Exogenous NGF Research Articles

"Turning up the heat": Role of neurotrophic batokines in the postnatal maturation and remodelling of brown adipose tissue in deer mice.

Activation of brown adipose tissue (BAT) thermogenesis impacts energy balance and must be tightly regulated. Several neurotrophic factors, expressed in BAT of adult laboratory rodents, have been implicated in remodelling the sympathetic neural network to enhance thermogenesis (e.g., NGF, NRG4 and S100b). Here, we compare for the first time the relative roles of three neurotropic 'batokines' in establishing/remodelling innervation during postnatal development and adult cold stress. We used lab-reared P. maniculatus, which rely heavily on BAT-based thermogenesis for survival in the wild, beginning between postnatal days (P) 8 and 10. BAT sympathetic innervation was enhanced from P6-10, and exogenous NGF, NRG4, and S100b stimulated neurite outgrowth from P6 sympathetic neurons. Endogenous BAT protein stores and/or gene expression of NRG4, S100b, and calsyntenin-3ß (which regulates S100b secretion), remained high and constant during development. However, endogenous NGF was low and ngf mRNA was undetectable. Conditioned media (CM) from cultured P10 BAT slices stimulated neurite outgrowth from sympathetic neurons in vitro that was inhibited by antibodies against all three growth factors. P10 CM had significant amounts of secreted NRG4 and S100b protein, but not NGF. By contrast, BAT slices from cold-acclimated adults released significant amounts of all three factors relative to thermoneutral controls. These data suggest that while neurotrophic batokines regulate sympathetic innervation in vivo, their relative contributions differ depending on life stage. They also provide novel insight into the regulation of BAT remodelling and BAT's secretory role, which are both critical to our understanding of mammalian energy homeostasis.

Single Cell Analysis of Reversibility of the Cell Death Program in Ethanol-Treated Neuronal PC12 Cells.

Neurodegenerative diseases are generally characterized clinically by the selective loss of a distinct subset of neurons and a slow progressive course. Mounting evidence in vivo indicates that large numbers of neurons pass through a long period of injury and dysfunction before the actual death of the cells. Whether these dying neurons can be rescued and return to a normal, functional state is uncertain. In the present study, we explored the reversibility of the neuronal cell death pathway at various stages by monitoring the dynamics of single cells with high-resolution live-cell spinning disk confocal microscopy in an in vitro neuronal cell death model. We exposed differentiated neuronal PC12 cells to ethanol as our cell death model. Results showed that exposure to 5% ethanol for 24 h induced cell death in >70% of the cells. Ethanol treatment for 3 h already induced cellular changes and damage such as reactive oxygen species generation, elevation of intracellular Ca2+ level, phosphatidylserine exposure, nuclear shrinkage, DNA damage, mitochondrial fragmentation and membrane potential loss, and retraction of neurites. These phenomena are often associated with programmed cell death. Importantly, after removing ethanol and further culturing these damaged cells in fresh culture medium, cells recovered from all these cell injuries and generated new neurites. Moreover, results indicated that this recovery was not dependent on exogenous NGF and other growth factors in the cell culture medium. Overall, our results suggest that targeting dying neurons can be an effective therapeutic strategy in neurodegenerative diseases.

Rita Levi-Montalcini, NGF Metabolism in Health and in the Alzheimer's Pathology.

This chapter relates biographic personal and scientific interactions with Rita Levi-Montalcini. It highlights research from our laboratory inspired by Rita's fundamental discovery. This workfromstudies on potentially neuro-reparative gangliosides, their interactions with NGF, the role of exogenous NGF in the recovery of degenerating cholinergic neurons of the basal forebrain to the evidence that endogenous NGF maintains the "day-to-day" cortical synaptic phenotype and the discovery of a novel CNS "NGF metabolic pathway." This brain pathway's conceptual platform allowed the investigation of its status during the Alzheimer's disease (AD) pathology. This revealed a major compromise of the conversion of the NGF precursor molecule (proNGF) into the most biologically active molecule, mature NGF (mNGF). Furthermore, in this pathology, we found enhanced protein levels and enzymatic activity of the proteases responsible for the proteolytic degradation of mNGF. A biochemical prospect explaining the tropic factor vulnerability of the NGF-dependent basal forebrain cholinergic neurons and of their synaptic terminals. The NGF deregulation of this metabolic pathway is evident at preclinical stages and reflected in body fluid particularly in the cerebrospinal fluid (CSF). The findings of a deregulation of the NGF metabolic pathway and its reflection in plasma and CSF are opening doors for the development of novel biomarkers for preclinical detection of AD pathology both in Alzheimer's and in Down syndrome (DS) with "silent" AD pathology.

