AB1526SP Sigma-AldrichAnti-Nerve Growth Factor-β Antibody

Neuroimmune networks in the thymic microenvironment are thought to be involved in the regulation of T cell development. Nerve growth factor (NGF) is increasingly recognized as a potent immunomodulator, promoting cross-talk between various types of immune system cells. The present study describes the expression of NGF during thymus regeneration following acute involution induced by cyclophosphamide in the rat. Immunohistochemical stain demonstrated not only the presence of NGF but also its upregulated expression mainly in the subcapsular, paraseptal, and perivascular epithelial cells, and medullary epithelial cells including Hassall's corpuscles in both the normal and regenerating thymus. Biochemical data obtained using Western blot and RT-PCR supported these results and showed that thymic extracts contain NGF protein and mRNA, at higher levels during thymus regeneration. Thus, our results suggest that NGF expressed in these thymic epithelial cells plays a role in the T lymphopoiesis associated with thymus regeneration during recovery from acute thymic involution.

Fibroblast growth factor-1 (FGF1 or acidic FGF) is highly expressed in motor neurons. FGF-1 is released from cells by oxidative stress, which might occur from SOD-1 aberrant function in amyotrophic lateral sclerosis (ALS). Although FGF-1 is known to be neuroprotective after spinal cord injury or axotomy, we found that FGF-1 could activate spinal cord astrocytes in a manner that decreased motor neuron survival in co-cultures. FGF-1 induced accumulation of the FGF receptor 1 (FGFR1) in astrocyte nuclei and potently stimulated nerve growth factor (NGF) expression and secretion. The FGFR1 tyrosine kinase inhibitor PD166866 prevented these effects. Previously, we have shown that NGF secretion by reactive astrocytes induces motor neuron apoptosis through a p75(NTR)-dependent mechanism. Embryonic motor neurons co-cultured on the top of astrocytes exhibiting activated FGFR1 underwent apoptosis, which was prevented by PD166866 or by adding either anti-NGF or anti-p75(NTR) neutralizing antibodies. In the degenerating spinal cord of mice carrying the ALS mutation G93A of Cu, Zn superoxide dismutase, FGF-1 was no longer localized only in the cytosol of motor neurons, while FGFR1 accumulated in the nuclei of reactive astrocytes. These results suggest that FGF-1 released by oxidative stress from motor neurons might have a role in activating astrocytes, which could in turn initiate motor neuron apoptosis in ALS through a p75(NTR)-dependent mechanism.

Adult visual cortex undergoes substantial functional change as a result of alterations in visual experience. Binocular retinal lesions lead to a reorganization of the visuotopic map in primary visual cortex. Associated with this change is a strengthening of an existing plexus of long-range horizontal connections by sprouting of axon collaterals and synaptogenesis. To explore the molecular substrate of this change, we studied the expression of potential factors involved in neural plasticity in the area of reorganization. We found elevation in a number of factors as early as 3 days following the lesion, including neurotrophins BDNF, NT3, NGF and the insulin-like growth factor IGF-1. Associated with the changes in neurotrophin levels was an elevation in their receptors. We also measured elevation of transcription factors, CaMKII, MAP2 and synapsins. These experiments provide evidence for a signal transduction cascade associated with cortical reorganization.

In the present study we applied an improved nerve growth factor (NGF) extraction method to examine the effects of denervation and sympathetic decentralization on NGF levels in vascular tissue. Adult male Wistar Kyoto rats underwent mesenteric arterial denervation or splanchnic nerve transection. Four days after operation, animals were killed, and the mesenteric artery and coeliac-superior mesenteric ganglia were removed. The arterial adventitia was stripped from the media to measure NGF levels in nerve and smooth muscle separately. A high concentration of NGF was detected in the normal artery, 90% of which was in the adventitial layer. Surgical denervation significantly reduced the NGF levels in the artery and ganglia by 78 and 71%, respectively. However, within the artery the level of NGF was reduced in the adventitia but not in the media. Thus, the large reduction of NGF content resulted from the loss of nerve plexus from the artery. In contrast, decentralization did not alter the NGF content in the artery, in either the adventitia or media. Our results are in marked contrast to previous studies reporting elevated levels of NGF following denervation. This discrepancy is explained by the ability of our new procedure to extract much greater amounts of NGF from the tissue.

Nerve growth factor (NGF) is a survival factor required by a number of neuronal populations including most post-ganglionic sympathetic neurones. NGF has been detected and quantified in many tissues but there is little information regarding its cellular localization. Although it has been argued that histological detection has proven difficult due to the low levels of NGF present, other factors may contribute to prevent its identification. In the present study, we report a method for the histological detection of NGF-like immunoreactivity in the rat superior cervical ganglia (SCG). Adult Wistar-Kyoto rats were perfused briefly with either a high or low pH buffer prior to fixation and routine immunohistochemistry. Polyclonal antibodies to native mouse NGF used in the present study recognized mouse NGF but not recombinant human neurotrophin 3 (rhNT3) or brain-derived neurotrophic factor (rhBDNF) by immunoblot analysis. NGF-like immunoreactivity was localized to most sympathetic neurones. Immunoreactivity was detected in the cytoplasm with dense labelling around nuclei. No stain was seen in sections incubated with normal sheep IgG or from animals perfused with phosphate buffer (pH 7.4) prior to fixation. In addition, axotomy resulted in the disappearance of NGF immunoreactivity which was confirmed by biochemical quantification. Finally, no NGF immunoreactivity was found in neurones of rats treated systemically with NGF antiserum 3 days earlier. Possible mechanisms underlying the improvement of NGF immunohistochemistry by pH manipulation before fixation are discussed.

Immunohistochemistry has been used to demonstrate the presence of nerve growth factor (NGF)-like immunoreactivity in normal and sectioned mouse sciatic nerves. In normal nerves, immunoreactive material was not visible unless a silk ligature had previously been applied to constrict the nerves, and only then in the segment of nerve immediately distal to the ligation. Immunoreactivity was visible as early as 2 h after application of the ligature. When nerves were sectioned prior to ligation to prevent the transport of material from nerve terminals within innervated tissues, the NGF-like immunoreactivity continued to accumulate. This accumulation also occurred when a portion of the proximal stump from sectioned nerves was removed from the animal and placed in culture. Quantitative estimate of NGF concentrations with a sensitive immunoassay showed that the amount of NGF present within a segment of the proximal stump of sectioned nerves more than doubled in a 24 h period. The findings indicate that NGF is produced by cells within sectioned nerves, and further suggest that in the normal intact nerve at least a proportion of the NGF being transported derives from these cells.