Infusion Of Nerve Growth Factor Research Articles

Effects of Aging and Nerve Growth Factor on Neuropeptide Expression and Cholinergic Innervation of the Rat Basolateral Amygdala.

The basolateral amygdala (BLA) contains interneurons that express neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP), both of which are involved in the regulation of functions and behaviors that undergo deterioration with aging. There is considerable evidence that, in some brain areas, the expression of NPY and VIP might be modulated by acetylcholine. Importantly, the BLA is one of the brain regions that has one of the densest cholinergic innervations, which arise mainly from the basal forebrain cholinergic neurons. These cholinergic neurons depend on nerve growth factor (NGF) for their survival, connectivity, and function. Thus, in this study, we sought to determine if aging alters the densities of NPY- and VIP-positive neurons and cholinergic varicosities in the BLA and, in the affirmative, if those changes might rely on insufficient trophic support provided by NGF. The number of NPY-positive neurons was significantly reduced in aged rats, whereas the number of VIP-immunoreactive neurons was unaltered. The decreased NPY expression was fully reversed by the infusion of NGF in the lateral ventricle. The density of cholinergic varicosities was similar in adult and old rats. On the other hand, the density of cholinergic varicosities is significantly higher in old rats treated with NGF than in adult and old rats. Our results indicate a dissimilar resistance of different populations of BLA interneurons to aging. Furthermore, the present data also show that the BLA cholinergic innervation is particularly resistant to aging effects. Finally, our results also show that the reduced NPY expression in the BLA of aged rats can be related to changes in the NGF neurotrophic support.

Infusions of Nerve Growth Factor Into the Developing Frontal Cortex Leads to Deficits in Behavioral Flexibility and Increased Perseverance.

In the pursuit of further establishing a neurodevelopmental animal model to investigate the mechanisms underlying impaired executive function, a core and severely debilitating symptom of schizophrenia, we sought to characterize the deficits in behavioral flexibility in adult rats following neonatal infusions of nerve growth factor (NGF) into the medial part of the developing frontal cortex. Our previous studies using this neonatal frontal cortical lesion model have shown that it leads to adult-onset positive and negative symptom-like features, and several neuropathological abnormalities of schizophrenia. In the present study, we used operant conditioning-based paradigms to investigate set-shifting ability and reversal learning performance in adult rats that received infusions of NGF into the developing frontal cortex on post-natal day 1. NGF-infusion caused apoptosis of cells in the subplate layer. Adult rats that received neonatal infusions of NGF showed decreased grey matter thickness, and decreased levels of parvalbumin in prelimbic and infralimbic areas of the medial prefrontal cortex (mPFC). NGF-treated rats had difficulty completing the set-shifting and reversal learning tasks due to increased perseverance (ie, a failure to disengage from the previously-learned strategy once the rule contingencies were changed) compared to the control group. Collectively, these results identify the crucial role of the frontal cortical subplate layer in the structural and functional development of the mPFC relevant to schizophrenia. Furthermore, the present findings substantially advance the face and construct validity of this putative preclinical model of schizophrenia based on developmental disruption of the frontal cortical subplate.

Subcutaneous nerve activity and spontaneous ventricular arrhythmias in ambulatory dogs.

Stellate ganglion nerve activity (SGNA) is important in ventricular arrhythmogenesis. However, because thoracotomy is needed to access the stellate ganglion, it is difficult to use SGNA for risk stratification. The purpose of this study was to test the hypothesis that subcutaneous nerve activity (SCNA) in canines can be used to estimate SGNA and predict ventricular arrhythmia. We implanted radiotransmitters to continuously monitor left stellate ganglion and subcutaneous electrical activities in 7 ambulatory dogs with myocardial infarction, complete heart block, and nerve growth factor infusion to the left stellate ganglion. Spontaneous ventricular tachycardia (VT) or ventricular fibrillation (VF) was documented in each dog. SCNA preceded a combined 61 episodes of VT and VF, 61 frequent bigeminy or couplets, and 61 premature ventricular contractions within 15 seconds in 70%, 59%, and 61% of arrhythmias, respectively. Similar incidence of 75%, 69%, and 62% was noted for SGNA. Progressive increase in SCNA [48.9 (95% confidence interval [CI] 39.3-58.5) vs 61.8 (95% CI 45.9-77.6) vs 75.1 (95% CI 57.5-92.7) mV-s] and SGNA [48.6 (95% CI 40.9-56.3) vs 58.5 (95% CI 47.5-69.4) vs 69.0 (95% CI 53.8-84.2) mV-s] integrated over 20-second intervals was demonstrated 60 seconds, 40 seconds, and 20 seconds before VT/VF (P <.05), respectively. The Pearson correlation coefficient for integrated SCNA and SGNA was 0.73 ± 0.18 (P <.0001 for all dogs, n = 5). Both SCNA and SGNA exhibited circadian variation. SCNA can be used as an estimate of SGNA to predict susceptibility to VT and VF in a canine model of ventricular arrhythmia and sudden cardiac death.