Nerve growth factor receptor role on rabbit sperm storage

The influence of NGF in male reproduction in some animal species and humans has already been assessed. Many of these effects are mediated by the distribution and abundance of tropomyosin receptor kinase A (TrKA) and p75 neurotrophin (p75NTR) receptors on sperm cells. The aim of this research was to investigate the role of NGF and its receptors, TrKA and p75NTR, in rabbit sperm outcomes during in vitro storage. Major semen traits (kinetic parameters, apoptotic, necrotic and live sperm) were recorded in rabbit semen samples from 0 to 12 h of storage (every 4 h). Three experimental hypotheses were formulated: i) sperm storage changes NGF receptor abundance in rabbit sperm; ii) TrKA and p75NTR differently modulate NGF signalling (assessed by the neutralisation of receptors); iii) NGF-receptor interactions show different responses during storage (evaluated by the addition of exogenous NGF). The results demonstrate that: (i) the receptor number changed in a time-dependent manner with a significant increase in p75NTR after 8–12 h of storage; ii) the neutralisation of NGF receptors largely affected VCL, apoptotic, necrotic and live cells during sperm storage, i.e. blockade of TrKA significantly increased speed, capacitation, necrosis and apoptosis, whereas blockade of p75NTR improved motility and live cells; iii) the addition of exogenous human NGF (100 ng/mL) at different time points of storage (0, 4, 8 h) differently influenced sperm traits i.e. NGF addition at time 0 positively affected all the pro-vital traits (kinetic, live cells) whereas, after 4–8 h, the effect of NGF was null or negative. In conclusion, NGF affects kinetic and other physiological traits (capacitation, apoptosis and necrosis) of rabbit sperm in a time-dependent manner. Most of these modifications are modulated by the receptors involved (TrKA or p75NTR), which changed considerably during sperm storage (increase of p75NTR).

Intrathecal lentivirus-mediated RNA interference targeting nerve growth factor attenuates myocardial ischaemia–reperfusion injury in rat

BackgroundNerve growth factor (NGF) has been implicated in hyperalgesia by sensitising nociceptors. A role for NGF in modulating myocardial injury through ischaemic nociceptive signalling is plausible. We examined whether inhibition of spinal NGF attenuates myocardial ischaemia–reperfusion injury and explored the underlying mechanisms. MethodsIn adult rats, lentivirus-mediated short-hairpin RNA targeted at reducing NGF gene expression (NGF-shRNA) or a transient receptor potential vanilloid 1 (TRPV1) antagonist (capsazepine) was injected intrathecally before myocardial ischaemia–reperfusion. Infarct size (expressed as the ratio of area at risk) and risk of arrhythmias were quantified. Whole-cell clamp patch electrophysiology was used to record capsaicin currents in primary dorsal root ganglion neurones. The co-expression of substance P (SP) and calcitonin gene-related peptide (CGRP), plus activation of TRPV1, protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) were also quantified. ResultsNGF levels increased by 2.95 (0.34)-fold in dorsal root ganglion and 2.12 (0.27)-fold in spinal cord after myocardial ischaemia–reperfusion injury. Intrathecal injection of NGF-shRNA reduced infarct area at risk from 0.58 (0.02) to 0.37 (0.02) (P<0.01) and reduced arrhythmia score from 3.67 (0.33) to 1.67 (0.33) (P<0.01). Intrathecal capsazepine was similarly cardioprotective. NGF-shRNA suppressed expression of SP/CGRP and activation of Akt/ERK and TRPV1 in spinal cord. NGF increased capsaicin current amplitude from 144 (42) to 840 (132) pA (P<0.05), which was blocked by the TRPV1 antagonist 5′-iodoresiniferatoxin. Exogenous NGF enhanced capsaicin-induced Akt/ERK and TRPV1 activation in PC12 neuroendocrine tumour cells in culture. ConclusionsSpinal NGF contributes to myocardial ischaemia–reperfusion injury by mediating nociceptive signal transmission.

