High Concentrations Of Nerve Growth Factor Research Articles

High serum nerve growth factor concentrations are associated with good functional outcome at 3 months following acute ischemic stroke

BackgroundPrevious studies in animal model have demonstrated that neurotrophins were associated with functional outcome following stroke. However, the relationship between serum nerve growth factor (NGF) and functional outcome in stroke patients has not been explored. Our objective was to investigate the association between serum NGF concentrations at admission and functional outcome of patients at 3 month after stroke. MethodsOne-hundred eight-five patients with acute ischaemic stroke were recruited in our study. Serum NGF concentrations were measured by ELISA at admission. The stroke severity at admission was assessed by the National Institute of Health Stroke Scale (NIHSS). The modified Rankin Scale (mRS) was used to assess the functional outcome of patients at 3 month after stroke. In addition, 100 healthy controls were recruited. ResultsSerum NGF concentrations were higher in good functional outcome group (mRS score of 0–2) than that in poor functional outcome group (mRS score of 3–6) (9.51 ± 2.33 vs. 8.12 ± 1.61, P < 0.001). Meanwhile, the serum NGF concentrations in healthy group were lower than that in acute ischemic stroke patients (7.17 ± 1.49 vs. 9.15 ± 2.24, P < 0.001). Moreover, our results demonstrated that high serum NGF concentrations (>9.21 ng/l) were independently associated with the better functional prognosis at 3 months following the occurrence of stroke (OR 0.048, 95% CI 0.012–0.185, P < 0.001). ConclusionsHigh concentrations of serum NGF at admission may predict good functional outcome of patients at 3 months after acute cerebral ischemia stroke.

NGF-dependent axon growth and regeneration are altered in sympathetic neurons of dystrophic mdx mice.

Duchenne muscular dystrophy (DMD) is a lethal disease, determined by lack of dystrophin (Dp427), a muscular cytoskeletal protein also expressed by selected neuronal populations. Consequently, besides muscular wasting, both human patients and DMD animal models suffer several neural disorders. In previous studies on the superior cervical ganglion (SCG) of wild type and dystrophic mdx mice (Lombardi et al. 2008), we hypothesized that Dp427 could play some role in NGF-dependent axonal growth, both during development and adulthood. To address this issue, we first analyzed axon regeneration potentials of SCG neurons of both genotypes after axotomy in vivo. While noradrenergic innervation of mdx mouse submandibular gland, main source of nerve growth factor (NGF), recovered similarly to wild type, iris innervation (muscular target) never did. We, therefore, evaluated whether dystrophic SCG neurons were poorly responsive to NGF, especially at low concentration. Following in vitro axotomy in the presence of either 10 or 50ng/ml NGF, the number of regenerated axons in mdx mouse neuron cultures was indeed reduced, compared to wild type, at the lower concentration. Neurite growth parameters (i.e. number, length), growth cone dynamics and NGF/TrkA receptor signaling in differentiating neurons (not injured) were also significantly reduced when cultured with 10ng/ml NGF, but also with higher NGF concentrations. In conclusion, we propose a role for Dp427 in NGF-dependent cytoskeletal dynamics associated to growth cone advancement, possibly through indirect stabilization of TrkA receptors. Considering NGF activity in nervous system development/remodeling, this aspect could concur in some of the described DMD-associated neural dysfunctions.

Nogo-p4 Suppresses TrkA Signaling Induced by Low Concentrations of Nerve Growth Factor Through NgR1 in Differentiated PC12 Cells

Background: Regeneration of injured axons in adult mammalian central nervous system (CNS) is not spontaneous. Nogo is a major inhibitory molecule contributing to axon regeneration failure. The molecular mechanisms of Nogo inhibition of axon regeneration are not completely understood. To further investigate the underlying mechanisms, we studied the effects of Nogo-p4, a 25-amino acid core inhibitory fragment of Nogo, on nerve growth factor (NGF)-induced TrkA signaling. Methods: NGF-differentiated PC12 cells were used as cell models. The effects of Nogo-p4 on two key components of TrkA signaling, phosphorylated Erk1/2 and Akt, were analyzed by western blot. Co-immunoprecipitation experiments were performed to detect the formation of NgR1/p75 complexes. Neurite growth was quantified by measuring the neurite length. Results: Nogo-p4 did not significantly affect TrkA signaling induced by 100 ng/ml NGF, but signaling was suppressed when an NGF concentration of 5 ng/ml was used. Further investigation demonstrated that Nogo-p4 affected TrkA signaling in an NGF concentration-dependent manner. Nogo-p4 suppression of TrkA signaling was strong at low (1 and 5 ng/ml), moderate at intermediate (25 ng/ml), but absent at high (50 and 100 ng/ml) NGF concentrations. NEP1-40 attenuated, and NgR1 overexpression enhanced, Nogo-p4 suppression of TrkA signaling induced by low concentrations of NGF. High but not low concentrations of NGF reduced the formation of NgR1/p75 complexes triggered by Nogo-p4. Nogo-p4 strongly inhibited neurite growth induced by low rather than high concentrations of NGF. Conclusion: Nogo-p4 binding with NgR1 suppresses TrkA signaling induced by low concentrations of NGF in differentiated PC12 cells. Suppression of NGF-induced TrkA signaling may be another mechanism by which Nogo inhibits neurite growth.

