Nerve Growth Factor Immunostaining Research Articles

Inhibitory effect of valproate sodium on pain behavior in diabetic mice involves suppression of spinal histone deacetylase 1 and inflammatory mediators

Anti-epileptic medications are included in the international guidelines for managing neuropathic pain. Valproate sodium (VPS) was recently described as “the forgotten analgesic” and has been reported to relief pain in various models of neuropathic pain. Some studies reported anti-inflammatory and histone deacetylase 1 (HDA1) inhibitory properties for sodium valproate. The aim of the current study was to investigate the modulatory effect of VPS on pain behavior and inflammatory reactions in alloxan-induced diabetic neuropathy focusing on HDA1 inhibition and glia reactivity. 28 Male Swiss albino mice were allocated into four groups, (1) vehicle group, (2) alloxan-diabetic group, (3 & 4) alloxan+VPS (25 or 50 mg/kg) groups. VPS was given daily for 5 weeks by oral gavage. Pain behavior demonstrated increased allodynia (von-Frey filaments) and hyperalgesia (hot-plate test) in alloxan-diabetic mice that was reduced significantly by at least one of VPS doses. Sciatic nerves in diabetic mice showed increased histopathology score, increased silver staining for the nerves-indicating myelopathy- and a decrease in immunostaining for nerve growth factor. Spinal cord of diabetic mice showed greater histopathologic score, increased CD11b and glia fibrillary acidic protein (GFAP) immunostaining than vehicle treated mice. Molecular investigations highlighted greater content of spinal histone deacetylases, tumor necrosis factor-α (TNF-α) and interlukin-1β (IL1β) that were favorably modified by VPS. Overall, the current data confirmed that the pain killing and anti-inflammatory activity of VPS is at least partly mediated through inhibition of spinal HDA1 and glia reactivity. These findings support the view of inviting antiepileptics for treating neuropathies.

Nerve Growth Factor Involves Mutual Interaction between Neurons and Satellite Glial Cells in the Rat Trigeminal Ganglion

Nerve growth factor (NGF) plays a critical role in the trigeminal ganglion (TG) following peripheral nerve damage in the oral region. Although neurons in the TG are surrounded by satellite glial cells (SGCs) that passively support neural function, little is known regarding NGF expression and its interactions with TG neurons and SGCs. This study was performed to examine the expression of NGF in TG neurons and SGCs with nerve damage by experimental tooth movement. An elastic band was inserted between the first and second upper molars of rats. The TG was removed at 0–7 days after tooth movement. Using in situ hybridization, NGF mRNA was expressed in both neurons and SGCs. Immunostaining for NGF demonstrated that during tooth movement the number of NGF-immunoreactive SGCs increased significantly as compared with baseline and reached maximum levels at day 3. Furthermore, the administration of the gap junction inhibitor carbenoxolone at the TG during tooth movement significantly decreased the number of NGF-immunoreactive SGCs. These results suggested that peripheral nerve damage may induce signal transduction from neurons to SGCs via gap junctions, inducing NGF expression in SGCs around neurons, and released NGF may be involved in the restoration of damaged neurons.

Nerve Growth Factor and Tyrosine Kinase A Receptor in Oral Squamous Cell Carcinoma: Is There an Association With Perineural Invasion?

Perineural invasion (PNI) in oral squamous cell carcinoma (SCC) is recognized as a significant predictor of outcome. PNI is associated with locoregional recurrence and decreased survival of patients with head and neck SCC. Nerve growth factor (NGF) has been shown to be involved in PNI in several malignancies, including breast, prostate, and pancreatic cancers. We investigated the hypothesis that NGF and its high-affinity receptor tyrosine kinase A (TrkA) are highly expressed in cases of oral SCC that have histologic evidence of PNI. We performed immunohistochemistry on archived oral tongue SCC specimens from the established oral and general pathology databases at the University of California, San Francisco. The following groups were evaluated: 1) 21 T1/T2 oral tongue SCC cases with PNI and 2) 21 T1/T2 oral tongue SCC cases without histologic evidence of PNI. Strong homogeneous cytoplasmic staining for NGF and TrkA was detected in the malignant cells in the PNI-positive group of tumors. In group II (PNI negative) NGF and TrkA were detected in the stroma cells or were very weakly expressed by the malignant cells. We were able to show the presence of NGF and TrkA in the cytoplasm of malignant squamous cells in tumors with histologic evidence of PNI. Immunostaining for NGF (P = .0001) and TrkA (P = .039) was significantly higher in the PNI-positive oral SCC group than in the PNI-negative oral SCC group. This study shows that oral SCC with evidence of PNI shows increased expression of NGF and TrkA and suggests that NGF and TrkA are involved with the mechanism leading to PNI. Further investigations are warranted to determine the potential for use of NGF and TrkA as candidate biomarkers to predict progression and outcome.