Behavioral Compensations and Neuronal Remodeling in a Rodent Model of Chronic Intervertebral Disc Degeneration

Low back pain is associated with degeneration of the intervertebral disc, but specific mechanisms of pain generation in this pathology remain unknown. Sensory afferent nerve fiber growth into the intervertebral disc after injury-induced inflammation may contribute to discogenic pain. We describe a clinically relevant behavioral phenotype in a rodent model of chronic intervertebral disc degeneration which provides a means to map sensory neuron changes to a single affected lumbar intervertebral disc. Unilateral disc puncture of one lumbar intervertebral disc revealed a bilateral behavioral phenotype characterized by gait changes and decreased activity. Moreover, neurons extracted from the dorsal root ganglia in animals with intervertebral disc injury demonstrated altered TRPV1 activation in vitro independent of exogenous NGF administration. Finally, neuronal nuclear hypertrophy and elevated expression of p75NTR provide evidence of active adaptation of innervating sensory neurons in chronic intervertebral disc degeneration. Therefore, this model and findings provide the template for future studies to establish specific mechanisms of nociceptive pain in chronic intervertebral disc degeneration.

Neurotrophin-dependent plasticity of neurotransmitter segregation in the rat superior cervical ganglion in vivo.

Neurons are able to segregate transmitters to different axon endings. Segregation is a plastic neuronal feature; it can be modulated by synaptic environment. We have demonstrated that neurotrophin and other cellular factors regulate segregation in sympathetic neurons in culture. Herein we tested the hypothesis that sympathetic neurons in vivo are also capable to exhibit neurotrophin-dependent plasticity of segregation. To explore the effect of neurotrophin on segregation, we reduced ganglionic NGF content by the transection of postganglionic nerves (axotomy) of the superior cervical ganglia. By immunohistochemistry, Western blot, and PCR analyses, we explored the effect of axotomy on the NGF and BDNF content of ganglionic neurons, and on the segregation extent of vesicular acetylcholine transporter (VAChT) and methionine enkephalin (mENK) in pre-ganglionic varicosities. We analyzed NGF-dependence of the changes found by applying exogenous NGF. Axotomy reduced ganglionic NGF and BDNF content, increased NGF transcripts, and increased VAChT-mENK segregation. Axotomy also increased the number of VAChT immunopositive varicosities, and caused the appearance of a population of VAChT-, mENK- or SV2-containing varicosities lacking Synaptophysin (Syn). Administration of NGF prevented changes in NGF content, kept NGF transcripts increased, and counteracted changes in segregation and in the number of cholinergic varicosities. The exogenous NGF did not preclude change in BDNF content or in the occurrence of the VAChT- or mENK-containing varicosities lacking Syn. Data demonstrate that segregation of transmitters in vivo is plastic and it is modulated by environmental signals like NGF. We propose a possible functional correlate of segregation plasticity in the sympathetic ganglia. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 832-846, 2016.

Expression of β-nerve growth factor and homeobox A10 in experimental cryptorchidism treated with exogenous nerve growth factor.

With the exception of standard inguinal orchidopexy, treatment of cryptorchidism with human chorionic gonadotropin has been performed for several years; however, its side effects have limited its application. The β‑nerve growth factor (NGF) and homeobox A10 (HoxA10) genes are closely associated with the development of the testes. To the best of our knowledge, whether exogenous NGF alters the endogenous levels of NGF and HoxA10 in cryptorchidism in rats remains to be elucidated. The aim of the present study was to evaluate the gene and protein expression of NGF and HoxA10 in experimental cryptorchidism following treatment with exogenous NGF. A unilateral mechanical cryptorchidism model in Sprague-Dawley rats was established and different concentrations of exogenous NGF were administered to observe the effects of NGF on cryptorchidism. Changes in the gene and protein expression levels of NGF and HoxA10 in the cryptorchid tissues of each group were identified using one step reverse transcription-quantitative polymerase chain reaction, in situ hybridization with digoxigenin‑labeled‑β‑NGF RNA probes, immunofluorescence and immunohistochemistry, respectively. The expression levels of NGF and HoxA10 were markedly higher in the group treated with a high dose of exogenous NGF compared with the group treated with a low dose of exogenous NGF and the group treated with human chorionic gonadotropin. These results confirmed the potential therapeutic effect of exogenous NGF in human cryptorchidism.