Intrathecal lidocaine neurotoxicity: Combination with bupivacaine and ropivacaine and effect of nerve growth factor

AimsThe study aims to investigate the neurotoxicity induced by combined use of intrathecal lidocaine with bupivacaine and ropivacaine, and to examine the effect of nerve growth factor (NGF) on lidocaine-induced neurotoxicity. Main methodsAll rats received intrathecal infusion of anesthetics and NGF. To study the neurotoxicity of combined use of lidocaine with bupivacaine and ropivacaine, rats received saline, 5% lidocaine, 1.065% bupivacaine, 1.5% ropivacaine, 5% lidocaine+bupivacaine, or 5% lidocaine+1.5% ropivacaine. To study the neurotoxicity of different proportions of lidocaine and bupivacaine, mixtures were made by mixing 10% lidocaine and 2.5% bupivacaine in ratios of 1:3, 1:2, 1:1, 2:1 and 3:1 by volume. To study the effect of NGF on lidocaine-induced neurotoxicity, rats received saline or 10μg NGF for 1, 2, 5 and 8days. Key findingsThe neurotoxicity of lidocaine was significantly increased when combined with ropivacaine. A mixture of lidocaine and bupivacaine in a ratio of ≤1:1 did not significantly increase lidocaine-induced neurotoxicity. NGF significantly reduced lidocaine-induced neurobehavioral and morphological damage in the spinal cord. This was accompanied by downregulation of caspase 3 expression. SignificanceRopivacaine is not safe when intrathecally administered with lidocaine at the concentrations used in this study. Bupivacaine may be safely used with lidocaine at a ratio of 1:1. NGF can reduce lidocaine-induced neurotoxicity, possibly via inhibition of caspase 3-mediated apoptosis.

Intracerebroventricular administration of nerve growth factor induces gliogenesis in sensory ganglia, dorsal root, and within the dorsal root entry zone.

Previous studies indicated that intracerebroventricular administration of nerve growth factor (NGF) leads to massive Schwann cell hyperplasia surrounding the medulla oblongata and spinal cord. This study was designed to characterize the proliferation of peripheral glial cells, that is, Schwann and satellite cells, in the trigeminal ganglia and dorsal root ganglia (DRG) of adult rats during two weeks of NGF infusion using bromodeoxyuridine (BrdU) to label dividing cells. The trigeminal ganglia as well as the cervical and lumbar DRG were analyzed. Along the entire neuraxis a small number of dividing cells were observed within these regions under physiological condition. NGF infusion has dramatically increased the generation of new cells in the neuronal soma and axonal compartments of sensory ganglia and along the dorsal root and the dorsal root entry zone. Quantification of BrdU positive cells within sensory ganglia revealed a 2.3- to 3-fold increase in glial cells compared to controls with a similar response to NGF for the different peripheral ganglia examined. Immunofluorescent labeling with S100β revealed that Schwann and satellite cells underwent mitosis after NGF administration. These data indicate that intracerebroventricular NGF infusion significantly induces gliogenesis in trigeminal ganglia and the spinal sensory ganglia and along the dorsal root entry zone as well as the dorsal root.

