NGF prevention of neurotoxicity induced by cisplatin, vincristine and taxol depends on toxicity of each drug and NGF treatment schedule:: In vitro study of adult rat sympathetic ganglion explants

The simultaneous administration of nerve growth factor (NGF) has been found to prevent experimental neuropathies induced by anti-cancer drugs such as cisplatin, vincristine and taxol. However, it is clinically important to know whether NGF is beneficial once the neuropathy is already manifest. We established a bioassay system to examine the preventive effects of NGF in various treatment schedules. NGF significantly prevented the inhibition of neurite outgrowth by vincristine and taxol regardless of treatment schedules. The pre-treatment and co-treatment schedules were effective against cisplatin, but the post-treatment schedule was not. With regard to the neurite and nerve cell population densities, only the cisplatin group treated with NGF showed lower values than the control. These results indicate that NGF-treatment is effective for the toxic sympathetic nerve injury induced by vincristine and taxol regardless of the treatment schedule, but is not protective against cisplatin-induced nerve cell injury.     

The effects of lead on transient outward currents of acutely dissociated rat dorsal root ganglia

The effects of Pb2+ on transient outward currents (TOCs) were investigated on rat dorsal root ganglia (DRG) neurons at postnatal days of 15 approximately 21, using the conventional whole-cell patch-clamp technique. In media-sized (35 approximately 40 microm) neurons and in the presence of 50 mM TEA, TOCs that preliminarly included an A-current (IA) and a D-current (ID), were clearly present and dominant. Application of Pb2+ lengthened the initial delay of TOCs and increased the onset-peak time in a concentration-dependent manner. The amplitudes of initial outward current peak were reduced with increasing Pb2+ concentrations. The inhibitory effects of Pb2+ on TOCs were reversible with 80 approximately 90% of current reversed in 2 approximately 10 min at 1 approximately 400 microM Pb2+. For the normalized activation curves fitted by a single Boltzmann equation under each condition, there was a shift to more depolarized voltages with increasing concentrations of Pb2+. The V1/2 and the slope factor (k) increased from 12.76+/-1.49 mV and 15.31+/-1.66 mV (n=10) under control condition to 39.91+/-5.44 mV (n=10, P<0.01) and 21.39+/-3.13 mV (n=10, P<0.05) at 400 microM Pb2+, respectively, indicating that Pb2+ decreased the activation of TOCs. For the normalized steady-state inactivation curves, the V1/2 and the k increased from -92.31+/-2.72 and 8.59+/-1.36 mV (n=10) to -55.65+/-3.67 (n=10, P<0.01) and 23.02+/-2.98 mV (n=10, P<0.01) at 400 microM Pb2+, respectively. The curves were shifted to more depolarized voltages by Pb2+, indicating that channels were less likely to be inactivated at higher concentrations of Pb2+ at any given potential. The fast (tf) and slow (ts) decay time-constants were both significantly increased by increasing concentrations of Pb2+ (n=10, P<0.05), indicating that Pb2+ increased the decay time-course of TOCs. These effects were concentration-dependent and partly reversible following washing. Ca2+ modulated the TOCs gating and might share same binding site with Pb2+, for which Ca2+ had very low affinity. In summary, the results demonstrated that Pb2+ was a dose- and voltage-dependent, and reversible blocker of TOCs in rat DRG neurons. After Pb2+ application, normal sensory physiology of DRG neurons was affected, and these neurons might display aberrant firing properties that resulted in abnormal sensations. This variation caused by Pb2+ could underlie the toxical modulation of sensory input to the central nervous system.     

Reversible effects of hypoxia on neurons in mouse dorsal root ganglia in vitro

Mouse dorsal root ganglia (DRG) were isolated and maintained in a tissue chamber. Membrane potential of ‘A-type’ neurons was recorded with intracellular electrodes. When the supply of oxygen was reduced, cells depolarized by a few mV and then maintained a stable membrane potential or partially repolarized. During depolarization the action potential was reduced in amplitude and the hyperpolarizing afterpotential was depressed. Reoxygenation within 15–88 min was followed by a brief period of hyperpolarization and then complete recovery. In about 60% of the cells, invasion of the cell soma by impulses triggered by dorsal root (DR) stimulation failed during hypoxia while action potentials could still be evoked by stimulation of the peripheral nerve and by direct intracellular stimuli. Conduction from DR into the peripheral nerve stump was unchanged indicating that the blockade of DR-evoked impulse conduction occurred at the bifurcation of the axon. Results with paired pulse stimulation indicated that impulses passing the axon bifurcation leave a long lasting ( 25 ms) post-spike subnormal period. In DRG cells treated with tetraethylammonium (TEA) the calcium-mediated ‘shoulder’ of the action potential was curtailed during oxygen withdrawal. In contrast to CNS neurons, DRG cells did not show early hypoxic hyperpolarization, nor the delayed hypoxic spreading depression-like depolarization. The findings support the suggestion that the reversible depression of synaptic potentials in the CNS during the early phase of hypoxia is caused by a combination of conduction failure at axon branch points and curtailment of voltage calcium currents of presynaptic terminals, both effects resulting in reduced transmitter output.     