The effect of P75 on Trk receptors in neuroblastomas

Neuroblastomas (NBs) with favorable outcome usually express TrkA, whereas unfavorable NBs frequently express TrkB and its cognate ligand BDNF. P75 (p75 LNTR, NGFR, TNFRSF16) binds NGF-related neurotrophins with low affinity and usually is coexpressed with Trk receptors in NBs. Here, we investigated the importance of p75 coexpression with Trk receptors in NBs. We transfected p75 into two Trk-null NB cell lines, SH-SY5Y and NLF that were also engineered to stably express TrkA or TrkB. Cell numbers were compared between single (Trk alone) and double (Trk + p75) transfectants, and proliferation was assessed by flow cytometry. P75 coexpression had little effect on cell growth in Trk NB cells in the absence of ligand, but it increased sensitivity and greatly enhanced the effect of cognate ligand. Exogenous NGF induced greater phosphorylation of TrkA and AKT. This was associated with increased cell number in TrkA/p75 cells compared to TrkA cells ( p < 0.01), which was due to increased proliferation in TrkA/p75 cells ( p < 0.05), followed by differentiation. Exogenous BDNF also increased cell number in TrkB/p75 compared to TrkB cells ( p < 0.01), due to an increase in proliferation, but without differentiation. Coexpression of p75 also increased specificity of Trk-expressing cells to ligand. NT3-induced phosphorylation of TrkA and AKT was reduced in TrkA/p75 cells. NT3-induced phosphorylation of TrkB (as well as AKT and MAPK) was also reduced with p75 coexpression. Our results suggest that p75 plays an important role in enhancing both the sensitivity of Trk receptors to low levels of ligand, as well as increasing the specificity of Trks to their cognate ligands. It also enhances ligand-induced differentiation in TrkA/p75 but not TrkB/p75 cells.

Nerve growth factor increases the size of intracortical cholinergic transplants.

The effects of continuous intracortical mouse Nerve Growth Factor on fetal rat basal forebrain transplants in denervated adult rat neocortex were investigated. Enzyme-linked immunoassay (ELISA) was used to measure the time course of endogenous NGF protein production in neocortex, hippocampus, and basal forebrain in a cohort of animals receiving unilateral ibotenic acid (IBO) lesions of the nucleus basalis magnocellularis (nBM). A second cohort of IBO-nBM lesioned animals received transplants of fetal basal forebrain followed by two to four weeks of continuous NGF or cytochrome-C infusion into the ipsilateral frontoparietal neocortex. To study the effects of abnormally high NGF doses on transplanted and host tissue, the cumulative dose of intracortical NGF was on the order of micrograms, compared with maximum picogram levels of neocortical NGF produced following IBO-nBM lesions. A four-fold increase in transplant size, and greater cell and fiber densities were observed in NGF-treated compared with NGF-untreated transplants. No adverse histological effects of long-term, high-dose NGF treatment were observed on transplanted basal forebrain or host neocortical tissue. These data indicate that cholinergic-rich mammalian brain tissue and intrinsic host tissue can be stimulated by high doses exogenous NGF without obvious deleterious effects.

Siatic nerve regeneration in rats stimulated by fibrin glue containing nerve growth factor: An experimental study

To study the effects of fibrin sealant containing nerve growth factor on the peripheral nerve regeneration. A four-group experimental design with repeated measures on one factor was used. Fibrin glue (FG) containing NGF was injected into the site of end-and-end sutured peripheral nerve (sciatic nerve) (group I: NGF+FG), meanwhile three control groups were set-up: group II (NGF), group III (FG), and group IV (normal saline). Observation to the function and morphology of the sciatic nerve was carried out at the end of 4, 8, 12 weeks postoperation. Data were analyzed using ANOVA, with the appropriate post hoc between-groups comparison test. Electrophysiological testing. The NAP and NCV of group I (NGF+FG) were greater than those of group II (NGF), group III (FG), or group IV (normal saline) (p<0.05). Sciatic functional index (SFI). It began to ameliorate at 4 weeks postoperation and SFI increased as time went on. And the SFI in group I (NGF+FG) was better than those in group II (NGF), group III (FG), or group IV (normal saline) (p<0.05). Morphology. In the MGF-stained sections, dissociated myelin debris was less and regenerated nerve fibres were in larger quantities in group I (NGF+FG) than in other groups. In the HE-stained sections, regenerated nerve fibres distal to anastomosis significantly increased, and axon and myelin had a clearer outline in group I (NGF+FG) than in other groups. Electron microscopy indicated that the regenerated nerve fibres were more mature and the development of the axons was greater in group I than in other control groups. FG can be used as carrier of exogenous NGF, and they can provide synergistic effects for the peripheral nerve regeneration after the integration of the two.