Nerve Growth Factor Promotes Cardiac Repair following Myocardial Infarction

Nerve growth factor (NGF) promotes angiogenesis and cardiomyocyte survival, which are both desirable for postinfarction myocardial healing. Nonetheless, the NGF potential for cardiac repair has never been investigated. To define expression and localization of NGF and its high-affinity receptor TrkA (tropomyosin-related receptor A) in the human infarcted heart and to investigate the cardiac roles of both endogenous and engineered NGF using a mouse model of myocardial infarction (MI). Immunostaining for NGF and TrkA was performed on heart samples from humans deceased of MI or unrelated pathologies. To study the post-MI functions of endogenous NGF, a NGF-neutralizing antibody (Ab-NGF) or nonimmune IgG (control) was given to MI mice. To investigate the NGF therapeutic potential, human NGF gene or control (empty vector) was delivered to the murine periinfarct myocardium. Results indicate that NGF is present in the infarcted human heart. Both cardiomyocytes and endothelial cells (ECs) possess TrkA, which suggests NGF cardiovascular actions in humans. In MI mice, Ab-NGF abrogated native reparative angiogenesis, increased EC and cardiomyocyte apoptosis and worsened cardiac function. Conversely, NGF gene transfer ameliorated EC and cardiomyocyte survival, promoted neovascularization and improved myocardial blood flow and cardiac function. The prosurvival/proangiogenic Akt/Foxo pathway mediated the therapeutic benefits of NGF transfer. Moreover, NGF overexpression increased stem cell factor (the c-kit receptor ligand) expression, which translated in higher myocardial abundance of c-kit(pos) progenitor cells in NGF-engineered hearts. NGF elicits pleiotropic beneficial actions in the post-MI heart. NGF should be considered as a candidate for therapeutic cardiac regeneration.

Macrophage depletion suppresses sympathetic hyperinnervation following myocardial infarction

Myocardial infarction induces sympathetic axon sprouting adjacent to the necrotic region, and this has been implicated in the etiology of arrhythmias resulting in sudden cardiac death. Previous studies show that nerve growth factor (NGF) is essential for enhanced post-infarct sympathetic sprouting, but the cell types necessary to supply this neurotrophic protein are unknown. The objective of the present study was to determine whether macrophages, which are known to synthesize NGF, are necessary for post-infarct cardiac sympathetic sprouting. Ovariectomized female rats received left coronary artery ligation or sham operation, followed by intravenous injection of liposomes containing saline vehicle or clodronate, which kills macrophages. Sham-operated myocardium contained some sympathetic axons, few myofibroblasts and T cells and no CD-68-positive macrophages. In rats receiving saline liposomes through 7 days post-ligation, the posterolateral infarct border contained numerous myofibroblasts, macrophages and T cells, and sympathetic innervation was increased twofold. Treatment with clodronate liposomes reduced macrophage numbers by 69%, while myofibroblast area was reduced by 23% and T cell number was unaffected. Clodronate liposome treatment reduced sympathetic axon density to levels comparable to the uninfarcted heart. NGF protein content measured in western blots was reduced to 33% of that present in infarcts where rats received saline-containing liposomes. Tissue morphometry confirmed that NGF immunostaining was dramatically reduced, and this was attributable primarily to reduced macrophage content. These results show that macrophage destruction markedly reduces post-infarction levels of NGF and that the presence of elevated numbers of macrophages is obligatory for development of sympathetic hyperinnervation following myocardial infarction.