Intermittent vs Continuous Administration of Nerve Growth Factor to Injured Medial Septal Cholinergic Neurons in Rat Basal Forebrain

Many medial septal neurons of the basal forebrain are dependent on nerve growth factor (NGF) from the hippocampus for survival and maintenance of a cholinergic phenotype. When deprived of their source of NGF by axotomy, medial septal neuronal cell bodies atrophy and lose their cholinergic markers. This is similar to what is observed in the basal forebrain during Alzheimer’s disease (AD). In the present study, medial septal neurons were axotomized in female rats by way of a fimbria/fornix lesion. For fourteen days following axotomy, varying NGF doses (1 – 250 μg/ml) were administered to the lateral cerebral ventricle with either mini-osmotic infusion or daily injection. The responsiveness of medial septal neurons was evaluated with choline acetyltransferase immunohistochemistry. Within the mini-osmotic pumps, NGF activity diminished greatly during the first five days of implantation, but increased dramatically in the CSF after five days of infusion. The responsiveness of medial septal neurons to NGF was dose dependent and the ED50 for NGF injection was determined to be 14.08 μg/ml compared to 27.60 μg/ml for NGF infusion. Intermittent injections at varying intervals were evaluated with 30 μg/ml NGF over a fourteen-day time period (2, 3, 6, or 12 injections). No differences occurred in the number of choline acetyltransferase neurons from rats that received weekly injections to those that received daily injections of NGF. NGF administration has been suggested as a therapy for AD. The results of these studies continue to highlight the need for NGF stability within the delivery system and AD patient CSF, the choice of delivery system, frequency of administration, and the NGF dose for maintaining basal forebrain cholinergic neurons during AD.

Neurotrophic factors rescue basal forebrain cholinergic neurons and improve performance on a spatial learning test

This study investigated whether animals sustaining experimental damage to the basal forebrain cholinergic system would benefit from treatment with exogenous neurotrophic factors. Specifically, we set out to determine whether neurotrophic factors would rescue damaged cholinergic neurons and improve behavioral performance on a spatial learning and memory task. Adult rats received bilateral injections of either saline (controls) or 192 IgG-saporin to damage basal forebrain cholinergic neurons (BFCNs). Two weeks later, animals received implants of an Alzet mini-pump connected to cannulae implanted bilaterally in the lateral ventricles. Animals received infusions of nerve growth factor (NGF), neurotrophin 3 (NT3), a combination of NGF and NT3, or a saline control over a 4-week period. Compared to saline-treated controls, animals sustaining saporin-induced damage to BFCNs took significantly more trials to learn a delayed match to position task and also performed more poorly on subsequent tests, with increasing delays between test runs. In contrast, animals infused with neurotrophins after saporin treatment performed significantly better than animals receiving saline infusions; no differences were detected for performance scores among animals infused with NGF, NT3, or a combination of NGF and NT3. Studies of ChAT immunnocytochemical labeling of BFCNs revealed a reduction in the numbers of ChAT-positive neurons in septum, nucleus of diagonal band, and nucleus basalis in animals treated with saporin followed by saline infusions, whereas animals treated with infusions of NGF, NT3 or a combination of NGF and NT3 showed only modest reductions in ChAT-positive neurons. Together, these data support the notion that administration of neurotrophic factors can rescue basal forebrain cholinergic neurons and improve learning and memory performance in rats.

Chronic intracerebroventricular infusion of nerve growth factor improves recognition memory in the rat

Nerve Growth Factor (NGF) plays pivotal roles in neuronal survival in the adult mammalian brain and may modulate forms of structural and functional plasticity, including neurogenesis. We have shown previously that six weeks of housing in an enriched environment (EE) that did not include access to running wheels resulted in improved recognition memory concomitant with increased NGF expression and neurogenesis in the hippocampus. Here we have attempted to probe a causal link between NGF and the observed enrichment-induced changes in hippocampal function by assessing the effects of six weeks continuous intracerebroventricular (i.c.v.) infusion of NGF on recognition memory and cell proliferation. We report that NGF-infused rats show enhanced recognition memory when compared with vehicle-treated controls. Expression of NGF and its receptor, TrkA, was increased in treated rats, as was expression of the synaptic vesicle protein, synapsin. Finally, we observed an increase in cell proliferation in the dentate gyrus of NGF-treated rats. These data indicate that chronic infusion of NGF can stimulate an improvement in learning and memory that is associated with specific cellular changes in the hippocampus, including synaptogenesis and cell proliferation.

NGF rescues hippocampal cholinergic neuronal markers, restores neurogenesis, and improves the spatial working memory in a mouse model of Huntington's Disease.