Implanted embryonic sensory neurons project axons toward adult auditory brainstem neurons in roller drum and Stoppini co-cultures

Previously we have shown in vivo the survival, migration and integration of embryonic dorsal root ganglion (DRG) neurons that were grafted into the inner ear and peripheral auditory nervous system. In order to evaluate relevant factors determining integration of sensory neurons further into the central auditory nervous system, complementary in vitro techniques are necessary. The advantages of in vitro systems are that a large number of factors including various grafts and different conditions can be efficiently examined for. Hence, we co-cultured 300 μm thick postnatal rat brainstem slices containing the cochlear nucleus including the central part of the 8th cranial nerve with mouse embryonic DRG neurons. The organotypic co-cultures were either grown on coverslips using the roller drum method described by Gähwiler or on membranes according to the interface method described by Stoppini. Neurons in the cochlear nucleus were labeled with DiI. The results demonstrate that (1) brainstem slices survive for up to 5 weeks in culture, and that (2) co-cultures of embryonic sensory neurons and brainstem show a high degree of neuronal survival, and that (3) survival and axonal outgrowth from the implanted embryonic neurons are dependant on the presence of the brainstem slice rather than on exogenous NGF and that (4) implanted embryonic neurons send axons toward neurons in the cochlear nucleus.

Exogenous overexpression of nerve growth factor in the urinary bladder produces bladder overactivity and altered micturition circuitry in the lumbosacral spinal cord

BackgroundExogenous NGF or saline was delivered to the detrusor smooth muscle of female rats for a two-week period using osmotic mini-pumps. We then determined: (1) bladder function using conscious cystometry; (2) organization of micturition reflexes using Fos protein expression in lumbosacral (L5-S1) spinal cord neurons; (3) calcitonin gene-related peptide (CGRP)-immunoreactivity (IR) in lumbosacral spinal cord segments.MethodsAn osmotic pump infused 0.9% NaCl (n = 6) or NGF (n = 6)(2.5 μg/μl solution; 0.5 μl/hr) for two weeks into the bladder wall. NGF bladder content was determined by enzyme-linked immunoassays. Bladder function was assessed with conscious cystometry. Immunohistochemical and imaging techniques were used to determine the distribution of Fos-IR cells and CGRP expression in the L5-S1 spinal cord in saline and NGF-treated rats two hours after intravesical saline distention. Fos expression and CGRP-IR in NGF-treated rats with bladder distention was compared to that observed in cyclophosphamide (CYP; 75 mg/kg; i.p.) treated rats with bladder distention.ResultsTwo-week infusion of NGF into the bladder wall increased bladder weight, reduced bladder capacity (60%), reduced the intercontraction interval (60%) and increased the amplitude of non-voiding contractions. NGF treatment and intravesical saline distention (2 hr) increased expression of Fos protein in L6-S1 spinal cord and altered the distribution pattern of Fos-IR cells. CGRP-IR in the lumbosacral spinal cord was also increased after NGF treatment.ConclusionThese data suggest that NGF infusion into the bladder wall induces bladder overactivity, can reveal a "nociceptive" Fos expression pattern in the spinal cord in response to a non-noxious bladder stimulus and increases CGRP-IR in the lumbosacral spinal cord.

Effects of Nerve Growth Factor for Retinal Cell Survival in Experimental Retinal Detachment