Reduced NGF secretion by Schwann cells under the high glucose condition decreases neurite outgrowth of DRG neurons

Background

Schwann cells (SCs) have been supposed to play prominent roles in axonal regeneration under various diseases. Here, to evaluate the direct interaction between SCs and dorsal root ganglion (DRG) neurons under a diabetic condition, the effects of Schwann cell-conditioned media on neurite outgrowth of DRG neurons were investigated.

Methods

Immortalized mouse Schwann cells (IMS) were cultured under 5.5 mM glucose (NG) or 30 mM glucose (HG) conditions for 4 days. IMS-conditioned media (IMS-media) were added to the culture media of neurons isolated from 8-week-old DDY mice. Neurons were cultured for 48 h with or without mouse recombinant NGF (mrNGF) or nerve growth factor (NGF) neutralizing antibody. The concentrations of NGF in IMS-media by ELISA and neurite outgrowth by a computed image analysis system were evaluated.

Results

Neurite outgrowth was significantly enhanced by IMS-media (IMS-media (–): 177 ± 177 µm, IMS-media (+): 1648 ± 726). The neurite outgrowth cultured with IMS-media obtained under the HG condition was significantly reduced compared with that under the NG condition (NG: 1474 ± 652, HG: 734 ± 331). The NGF concentrations were significantly lower in IMS-media under the HG condition than in those under the NG condition. The accelerated neurite outgrowth by IMS-media was inhibited by NGF neutralizing antibody.

Conclusions

These results suggest that SCs play important roles in neurite outgrowth of DRG neurons, and that the decreased NGF secretion by SCs under the diabetic condition would cause a defect of axonal regeneration, resulting in the development of diabetic neuropathy.     

Hematogenous metastases of carcinoma to dorsal root ganglia

Summary Hematogenous metastases of carcinoma to dorsal root ganglia was found in 2 of approximately 500 consecutive autopsies in which a lumbar dorsal root ganglion was routinely examined microscopically. The primary tumors were poorly differentiated colonic adenocarcinoma and oat cell carcinoma of the lung, both with widespread hematogenous metastases which spared the central nervous system. No symptoms were detected clinically. In the same series of patients the sural nerve as well as the lumbar plexus were histologically sampled but no examples of distant endoneurial metastases were found. The vascular endothelium of dorsal root ganglia is fenestrated and, presumably as a consequence, provides no bloodganglion barrier. This microvascular difference may account for the susceptibility of the ganglia to metastases when compared to nerve trunks which possess unfenestrated endothelium and blood-nerve barrier.     

Transthyretin is not expressed by dorsal root ganglia cells

Several mutations in transthyretin (TTR) are related to familial amyloidotic polyneuropathy (FAP), a neurodegenerative disorder caused by extracellular deposition of TTR fibrils, particularly in the peripheral nervous system (PNS). TTR is mainly synthesized by the liver and choroid plexus of the brain that contribute to the plasma and cerebrospinal fluid (CSF) pools of the protein, respectively. It has recently been reported that TTR is additionally expressed in the PNS, namely by peripheral glial cells of dorsal root ganglia (DRG). This lead to the hypothesis that TTR synthesis in the DRG might contribute to the PNS involvement in FAP. In this report we clarify this issue by showing that TTR synthesis is absent in both human and mouse DRG. Moreover, by using TTR KO mouse DRG as controls, we demonstrate that TTR-like immunoreactivity in the perineurium is an artifact. As such, and similarly to what has been previously shown in the central nervous system (CNS), TTR amplification by RT-PCR in the DRG most probably results from contamination by the meninges. In conclusion, TTR deposited in the PNS of FAP patients should still be regarded as having blood and/or CSF origin.     