Recent studies in Huntington's disease (HD) mouse models and patients suggest that hippocampal neurons and their cholinergic afferents are involved in the cognitive deficits seen in the disease. Nerve growth factor (NGF) is an essential regulator of cholinergic neuronal survival and neurotransmission. We asked whether NGF might be involved in HD and if intra-cerebroventricular infusion of NGF can rescue hippocampal cholinergic neuronal markers, restore neurogenesis, and improve the spatial working memory in R6/1 mouse model of HD. We quantified NGF protein level by enzyme-linked immunosorbent assay (ELISA), intracerebroventricularly infused NGF, assessed cholinergic neuronal markers by Western blotting and quantitative RT-PCR, evaluated neurogenesis by immunohistochemistry, and studied spatial working memory using radial maze. By quantifying NGF protein in the hippocampus of the R6/1 mice at different ages, we found progressive decreases in NGF protein levels. We then increased NGF levels in the R6/1 mice through intra-cerebroventricular infusion. We observed elevations of the cholinergic neurochemical markers vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT) in the hippocampus and in the septal region, which contain the cell bodies of basal forebrain cholinergic neurons (BFCNs), but not in the striatum that harbors cholinergic interneurons. Finally, we found that NGF infusion also restored hippocampal neurogenesis and improved spatial working memory. Our results suggest that intracerebral injections of NGF might be a valuable therapy against cognitive symptoms in HD and should be further studied in HD animal models and patients.

Contribution of nerve growth factor to upregulation of P2X3 expression in DRG neurons of rats with femoral artery occlusion

Femoral artery occlusion augments the sympathetic nerve and pressor responses to muscle contraction and muscle metabolites injected into the arterial blood supply of the hindlimb muscles in rats. The underlying mechanism by which these reflex responses are enhanced after muscle vascular insufficiency is unclear. Purinergic P2X(3) receptor has been reported to contribute to the metabolic component of the exercise pressor reflex. Thus the purpose of this study was to examine if chronic femoral occlusion would alter the expression of P2X(3) in dorsal root ganglion (DRG) neurons of rats. Also, P2X(3)-mediated sympathetic responsiveness was examined after femoral occlusion. In addition, the role played by nerve growth factor (NGF) in regulating the expression and response of P2X(3) was examined. Western blot analysis showed that 24 h of femoral ligation increased the levels of P2X(3) (optical density: 0.93 ± 0.07 in control and 1.37 ± 0.10 after occlusion; P < 0.05 vs. control). The fluorescence immunohistochemistry further demonstrated that the occlusion elevated P2X(3) expression in DRG neurons (percentage of P2X(3)-positive cells: 33 ± 3% in control and 51 ± 3% in occlusion; P < 0.05 vs. control). Furthermore, the results showed that responses of renal sympathetic nerve activity and blood pressure to stimulation of P2X were greater in occluded rats than responses in control rats by injection of α,β-methylene ATP into the arterial blood supply of the hindlimb muscle. Finally, infusion of NGF in the hindlimb muscles of healthy rats increased P2X(3) (optical density: 0.98 ± 0.12 in control and 1.37 ± 0.16 with NGF; P < 0.05 vs. control). The pressor response to injection of α,β-methylene ATP was increased in the rats with NGF infusion. Likewise, blocking NGF attenuated exaggeration of the reflex response induced by α,β-methylene ATP in occluded rats. The findings of this study suggest that the levels of P2X(3) in primary afferent neurons are upregulated as the blood supply to the hindlimb is deficient under ischemic conditions, leading to augmentation of the muscle reflex. NGF is closely related to increases in P2X(3) receptor expression and response.

Nerve growth factor contributes augmented P2X3 expression of primary afferent neurons by femoral occlusion

Femoral artery occlusion augments the reflex sympathetic nerve and pressor responses to muscle metabolites and to muscle contraction in rats. Also, P2X3 receptors contribute the metabolic component of the exercise pressor reflex. Thus, the purpose of this study was to examine if femoral occlusion alters expression of P2X3 in dorsal root ganglion (DRG) neurons and its responsiveness in rats. In addition, we examined role of nerve growth factor (NGF) in the levels of P2X3 proteins in sensory neurons. Our western blot results showed that 24hrs of femoral ligation increased the levels of P2X3 (optical density: 0.93±0.07 in control vs. 1.37±0.10 after occlusion, P<0.05). The fluorescence immunohistochemistry further demonstrated that the occlusion elevated P2X3 expression in DRG neurons (percentage of P2X3 positive cells: 33±3% in control vs.51±3% in occlusion, P<0.05). Furthermore, our results showed that responses of renal sympathetic nerve activity and blood pressure to stimulation of P2X were greater in occluded rats than in control rats by injection of α,β me-ATP into the arterial blood supply of hindlimb muscle. Finally, infusion of NGF in the hindlimb muscles of healthy rats increased P2X3 (optical density: 0.98±0.12 in control vs. 1.37±0.16 with NGF, P<0.05). Our findings suggest that the levels of P2X3 in primary afferent neurons are increased as the blood supply to the hindlimb is insufficient, and NGF is likely responsible for enhanced P2X3. (Supported by NIH R01 HL-090720, P01HL-096570, and AHA grant 0840130N).