Purpose: To investigate the neuron protective effect of recombined nerve growth factor (rNGF) on retinal cell damage induced by experimental retinal detachment. Methods: Experimental retinal detachment models were created in Sprague-Dawley rats by subretinal injection of sodium hyaluronate. Intravitreal injection of rNGF (5 μ g/eye) or phosphate-buffered saline (PBS) was separately applied every 4 days after retinal detachment. The rat eyes were then observed and sacrificed at various time points. Morphologic changes were observed by light microscopy, electron microscopy, and cell counts. Apoptosis of retinal cells was detected by TUNEL assay. Results: After retinal detachment, most eyes from NGF-treated groups showed better organized structure of retinal cells than those from the PBS-treated control groups. Cell counts indicated that the nuclei numbers in the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL) of NGF-treated groups were significantly more than those from PBS-treated control group (p < 0.05) after retinal reattachment. TUNEL-positive cells were identified in ONL, INL, and GCL. They peaked at the fourth day after retinal detachment in both the NGF-treated groups and the control groups. But the cell counts of apoptosis revealed that the NGF-treated retina had less TUNEL-positive cells than the control groups (p < 0.05). Conclusions: The results showed that intravitreal injection of exogenous NGF can protect retinal cells from degeneration and apoptosis in experimental retinal detachment. It may exert its neuroprotection effect by preventing the apoptosis of retinal cells after retinal detachment.

The Effects of Decompression and Exogenous NGF on Compressed Cerebral Cortex

Using a rat epidural bead implantation model, we found that compression alone could reduce the overall and individual layer thicknesses of cerebral cortex with no apparent cell death. The dendritic lengths and spine densities of layer II/III and V pyramidal neurons started to decrease within 3 days of compression. Decompression for 14 days resulted in near complete to partial recovery of the cortical thickness and of the dendritic lengths of layer II/III and V pyramidal neurons, depending on the duration of the preceding compression. The recoverability was better following short (3-day) than long (1- or 3-month) periods of compression. The loss of dendritic spines nevertheless persisted. An intraventricular infusion of NGF was performed after decompressing the lesions following 3 days of cortical compression, and this increased the recovery of the spines but not the dendritic length of the cortical pyramidal neurons, nor did it alter the recovery of the cortical thickness. NGF also promoted the increase of the dendritic spines, but not the dendritic length of the cortical pyramidal neurons of normal animals. In short, the data show that a few days of compression alone can cause permanent cortical damage. Exogenous NGF, if applied topically, may restore the dendritic spine density of cortical neurons subjected to compression.

The Effects of Decompression and Exogenous NGF on Compressed Cerebral Cortex

The Effects of Decompression and Exogenous NGF on Compressed Cerebral Cortex

A novel neuroimmune mechanism in cannabinoid-mediated attenuation of nerve growth factor-induced hyperalgesia.

Nerve growth factor (NGF) is central to processes involved in an inflammatory hyperalgesia. Administration of exogenous NGF induces a hyperalgesia that is dependent on local neutrophil influx. The effects of administration of the cannabinoid anandamide and the cannabimimetic palmitoylethanolamide on an NGF-induced hyperalgesia and neutrophil accumulation were examined in this study. Baseline hind limb withdrawal latencies to a noxious heat stimulus were recorded before intraplantar administration of NGF (1 microg in 0.05 ml) to the hind paw of 75 male Wistar rats. Anandamide or palmitoylethanolamide (a substance that has cannabinoid-like actions but little affinity for cannabinoid receptors) at doses of 10 and 25 mg/kg were given (intraperitoneally) immediately after NGF. CB1 (SR141716A) and CB2 (SR144528) receptor antagonists were coadministered with the higher dose of cannabinoids. Withdrawal latencies were expressed as difference from baseline. Seventy rats received intraplantar NGF and intraperitoneal treatments. Neutrophil accumulation in the injected paw was assessed using a myeloperoxidase assay. Administration of NGF reduced latencies consistent with hyperalgesia. Anandamide and palmitoylethanolamide significantly reduced this hyperalgesia. The action of anandamide was CB1 receptor-mediated. SR144528 abrogated the action of palmitoylethanolamide. NGF also provoked neutrophil accumulation in the injected paw, denoted by an increase in myeloperoxidase. Palmitoylethanolamide significantly reduced neutrophil accumulation by an SR144528-sensitive action, whereas anandamide was without effect. NGF induced a thermal hyperalgesia that was attenuated by anandamide and palmitoylethanolamide. Only palmitoylethanolamide reduced neutrophil influx. Thus, cannabinoids show a neuronal CB1 receptor-mediated antihyperalgesic action and a separate inhibition of a proinflammatory neuroimmune process. Such a mechanism suggests a therapeutic site of analgesic action separable from central side effects.

Exogenous NT-3 and NGF differentially modulate PACAP expression in adult sensory neurons, suggesting distinct roles in injury and inflammation.