Increased brain-derived neurotrophic factor immunoreactivity in rat dorsal root ganglia and spinal cord following peripheral inflammation

Our recent study showed that peripheral inflammation induced an increased expression of brain-derived neurotrophic factor (BDNF) mRNA which was mediated by nerve growth factor (NGF) in the dorsal root ganglion (DRG). In the present study, we evaluated the change of BDNF immunoreactivity in the DRG and spinal cord following peripheral inflammation by means of immunohistochemistry. Significant increases in the percentage of BDNF-immunoreactive (IR) neuron profiles in the L5 DRG and marked elevation in the expression of BDNF-IR terminals in the spinal dorsal horn were observed following peripheral tissue inflammation produced by an intraplantar injection of Freund's adjuvant into the rat paws. These findings suggest that peripheral tissue inflammation induces an increased BDNF synthesis in the DRG and an elevated anterograde transport of BDNF to the spinal dorsal horn. The functional role of this increased BDNF was discussed briefly.     

Development of inflammatory hypersensitivity and augmentation of sodium channels in rat dorsal root ganglia

The development of thermal allodynia in relationship to sodium channel augmentation in dorsal root ganglia (DRGs) was studied in albino rats. Paw withdrawal latencies were measured hourly following complete Freund's adjuvant (CFA) injections. Sodium channels were demonstrated with immunocytochemistry. Sustained minimum latencies were attained between 10 and 12 h post-injection. Sodium channel labeling began to increase at 23 h post-injection and reached maximum levels by 24 h. Thermal hypersensitivity is thus established 12 h before sodium channel augmentation can be demonstrated.     

trkB-like immunoreactivity in rat dorsal root ganglia following sciatic nerve injury

The expression of full-length trkB protein, the functional high affinity receptor for BDNF and NT-4, was examined by immunohistochemistry in adult rat L4–L5 dorsal root ganglia after different types of sciatic nerve lesions. In normal ganglia, 52.5% of the neurons showed trkB-like immunoreactivity. Size measurements demonstrated that trkB-like immunoreactivity was seen predominantly in small- and medium-sized cells. This was confirmed by the finding that 28% of all trkB-positive neurons showed affinity to RT97, an antibody which lanels a neurofilament epitope specific for medium-sized and large primary afferent neurons. After crush, section or neuroma formation of the sciatic nerve, the proportion of trkB-positive cells was 64.5%, 58% and 61.9%, respectively. Since trkB-receptors are present in regenerating primary afferent neurons, these data could indicate that BDNF and/or NT-4 are involved in sensory nerve fiber regeneration after adult injury.     

Morphological effects of neuropathy-inducing organophosphorus compounds in primary dorsal root ganglia cell cultures

Chick embryo dorsal root ganglia (DRG) cultures were used to explore early pathological events associated with exposure to neuropathy-inducing organophosphorus (OP) compounds. This approach used an in vitro neuronal system from the species that provides the animal model for OP-induced delayed neuropathy (OPIDN). DRG were obtained from 9-day-old chick embryos, and grown for 14 days in minimal essential medium (MEM) supplemented with bovine and human placental sera and growth factors. Cultures were then exposed to 1 microM of the OP compounds phenyl saligenin phosphate (PSP) or mipafox, which readily elicit OPIDN in hens, paraoxon, which does not cause OPIDN, or the DMSO vehicle. The medium containing these toxicants was removed after 12 h, and cultures maintained for 4-7 days post-exposure. Morphometric analysis of neurites was performed by inverted microscopy, which demonstrated that neurites of cells treated with mipafox or PSP but not with paraoxon had decreased length-to-diameter ratios at day 4 post-exposure. Ultrastructural alterations of neurons treated with PSP and mipafox included dissolution of microtubules and neurofilaments and degrading mitochondria. Paraoxon-treated and DMSO control neuronal cell cultures did not show such evident ultrastructural changes. This study demonstrates that chick DRG show pathological changes following exposure to neuropathy-inducing OP compounds.     