Nestin(+) stem cells independently contribute to neural remodelling of the ischemic heart

Recent studies have revealed the existence of multipotent nestin-immunoreactive cells in the adult mammalian heart. These cells were recruited to infarct site following ischemic injury and differentiated to a vascular lineage leading to de novo blood vessel formation. Here, we show that a sub-population of cardiac resident nestin((+)) cells can further differentiate to a neuronal-like fate in vivo following myocardial infarction. In the ischemically damaged rat heart, neurofilament-M((+)) fibres were detected innervating the peri-infarct/infarct region and the preponderance of these fibres were physically associated with processes emanating from nestin((+)) cells. One week after isogenic heterotopic cardiac transplantation, the beating transplanted rat heart was devoid of neurofilament-M((+)) fibre staining. The superimposition of an ischemic insult to the transplanted heart led to the de novo synthesis of neurofilament-M((+)) fibres by cardiac resident nestin((+)) cells. Nerve growth factor infusion and the exposure of normal rats to intermittent hypoxia significantly increased the density of neurofilament-M((+)) fibres in the heart. However, these newly formed neurofilament-M((+)) fibres were not physically associated with nestin((+)) processes. These data highlight a novel paradigm of reparative fibrosis as a subpopulation of cardiac resident nestin((+)) cells directly contributed to neural remodelling of the peri-infarct/infarct region of the ischemically damaged rat heart via the de novo synthesis of neurofilament-M fibres.

Long-term Delivery of Nerve Growth Factor by Encapsulated Cell Biodelivery in the Göttingen Minipig Basal Forebrain

Nerve growth factor (NGF) prevents cholinergic degeneration in Alzheimer's disease (AD) and improves memory in AD animal models. In humans, the safe delivery of therapeutic doses of NGF is challenging. For clinical use, we have therefore developed an encapsulated cell (EC) biodelivery device, capable of local delivery of NGF. The clinical device, named NsG0202, houses an NGF-secreting cell line (NGC-0295), which is derived from a human retinal pigment epithelial (RPE) cell line, stably genetically modified to secrete NGF. Bioactivity and correct processing of NGF was confirmed in vitro. NsG0202 devices were implanted in the basal forebrain of Göttingen minipigs and the function and retrievability were evaluated after 7 weeks, 6 and 12 months. All devices were implanted and retrieved without associated complications. They were physically intact and contained a high number of viable and NGF-producing NGC-0295 cells after explantation. Increased NGF levels were detected in tissue surrounding the devices. The implants were well tolerated as determined by histopathological brain tissue analysis, blood analysis, and general health status of the pigs. The NsG0202 device represents a promising approach for treating the cognitive decline in AD patients.

Mechanisms of Fibrillation: Neurogenic or Myogenic? Reentrant or Focal? Multiple or Single?: Still Puzzling After 160 Years of Inquiry

Mechanisms of Fibrillation: Neurogenic or Myogenic? Reentrant or Focal? Multiple or Single?: Still Puzzling After 160 Years of Inquiry

B16 NGF improves the spatial working memory in R6/1 Huntington's disease transgenic mice through the augmentation of cholinergic function and neurogenesis

BackgroundOur previous studies demonstrated that cholinergic dysfunction occurred in transgenic mouse models and patients with Huntington's disease (HD). This may contribute to the cognitive deficits in the disease. Nerve growth...