Nerve growth factor localization in the nasal mucosa of patients with persistent allergic rhinitis

Nerve growth factor (NGF) and NGF receptors have been shown to be expressed by structural and infiltrating inflammatory cells in the human allergic bronchial mucosa and conjunctiva. In the nose, a positive immunostaining for NGF was recently reported in biopsies of subjects undergoing surgery for refractory nasal obstruction. This study was aimed at studying by immunohistochemistry NGF expression and localization in the nasal mucosa from subjects with moderate/severe persistent allergic rhinitis and natural allergen exposure. Immunostaining for NGF, tryptase and eosinophil cationic protein was performed in human nasal turbinate sections of 25 patients affected by persistent allergic rhinitis and sensitization to Dermatophagoides pteronyssinus. NGF was consistently expressed in the epithelium and in the submucosa of allergic rhinitic subjects, preferentially localized in eosinophils and mast cells. A strong NGF immunostaining was found in mucous cells of the epithelial lining and in the submucosal glands. As previously shown for allergic asthma and allergic conjunctivitis, NGF is also detectable in the nasal mucosa of patients with persistent allergic rhinitis. The preferential NGF localization in mucous cells of the epithelial lining and in the submucosal glands suggests a possible role for NGF in modulating secretion in allergic rhinitis and possibly other allergic diseases.

Cardiac NGF and GDNF expression during Trypanosoma cruzi infection in rats

In rats, autonomic nerve endings are damaged during Trypanosoma cruzi-induced myocarditis. Gradual recovery occurs after the acute phase. The present work shows the cardiac levels of glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF), and their cellular sources during T. cruzi infection in rats. Atrial and ventricular NGF levels (ELISA) increased significantly at day 20 post inoculation, the time-point of maximal sympathetic denervation. ELISA failed to show significant increase of cardiac GDNF levels. However immunohistochemistry showed a significant increase of anti-GDNF gold particles over atrial granules at day 20. Light microscopy showed stronger NGF immunostaining in atrial cardiomyocytes and several blood capillaries. In situ hybridization showed NGF and GDNF mRNAs in atrial and ventricular myocytes of both infected and uninfected animals. Endothelial cells exhibited NGF mRNA and protein only in infected rats. No evidence of neurotrophic factor expression by the infiltrating mononuclear cells was found. This is the first report on neurotrophic factor expression during T. cruzi infection. Our findings indicate an important role for NGF in the regenerative phenomena subsequent to a myocarditis able to damage sympathetic nerve endings, with preservation of preterminals and nerve trunks. GDNF could have a minor or a more transient participation.

Topical 0.1% Prednisolone Lowers Nerve Growth Factor Expression in Keratoconjunctivitis Sicca Patients

To compare nerve growth factor (NGF) levels in tears and on the ocular surfaces of normal control and non-Sjögren's type keratoconjunctivitis sicca subjects, and to investigate the effect of 0.1% prednisolone eyedrops on NGF levels in keratoconjunctivitis sicca patients. Prospective, double-masked, randomized, comparative clinical trial. Forty-one keratoconjunctivitis sicca patients and 23 age- and gender-matched healthy subjects. Baseline tear NGF levels were measured in keratoconjunctivitis sicca patients and healthy control subjects using enzyme-linked immunosorbent assays. Keratoconjunctivitis sicca patients received 0.1% prednisolone drops in one eye and 0.1% hyaluronic acid drops in the other, 3 times a day for 28 days. Also, impression cytology (IC) and immunostaining for NGF on conjunctival epithelium were performed on both groups. Tear NGF/total tear protein (TP) concentration ratio, IC and NGF immunocytologic staining, subjective symptom scale, tear breakup time, and Schirmer values. Keratoconjunctivitis sicca patients were found to have baseline tear NGF concentrations higher than those of age- and gender-matched healthy control subjects (65.9+/-14.5 vs. 122.1+/-45.3 pg/mug, P<0.0001). In keratoconjunctivitis sicca patients, prednisolone treatment for 28 days resulted in a decrease in tear NGF levels, symptom scores, and IC scores, whereas hyaluronic acid treatment had no such effect (68.2+/-25.0 pg/mug vs. 108.0+/-43.4 pg/mug, P<0.0001 for tear NGF/TP ratio; 2.16+/-1.01 vs. 3.39+/-1.50, P = 0.0014 for symptom scale; 1.05+/-0.67 vs. 1.61+/-0.86, P = 0.0317 for IC). Measurements taken at both 14 and 28 days indicated that neither prednisolone nor hyaluronic acid treatment affected breakup time or Schirmer values. Keratoconjunctivitis sicca patients showed elevated levels of tear NGF, which were decreased by treatment with 0.1% prednisolone. These data suggest that ocular surface NGF may play an important role in ocular surface inflammation processes associated with dry eyes.