Exogenous silver in dorsal root ganglia,peripheral nerve,enteric ganglia,and adrenal medulla

Summary Following intraperitoneal (i.p.) or oral administration of silver salts, the anatomic distribution of silver in the peripheral nervous system (PNS) has been studied. The structures examined were dorsal root ganglia, peripheral nerve (N. ischiadicus), enteric ganglia, and adrenal medulla.Four days after an i.p. injection of silver lactate, silver deposits were found in these structures. The silver content remained stable during the observation period (45 days).The localization of silver deposits in the orally treated animals was independent of the administered silver salt (silver nitrate or silver lactate).The silver deposits in neurons and chromaffin cells were located in the cytoplasm. In all organs silver was present in large amounts in connective tissue membranes, macrophage-like cells, vascular basal laminae, and supporting cells. Satellite cells of the dorsal root ganglia were always heavily stained, white less stain was present in Schwann cells of the peripheral nerves.Intracellular deposits were invariably located in lysosomes, whereas extracellular grains were found in connective tissue fibers and basement membranes.     

Distribution of calretinin mRNA in rat spinal cord and dorsal root ganglia: a study using non-radioactive in situ hybridization histochemistry

Using non-radioactive in situ hybridization calretinin mRNA was detected in numerous small neurons within lamina II and IV of the dorsal horn. Many labelled cells are distributed over the whole ventral horn; however, no motorneurons contained the mRNA. In dorsal root ganglia4.9 ± 1.7% (mean ± S.D., n = 5 animals) of the primary afferent neurons contained calretinin mRNA. Labelled cells were of intermediate and large size with diameters ranging from 36 to 68 μm indicating that calretinin is synthesized in neurons with myelinated afferetn fibers and presumably a corpuscular ending.     

Nerve growth factor receptors: analysis of the interaction of ßNGF with membranes of chick embryo dorsal root ganglia

The binding of the β subunit of Nerve Growth Factor (ßNGF) to membrane preparations of 8-day chick embryo dorsal root ganglia (DRG) has been investigated under conditions similar to those used to study the binding of ßNGF to intact single cell dissociates of DRG²³.The equilibrium binding data reveal heterogeneity of binding that is more complex than that seen with intact cells. Binding is not saturable up to¹²⁵IßNGF concentrations of 10⁻⁸ M. Steady-state and kinetic binding data show two sites with dissociation constants similar to those found on DRG cells. In addition, displacement data reveal a binding component with lower affinity (K_d = 10⁻⁶M) which is not found on intact cells.As with intact cells, the difference in the affinities of the two high affinity sites has been shown to be due to different rate constants of dissociation. The kinetics of dissociation of NGF are slower with membranes than with cells, and dissociation characteristics of¹²⁵IßNGF change with increasing time of exposure to membranes. Degradation of¹²⁵IßNGF during incubation with membranes is minimal and does not complicate the analysis of steady-state binding. Insulin does not to either of the two high affinity sites.Heterogeneity of the¹²⁵IßNGF preparation and cooperativity of binding as a cause for the heterogeneity of the binding of NGF has been ruled out. Although there was an apparent increase in the rate of dissociation of¹²⁵IßNGF in the presence of unlabelled NGF, a finding previously interpreted as evidence for negative cooperativity⁷, this was shown to be independent of receptor site occupancy by NGF, and in part due to isotopic dilution within a diffusion barrier around the membranes.     

NGF对鸡胚背根神经节神经突起生长作用的机制研究

目的探讨神经生长因子(nerve growth factor,NGF)促进鸡胚背根神经节(dorsal root ganglion,DRG)神经突起生长的作用机制。方法实验采用9 d的鸡胚分离背根神经节,原代培养法,观察鸡胚DRG的体外生长情况。通过半定量PCR检测诱导型一氧化氮合酶(iNOS)mRNA表达,采用NO检测试剂盒检测NO释放水平。结果 NGF能明显促进鸡胚背根神经节神经突起生长,同时可见NGF抑制iNOS mRNA表达,NO检测结果显示,添加NGF培养的背根神经节上清NO分泌水平明显降低,与阴性对照组比较差异显著(P0.05)。结论 NGF可促进鸡胚背根神经节神经突起生长,其作用与其下调iNOS mRNA表达及抑制神经损伤因子NO释放有关。     

Effects of nerve growth factor on electrical membrane properties of cultured dorsal root ganglia neurons from normal and trisomy 21 human fetuses