The p75 neurotrophin receptor, semaphorins, and sympathetic traffic in the heart

the sympathetic and parasympathetic branches of the autonomic nervous system control cardiac function through the coordinated activity generated by neurons within the intracardiac autonomic ganglia and central nervous system (3). Activation of the sympathetic efferent projections to the heart increases heart rate, modifies the pattern and conduction velocity of the impulse through the heart, and augments both atrial and ventricular contractility while hastening myocardial relaxation. In the vasculature, the sympathetic nervous system (SNS) reduces venous capacitance and constricts resistance vessels, increasing blood pressure. Conversely, the parasympathetic nervous system (PSNS) decreases heart rate and cardiac impulse conduction. More recent evidence has suggested that the PSNS may also affect ventricular function, indirectly by countering β-adrenergic action or directly via inhibition of L-type Ca2+ channels (34). A transient imbalance between these two branches can lead to increased heart rate and blood pressure when cardiac output is increased. When subnormal cardiac PSNS activity concomitant with sympathetic hyperactivation persists, such as after myocardial infarction, this disequilibrium may increase the risk for cardiac adverse events, including arrhythmias, hypertension, cardiac hypertrophy, and, ultimately, heart failure. This condition is called “autonomic neural remodeling” and can greatly contribute to the induction of ventricular fibrillation, thus increasing the patient's susceptibility to sudden cardiac death (41, 47). It is plausible that neurotrophic factors, target organ-secreted proteins essential for the development and differentiation of neurons, are involved in the remarkable neuroplasticity exhibited by the SNS in the adult life, including autonomic remodeling after infarction and/or in the onset and progression of congestive heart failure (17, 30, 32).

Axotomy-induced neurotrophic withdrawal causes the loss of phenotypic differentiation and downregulation of NGF signalling, but not death of septal cholinergic neurons.

BackgroundSeptal cholinergic neurons account for most of the cholinergic innervations of the hippocampus, playing a key role in the regulation of hippocampal synaptic activity. Disruption of the septo-hippocampal pathway by an experimental transection of the fimbria-fornix drastically reduces the target-derived trophic support received by cholinergic septal neurons, mainly nerve growth factor (NGF) from the hippocampus. Axotomy of cholinergic neurons induces a reduction in the number of neurons positive for cholinergic markers in the medial septum. In several studies, the reduction of cholinergic markers has been interpreted as analogous to the neurodegeneration of cholinergic cells, ruling out the possibility that neurons lose their cholinergic phenotype without dying. Understanding the mechanism of cholinergic neurodegeneration after axotomy is relevant, since this paradigm has been extensively explored as an animal model of the cholinergic impairment observed in neuropathologies such as Alzheimer's disease.The principal aim of this study was to evaluate, using modern quantitative confocal microscopy, neurodegenerative changes in septal cholinergic neurons after axotomy and to assess their response to delayed infusion of NGF in rats.ResultsWe found that there is a slow reduction of cholinergic cells labeled by ChAT and p75 after axotomy. However, this phenomenon is not accompanied by neurodegenerative changes or by a decrease in total neuronal number in the medial septum. Although the remaining axotomized-neurons appear healthy, they are unable to respond to delayed NGF infusion.ConclusionsOur results demonstrate that at 3 weeks, axotomized cholinergic neurons lose their cholinergic phenotype without dying and down-regulate their NGF-receptors, precluding the possibility of a response to NGF. Therefore, the physiological role of NGF in the adult septal cholinergic system is to support phenotypic differentiation and not survival of neurons. This evidence raises questions about the relationship between transcriptional regulation of the cholinergic phenotype by retrograde-derived trophic signaling and the transcriptional changes experienced when retrograde transport is impaired due to neuropathological conditions.

Intrathecal NGF Administration Reduces Reactive Astrocytosis and Changes Neurotrophin Receptors Expression Pattern in a Rat Model of Neuropathic Pain