Expression of Nerve Growth Factor (NGF) Isoforms in the Rat Uterus during Pregnancy: Accumulation of Precursor proNGF

The mechanisms that promote the transient degenerative changes in the uterus innervation during pregnancy remain incompletely understood. Signaling by the nerve growth factor (NGF)-beta is important for maintaining the density of peripheral sympathetic innervation. Here, we analyzed the spatial and temporal expression of NGF isoforms in the rat uterus using RT-PCR, immunoblot analysis, and immunohistochemistry during pregnancy (d 7, 14, and 21), and postpartum (d 1, 8, and 22). Western blot analysis using antibodies to mature NGF-beta and to proNGF domain demonstrated a significant decrease in mature NGF-beta at gestational d 14 and 21 (term pregnancy) and 1 d postpartum, which paralleled a remarkable accumulation of the 26-28-, 32-, and 60-kDa proNGF forms. There were diminished ratios of mature NGF-beta to proNGF independent of uterus growth on the same gestational days. Immunohistochemistry revealed a progressive NGF-beta decline throughout pregnancy in the myometrium and a near absence at term pregnancy, which contrasted with increased NGF immunostaining in the intermyometrial connective tissue layers. More importantly, proNGF-specific antibodies identified the increased NGF immunoreactivity in the intermyometrial layers at term pregnancy as proNGF and not mature NGF-beta. Alterations in the processing of NGF and accumulation of proNGF in the intermyometrial layers, where axonal degeneration occurs, may contribute significantly to the pregnancy-related uterine denervation and to the control of myometrial activity.

Expression of NGF in hepatocellular carcinoma cells with its receptors in non-tumor cell components

Nerve growth factor (NGF) is suggested to have a role in tumor progression in addition to its role in differentiation and survival of neuronal cells. We investigated expression of NGF and its receptors, TrkA and p75NTR, in hepatocellular carcinomas (HCCs). Although hepatocytes and hepatic stellate cells (HSCs) showed respectively weak and intense NGF immunostaining in the background livers of patients suffering from liver cirrhosis (LC) or chronic hepatitis (CH), intense staining was demonstrated in HCC cells of 33 of 54 (61.1%) tumors. RT-PCR detected NGF mRNA in 7 freshly-isolated HCC samples, and in 2 of 4 cases, in which both background livers and tumors could be analyzed, NGF mRNA was more abundant in the tumors than the background livers. TrkA was detected in the smooth muscle cells of hepatic arteries, but it was negative in tumor cells as well as non-neoplastic hepatocytes. p75NTR and alpha-smooth muscle actin (alphaSMA) was expressed in HSCs in the background liver and fibroblast-like cells in stromal septa, whereas HSCs within the HCC tissues were mostly negative for p75NTR but positive for alphaSMA. This suggests that HSCs in HCC have a different property from those in background livers. Furthermore, the stromal septa contained abundant nerve fibers, which may be related to the increased NGF expression in HCC cells. NGF and its receptors are then thought to have a role in cellular interactions involving HCC cells, HSCs, arterial cells and nerve cells in HCC tissues.

Nerve growth factor mediates alterations of colonic sensitivity and mucosal barrier induced by neonatal stress in rats

Background & Aims: Maternal deprivation (MD) increases nerve growth factor (NGF) expression and colonic mast cell density and alters visceral sensitivity. This study aimed to establish whether NGF overexpression induced by neonatal stress is involved in altered visceral sensitivity and gut mucosal integrity in adult rats. Methods: Male Wistar rat pups were either submitted to MD and treated with anti-NGF antibodies or left with their dam and treated daily with NGF. All rats were tested 10 weeks later for visceral sensitivity and 12 weeks later for gut permeability, myeloperoxidase activity, and mast cell numbers. Colonic NGF and NGF receptor expression were determined at 14 days and 12 weeks of age. To determine the involvement of colonic NGF overexpression and mast cell hyperplasia in visceral hyperalgesia induced by MD, neonatally deprived adult rats received anti-NGF antibodies or doxantrazole. Results: MD increased visceral sensitivity to rectal distention, gut permeability, colonic myeloperoxidase activity, and mast cell density, and anti-NGF antibodies abolished these effects. Neonatal daily treatment with NGF mimicked the alterations induced by MD on both rectal sensitivity and mucosal barrier. In deprived compared with nondeprived rats, colonic NGF immunostaining and NGF messenger RNA were increased at 14 days and 12 weeks. Overexpression of NGF receptor messenger RNA, present at 14 days, was not observed later. Moreover, adult deprived rats treated with doxantrazole or anti-NGF antibodies exhibited normal gut permeability and visceral sensitivity to rectal distention. Conclusions: These data indicate that NGF triggers and maintains long-term alterations of visceral sensitivity and gut mucosal integrity induced by MD.