Expression of pituitary adenylate cyclase-activating polypeptide in sensory neurons varies with injury or inflammation. The neurotrophins NGF and NT-3 are profound regulators of neuronal peptidergic phenotype in intact and injured sensory neurons. This study examined their potential for modulation of PACAP expression in adult rat with intact and injured L4-L6 spinal nerves with or without immediate or delayed intrathecal infusion of NT-3 or NGF. Results indicate that in L5 DRG, few trkC neurons express high levels of PACAP mRNA in the intact state, but many do following injury. The elevated expression in injured neurons is mitigated by NT-3 infusion, suggesting a role for NT-3 in returning the 'injured phenotype' back towards an 'intact phenotype'. NGF dramatically up-regulated PACAP expression in trkA-positive neurons in both intact and injured DRGs, implicating NGF as a positive regulator of PACAP expression in nociceptive neurons. Surprisingly, NT-3 modulates PACAP expression in an antagonistic fashion to NGF in intact neurons, an effect most evident in the trkA neurons not expressing trkC. Both NT-3 and NGF infusion results in decreased detection of PACAP protein in the region of the gracile nuclei, where central axons of the peripherally axotomized large sensory fibers terminate. NGF infusion also greatly increased the amount of PACAP protein detected in the portion of the dorsal horn innervated by small-medium size DRG neurons, while both neurotrophins appear able to prevent the decrease in PACAP expression observed in these afferents with injury. These results provide the first insights into the potential molecules implicated in the complex regulation of PACAP expression in sensory neurons.

Delayed administration of NGF reverses nerve injury induced central alterations of primary afferents.

We have examined whether delayed exogenous NGF administered to an axotomised peripheral nerve reverses the increased transganglionic choleragenoid (CTB) labelling in lamina II. Two, four, eight or 18 weeks after bilateral sciatic nerve section, NGF was applied unilaterally for an additional 2-week period to the transected nerve stump. The transganglionic choleragenoid labelling and substance P (SP) expression were determined and compared to the contralateral axotomised side in the spinal cord dorsal horn. Delayed NGF administration reversed the transganglionic choleragenoid labelling in lamina II when administered 2 or 18 weeks after the sciatic nerve lesion, but not at 4 or 8 weeks. There was also a clear recovery of SP on the axotomised, NGF-treated side 2 or 18 weeks after the sciatic nerve lesion, but not at the intermediate survival times. At the longer survival time, however, there was a recovery of SP regardless of NGF treatment. These results suggest that there is a critical window as to when NGF administration can be effective in reversing axotomy-induced changes in the spinal cord.

Effect of NGF and neurotrophin-3 treatment on experimental diabetic autonomic neuropathy.

Peripheral neuropathy is a significant complication of diabetes resulting in increased patient morbidity and mortality. Deficiencies of neurotrophic substances (e.g. NGE NT-3, and IGF-I) have been proposed as pathogenetic mechanisms in the development of distal symmetrical sensory diabetic polyneuropathy, and salutary effects of exogenous NGF administration have been reported in animal models. In comparison, relatively little is known concerning the effect of NGF on experimental diabetic sympathetic autonomic neuropathy. We have developed an experimental animal model of diabetic autonomic neuropathy characterized by the regular occurrence of pathologically distinctive dystrophic axons in prevertebral sympathetic ganglia and ileal mesenteric nerves of rats with chronic streptozotocin (STZ)-induced diabetes. Treatment of STZ-diabetic rats for 2-3 months with pharmacologic doses of NGF or NT-3, neurotrophic substances with known effects on the adult sympathetic nervous system, did not normalize established neuroaxonal dystrophy (NAD) in diabetic rats in the prevertebral superior mesenteric ganglia (SMG) and ileal mesenteric nerves as had pancreatic islet transplantation and IGF-I in earlier experiments. NGF treatment of control animals actually increased the frequency of NAD in the SMG. New data suggests that, in adult sympathetic ganglia. NGF may contribute to the pathogenesis of NAD rather than its amelioration, perhaps as the result of inducing intraganglionic axonal sprouts in which dystrophic changes are superimposed. NT-3 administration did not alter the frequency of NAD in diabetic animals, although it resulted in a significant decrease in NAD in control SMG. Although deficiencies of neurotrophic substances may represent the underlying pathogenesis of a variety of experimental neuropathies, delivery of excessive levels of selected substances may produce untoward effects.