Nerve growth factor (NGF), an essential peptide for sensory neurons, seems to have opposite effects when administered peripherally or directly to the central nervous system. We investigated the effects of 7-days intrathecal (i.t.) infusion of NGF on neuronal and glial spinal markers relevant to neuropathic behavior induced by chronic constriction injury (CCI) of the sciatic nerve. Allodynic and hyperalgesic behaviors were investigated by Von Frey and thermal Plantar tests, respectively. NGF-treated animals showed reduced allodynia and thermal hyperalgesia, compared to control animals. We evaluated on lumbar spinal cord the expression of microglial (ED-1), astrocytic (GFAP and S-100beta), and C- and Adelta-fibers (SubP, IB-4 and Cb) markers. I.t. NGF treatment reduced reactive astrocytosis and the density of SubP, IB4 and Cb positive fibers in the dorsal horn of injured animals. Morphometric parameters of proximal sciatic nerve stump fibers and cells in DRG were also analyzed in CCI rats: myelin thickness was reduced and DRG neurons and satellite cells appeared hypertrophic. I.t. NGF treatment showed a beneficial effect in reversing these molecular and morphological alterations. Finally, we analyzed by immunohistochemistry the expression pattern of neurotrophin receptors TrkA, pTrkA, TrkB and p75(NTR). Substantial alterations in neurotrophin receptors expression were observed in the spinal cord of CCI and NGF-treated animals. Our results indicate that i.t. NGF administration reverses the neuro-glial morphomolecular changes occurring in neuropathic animals paralleled by alterations in neurotrophin receptors ratio, and suggest that NGF is effective in restoring homeostatic conditions in the spinal cord and maintaining analgesia in neuropathic pain.

Contribution of nerve growth factor to augmented TRPV1 responses of muscle sensory neurons by hindlimb ischemia

When metabolite‐sensitive transient receptor potential vanilloid type 1 (TRPV1) receptors are stimulated a reflex is engaged that increases sympathetic nerve activity. In rats, hindlimb ischemia enhances this response. The underlying mechanism by which TRPV1 receptor responses are increased after ischemic insult is unclear. In this experiment we tested the hypothesis that muscle ischemia/vascular insufficiency elevates nerve growth factor (NGF) levels in primary afferent neurons and thereby increasing TRPV1 responsiveness. Hindlimb muscle ischemia/vascular insufficiency induced by the femoral artery ligation significantly increased NGF in the dorsal root ganglion (DRG) as compared with sham‐controls. Furthermore, when NGF was infused into hindlimb muscles of healthy rats (72 hours using an osmotic minipump), the magnitude of the DRG neuron TRPV1 response increased (5.4±0.54 nA with NGF infusion vs. 2.5±0.55 nA in control, P&lt;0.05). After adding NGF, the magnitude of the DRG neuron TRPV1 response induced by capsaicin was greater (5.5±0.33 nA, P&lt;0.05 vs. control) than that seen in controls (2.2±0.37 nA). This NGF effect was seen in isolectin B4‐negative DRG neurons, a group of thin fiber nerves that contain neuropeptides, and depend on NGF for survival. Thus NGF plays an important role in augmented TRPV1 responses after femoral artery occlusion. (All procedures outlined in this study were performed in compliance with the rules and regulations described in the NIH Guide for the Care and Use of Laboratory Animals).

Contribution of nerve growth factor to augmented TRPV1 responses of muscle sensory neurons by femoral artery occlusion

In rats, hindlimb muscle ischemia induced by femoral artery occlusion augments the sympathetic nervous response to stimulation of transient receptor potential vanilloid type 1 (TRPV1) by injection of capsaicin into the arterial blood supply of the hindlimb muscles. The enhanced sympathetic response is due to alterations in TRPV1 receptor expression and its responsiveness in sensory neurons. The underlying mechanism by which TRPV1 receptor responses are increased after muscle vascular insufficiency/ischemia is unclear. In this report we tested the hypothesis that muscle ischemia elevates nerve growth factor (NGF) levels in primary afferent neurons, thereby increasing TRPV1 responsiveness. Muscle vascular insufficiency induced by the femoral artery ligation significantly increased NGF in the dorsal root ganglion (DRG) compared with sham controls. Furthermore, when NGF was infused in the hindlimb muscles of healthy rats (72 h using an osmotic minipump), the magnitude of the DRG neuron response to capsaicin was augmented (5.4 +/- 0.54 nA with NGF infusion vs. 3.0 +/- 0.17 nA in control; P < 0.05). With the addition of NGF in the culture dish containing the DRG neurons, the magnitude of the DRG neuron response to capsaicin was greater (6.4 +/- 0.27 nA; P < 0.05 vs. control) than that seen in control (2.9 +/- 0.16 nA). Note that this NGF effect was seen in isolectin B(4)-negative DRG neurons, a group of thin fiber nerves that contain neuropeptides and depend on NGF for survival. These data suggest that NGF affects a selective subpopulation of the afferent neurons in mediating augmented TRPV1 responses after femoral artery occlusion.