Evidence for reduced accumulation of exogenous neurotrophin by aged sympathetic neurons

The present study investigated the potential for neurotrophin uptake by cerebrovascular axons and subsequent accumulation in the aged superior cervical ganglion (SCG) following a two week intracerebroventricular infusion of nerve growth factor (NGF). In the SCG from aged rats, NGF protein levels declined significantly compared with the SCG from young adult rats. Following NGF infusion, perivascular axons from both young adult and aged rats showed intense NGF immunostaining. In addition, significant increases in NGF protein were shown using enzyme-linked immunosorbent assay (ELISA) and in counts of NGF immunopositive cell bodies in the SCG when compared with age-matched controls. NGF accumulation in ganglia from aged rats, however, was significantly less when compared with ganglia from young adult rats. The results of the present study suggest that NGF protein is significantly reduced in aged ganglia with the neurons retaining some capacity to take up and transport exogenous neurotrophin. Even so, the potential for NGF accumulation is dramatically reduced in aged rats when compared with that of young adult rats. While previous results have shown robust NGF-induced neurotransmitter responses by sympathetic neurons from the aged animal, the present finding of reduced accumulation of NGF in aged sympathetic neurons suggests an age-related difference in the utilization or transport of NGF.

Expression of nerve growth factor in astrocytes of the hippocampal CA1 area following transient forebrain ischemia

We have examined by immunoassay and immunohistochemistry, the expression of nerve growth factor in the rat hippocampus, one to 28 days after transient forebrain ischemia. In the CA1 area, the overall level of nerve growth factor expression remained constant over the first three days of reperfusion while it increased by about 45% of control levels after longer postischemic periods. In contrast, a slight decrease in nerve growth factor levels, which was most prominent at three days postlesion, was observed in the other hippocampal regions. Immunohistochemical analysis of the distribution of nerve growth factor showed that its expression was up-regulated in astrocytes but not in microglia of the postischemic CA1 region and that the intensity and temporal profile of the changes in nerve growth factor immunostaining in these cells, was consistent with that observed in the immunoassay. Interestingly, the regulation of the nerve growth factor expression in reactive astrocytes of the postischemic CA1 area closely parallels that of kainate receptor subunits GluR5–7, raising the possibility of a cause–effect relationship. These results indicate that after ischemia nerve growth factor expression is up-regulated in reactive astrocytes suggesting that these cells may contribute to rescuing damaged neurons by means of increasing nerve growth factor production.

Changes in nerve growth factor immunoreactivity following entorhinal cortex lesions: possible molecular mechanism regulating cholinergic sprouting.

To assess the possible role of trophic factors in lesion-induced plasticity, we have used a sensitive immunohistochemical technique to evaluate changes in nerve growth factor (NGF) staining in the hippocampal formation 3, 8, 16, and 30 days following entorhinal cortex lesions. Our results indicate that a band of NGF immunoreactivity appears in the outer molecular layer of the ipsilateral dentate gyrus following entorhinal ablation. The distribution of the NGF-immunoreactive band exactly coincides with the distribution of sprouting cholinergic terminals revealed by acetylcholinesterase histochemistry or NGF-receptor immunostaining. Increased NGF-immunoreactivity is detectable at 3 days postlesion, is most intense at 8 days, and decreases to near control levels by 30 days. Lesion-induced increases in NGF immunostaining also occur in animals in which septohippocampal fibers had been removed by prior destruction of the fimbria-fornix. Increases in NGF-immunoreactivity, however, are substantially reduced in animals receiving intraventricular injections of colchicine, which presumably blocks NGF release. These results indicate that 1) increases in NGF immunostaining, which occur following entorhinal lesions, precede any changes in cholinergic sprouting parameters and are greatest during the period of maximal cholinergic sprouting; 2) increased NGF-immunoreactivity is not due to NGF binding by septohippocampal fibers; and 3) increased NGF-immunoreactivity appears to depend on the release of NGF by neurons that produce it. We hypothesize that, following entorhinal lesions, NGF immunostaining within the hippocampal formation may represent NGF "anchored" within the tissue and that NGF accumulation by such a mechanism may direct the sprouting response of NGF-sensitive cholinergic neurons.