57% to 32%. This was accompanied by a rightward shift and a reduced slope of the stimulus response function relating the intracutaneous temperature to the neural response. The number of nociceptors responding to application of bradykinin was also significantly reduced from a normal of 28% to 8%. In contrast, the threshold for mechanical stimuli and the response to suprathreshold stimuli remained unaltered, as did the percentage of nociceptors responding to noxious cold. The reduced sensitivity of primary afferent nociceptors was accompanied by a reduction in the innervation density of the epidermis by 44% as assessed with quantitative immunocytochemical analysis of the panaxonal marker PGP 9.5. This demonstrates that endogenous NGF in the adult specifically modulates the terminal arborization of unmyelinated fibres and the sensitivity of primary afferent nociceptors to thermal and chemical stimuli in vivo.     

Intrathecal administration of nerve growth factor delays GAP 43 expression and early phase regeneration of adult rat peripheral nerve

Whether nerve growth factor (NGF) promotes peripheral nerve regeneration in vivo, in particular in adults, is controversial. We therefore examined the effect of exogenous NGF on nerve regeneration and the expression of GAP 43 (growth-associated protein 43) in adult rats. NGF was infused intrathecally via an osmotic mini-pump, while control rats received artificial cerebrospinal fluid. Two days after the infusion was initiated, the right sciatic nerves were transected or crushed, and the animals allowed to survive for 3 to 11 days. The right DRG, the right proximal stump of the transected sciatic nerve, and the posterior horn of the spinal cord were examined by Western blotting, immunohistochemistry, and electron microscopy. GAP 43 immunoreactivity in the NGF-treated animals was significantly lower than in the aCSF-treated controls. Electron microscopy showed that the number of myelinated and unmyelinated axons decreased significantly in the NGF-treated rats as compared with the controls. These findings are indicative that exogenous NGF delayed GAP 43 induction and the early phase of peripheral nerve regeneration and supports the hypothesis that the loss of NGF supply from peripheral targets via retrograde transport caused by axotomy serves as a signal for DRG neurons to invoke regenerative responses. NGF administered intrathecally may delay the neurons' perception of the reduction of the endogenous NGF, causing a delay in conversion of DRG neurons from the normal physiological condition to regrowth state.     

Dependence on nerve growth factor of early human fetal dorsal root ganlion neurons in organotypic cultures

Dorsal root ganglion (DRG) neurons explanted from human embryos, at stages less than about 8 weeks in utero, appeared to be strongly dependent on nerve growth factor (NGF) for their long-term survival. In cultures containing a high concentration of NGF (1000 units/ml, added only at explantation), most of the DRG neurons survived and developed for many weeks in vitro. In contrast, extensive degeneration of DRG neurons was evident within the 1st week after explantation of these immature ganglia in our normal culture medium without added NGF. On the other hand, although introduction of NGF in cultures of 10- to 12-week-old human fetal DRG neurons enhanced the early outgrowth of neurites, these ganglia showed relatively good growth and maintenance in long-term culture even when NGF was omitted from the medium. DRGs from human fetuses estimated to be between 9 and 10 weeks in utero showed intermediate degrees of survival when NGF was omitted from the culture medium (about 10 to 25% of the DRG neurons survived compared with those in paired cultures treated with NGF). The data demonstrate the existence of a critical period during which human DRG neurons may require high NGF concentrations to ensure long-term survival and maturation. Human fetal DRG cultures may provide a useful model system for studies related to familial dysautonomia where drastic deficits in sensory and sympathetic ganglia occur in utero.     

Administration or deprivation of nerve growth factor during development permanently alters neuronal geometry.

We investigated whether the administration or deprivation of a neuronal growth factor during development can permanently alter the dendritic architecture of sensitive neurons. Nerve growth factor (NGF) or NGF antiserum treatment in the first 2-3 postnatal weeks markedly affected the survival, size, and dendritic arborization of mouse sympathetic ganglion cells acutely. Six months after the completion of treatment, the number of surviving neurons, cell body size, and higher order dendritic branching had changed considerably from their values at 3 weeks, suggesting that these parameters remain malleable throughout postnatal life. However, the number of primary dendrites, a fundamental determinant of organization within sympathetic ganglia, was permanently altered by the neonatal treatment protocol. The idea emerging from this study is that NGF influences the elaboration of primary dendrites by sympathetic ganglion cells only during a critical developmental period. In maturity, NGF acts as a "maintenance" factor necessary for normal neuronal function and survival, but neurons lose the capacity to respond with wholesale rearrangements of dendritic architecture.     

Nerve growth factor effects on protein synthesis after nerve damage

Axonal damage can induce a variety of changes in the cell bodies of neurons and in neighboring cells. Such changes, termed the axon reaction, can include neuronal chromatolysis, synaptic disconnection, and altered protein synthesis. Nerve growth factor (NGF) treatment of sympathetic ganglia after nerve damage has been reported to block partially both chromatolysis and synaptic disconnection. We examined the proteins synthesized in rat superior cervical ganglia using two-dimensional polyacrylamide gel electrophoresis. Axotomy induces changes in the relative rates of synthesis of a number of the proteins. The NGF treatment after axotomy does not reverse most of these, and induces other changes. It thus appears that the absence of NFG in retrograde transport from target tissues cannot alone be the signal for the axon reaction.     

Behavioral effects of early deprivation of nerve growth factor: Some similarities with familial dysautonomia

Female rats immunized with mouse nerve growth factor develop an antibody (anti-NGF) which reaches offspring through the placenta and via the milk. Pups exposed to maternal anti-NGF have fewer dorsal root and sympathetic neurons. When the offspring are examined on a wide variety of behavioral tests, they exhibit severe deficits in response to stress (ulceration, corticosterone levels), and mild deficits on some sensory and cognitive tasks. Explaratory and motor functions, however, are relatively normal. The pathologic and behavioral profiles of the animals closely mimic the sensory and sympathetic aspects of familial dysautonomia.