Trisomy 21 (Down syndrome) results in abnormalities of electrical membrane properties of cultured human fetal dorsal root ganglion (DRG) neurons; namely, faster rates of depolarization and repolarization of the action potential, and a shortened spike duration. A possible role of nerve growth factor (NGF) in the expression of abnormal electrical membrane properties fetal human DRG neurons from trisomy 21 subjects was examined. DRG neurons obtained from normal and trisomy 21 abortuses of 16–20 weeks gestation were cultured in the presence or absence of 40 nM 7S NGF. After 1 week in culture, action potentials were recorded using the whole cell patch-clamp technique, in current clamp mode. At the testing membrane potential, normal (diploid) neurons grown without NGF showed reduced maximal rates of depolarization (−41.3%) and of repolarization (−31.4%), a decreased spike amplitude (−14.2%) and a prolonged action potential (+49.2%), when compared to normal cells cultured with NGF. Trisomy 21 neurons showed similar changes, but had a greater relative decrease in the rates of action potential depolarization and repolarization. These changes were evident at different membrane potentials. Normal and trisomic DRG neurons cultured without NGF showed differences in action potential parameters similar to those previously described using NGF-supplemented culture medium. These data indicate that NGF can regulate electrical membrane properties in cultured human fetal DRG neurons, but apparently is not responsible for the abnormalities observed in trisomy 21 neurons.     

Selective degeneration of dorsal root ganglia and dorsal nerve roots in methyl mercury-intoxicated rats: a stereological study

The components of the nervous system of rats that are most critically affected by methyl mercury are still a matter of debate. A recent stereological study of rats with typical symptoms resulting from methyl mercury intoxication demonstrated that the morphology of cerebellar granule cells and Purkinje cells were unchanged at the light microscopic level, even though there was pronounced degeneration of myelinated axons in dorsal nerve root nerves. In the present study, unbiased stereological methods were used to quantify morphological changes in the dorsal root ganglion, and dorsal and ventral nerve roots of the rats used in the previous study. The rats were treated with methyl mercury (2 mg daily/kg, per os) for a 19-day period that was followed by a 32-day period without treatment. The means of the total numbers of A-cell and B-cell perikarya in the dorsal root ganglion of the intoxicated rats were reduced by 60% and 24%, respectively. The mean volume of A-cell perikarya in rats of the experimental group was reduced by 22%, whereas the mean volume of B-cell perikarya was the same in the two groups. In the experimental group, the total number of myelinated axons in the dorsal nerve roots was reduced by 60%, whereas no difference was found in the ventral nerve roots. The areas of axon and myelin sheath, dorsal and ventral nerve roots were not affected. This study demonstrates that extensive loss of dorsal root ganglion cells and myelinated axons in dorsal nerve roots precedes light microscopical changes in the ventral nerve roots and the cerebellum of rats intoxicated with methyl mercury. Received: 16 January 1998 / Revised, accepted: 23 February 1998     

Expression of mRNAs for preprotachykinin and nerve growth factor receptors in the dorsal root ganglion following peripheral inflammation

Nerve growth factor (NGF) plays a dynamic role in the control of substance P (SP) levels and synthesis in the dorsal root ganglion (DRG). In the present study, in situ hybridization was used to examine the change of preprotachykinin (PTT), trkA and p75 mRNAs levels in the DRG after the injection of complete Freund's adjuvant into the hindpaws of rats. Peripheral tissue inflammation increased PTT and p75 mRNAs levels in the DRG, while trkA mRNA levels showed no change. These findings suggest that p75, in addition to trkA, also may be important in mediating the action of NGF on the synthesis of SP in the DRG following peripheral inflammation.     

Evidence for the direct role of acetylcholinesterase in neurite outgrowth in primary dorsal root ganglion neurons

Dorsal root ganglion (DRG) neurons show a transient peak expression of acetylcholinesterase (AChE) during periods of axonal outgrowth prior to synaptogenesis, suggesting that AChE has a non-enzymatic role during development. We have previously shown that perturbation of cell surface AChE in cultured embryonic rat DRG neurons results in decreased neurite outgrowth and neurite detachment. In this report, we demonstrate a direct correlation between endogenous AChE content and neurite outgrowth in primary DRG neurons. Adenoviral vectors were constructed using full-length rat AChE(T) cDNA in either the sense or antisense orientations to overexpress or knock down AChE expression, respectively. Treatment with the sense-expressing vector produced a 2.5-fold increase in AChE expression and a 2-fold increase in neurite length compared with either untreated or null virus-treated control cells. Conversely, treatment with the antisense-expressing vector reduced AChE expression by 40% and resulted in a reduction in neurite length of similar magnitude. We also observed that overexpression of AChE resulted in greater branching at the distal tips of each primary neurite as well as an increase in cell body size. These findings further indicate that AChE expressed on the axonal surface of developing DRG neurons may modulate their adhesive properties and thereby support axonal development.