Capillary electrophoresis of induced pluripotent stem cells during differentiation toward neurons

Understanding of electrical properties of neurons, derived from induced pluripotent stem cells (iPSCs), is an emerging issue in treating human neurological disorders. This study presents the variation in zeta potential and electrophoretic mobility of iPSCs during differentiation toward neurons. The fixed charge density of differentiating iPSCs was also evaluated using Ohshima's soft particle theory. The optical images demonstrated that in passage 12, a cluster of iPSCs emerged at day 5. The colonial expansion enlarged the cluster size and an increasing culture period led to a mature multiplication. In addition, immunochemical staining with stage-specific embryonic surface antigen-1, Oct4, Sox2, and Nanog assured the starting iPSCs of phenotypic pluripotency. A longer period of induction with nerve growth factor (NGF) produced a higher quantity of neuron-like cells. Moreover, a high concentration of NGF yielded short migration time of differentiating iPSCs, suggesting a raised surface charge and accelerated neuronal differentiation. An increasing NGF concentration and inductive span enhanced the absolute value of zeta potential, electrophoretic mobility, and fixed charge density of differentiating iPSCs. The propagated colony of embryonic phenotype and membrane charge of differentiating iPSCs can be identified and controlled as a biophysical foundation for future clinical application.

Nerve growth factor increases sodium current via interferon regulatory factor-1 pathway in rat pheochromocytoma cells

To explore the effect of nerve growth factor(NGF) and interferon regulatory factor-1(IRF-1) on sodium current change of sensory neuron in rat pheochromocytoma cells. Sensory neuron rat pheochromocytoma cells were stimulated by different concentrations of NGF(0-200 ng/mL), the IRF-1 mRNA levels were examined by real-time PCR, and the activation of IRF-1 was examined by Western blot. The sodium current change was recorded by patch clamp. Low concentration of NGF improved the sodium current, which was concentration dependent. When exposed to high concentration of NGF, the expression of IRF-1 mRNA in PC-12 was improved. Low concentration of NGF resulted in IRF-1 intronuclear transporting, and the expression was not affected. Sodium current did not occur in PC-12 cells when IRF-1 was blocked. NGF can improve the sodium current in PC-12 cells concentration-dependently, and the improvement is regulated by IRF-1.

Concentration-Dependent Effect of Nerve Growth Factor on Cell Fate Determination of Neural Progenitors

Stem cell-based spiral ganglion neuron (SGN) replacement therapy has been proposed to be a promising strategy to restore hearing either via replacing degenerated neurons or by improving the efficacy of cochlear implants which rely on functional neurons. However, lack of suitable donor cells and low survival rate of implanted cells are the major obstacles to successful implementation of therapeutic transplantation. The present study investigated the potential of mouse inner ear statoacoustic ganglion (SAG)-derived neural progenitors (NPs) to differentiate toward SGN-like glutamatergic cells and the influence to cell survival and differentiation when nerve growth factor (NGF) was supplied. We found that SAG-NPs could form neurospheres, proliferate, and differentiate into cells expressing neuronal protein neurofilament and β-III tubulin. NGF affected the cell fate of SAG-NP in a concentration-dependent manner in vitro. Low concentration of NGF (2-5 ng/mL) promoted cell proliferation. Medium concentration of NGF (20-40 ng/mL) stimulated cells to differentiate into bi-polar SGN-like cells expressing glutamatergic proteins. High concentration of NGF (100 ng/mL) could rescue cells from induced apoptosis. In the in vivo study, NGF (100 ng/mL) dramatically enhanced SAG-NP survival rate after implantation into adult mammalian inner ear. This finding raises the possibility to further induce these NPs to differentiate into SGN-like neurons in future in vivo study. In conclusion, given the capability of proliferation and differentiation into SGN-like cells with the supplement of NGF in vitro, SAG-NPs can serve as donor cells in stem cell-based SGN replacement therapy. NGF improved the survival of SAG-NPs not only in vitro but also in vivo.

Green tea polyphenols potentiate the action of nerve growth factor to induce neuritogenesis: Possible role of reactive oxygen species

Exogenously administered nerve growth factor (NGF) repairs injured axons, but it does not cross the blood-brain barrier. Thus, agents that could potentiate the neuritogenic ability of endogenous NGF would be of great utility in treating neurological injuries. Using the PC12 cell model, we show here that unfractionated green tea polyphenols (GTPP) at low concentrations (0.1 μg/ml) potentiate the ability of low concentrations of NGF (2 ng/ml) to induce neuritogenesis at a level comparable to that induced by optimally high concentrations of NGF (50 ng/ml) alone. In our experiments, GTPP by itself did not induce neuritogenesis or increase immunofluorescent staining for β-tubulin III; however, it increased expression of mRNA and proteins for the neuronal markers neurofilament-L and GAP-43. Among the polyphenols present in GTPP, epigallocatechin-3-gallate (EGCG) alone appreciably potentiated NGF-induced neurite outgrowth. Although other polyphenols present in GTPP, particularly epigallocatechin and epicatechin, lack this activity, they synergistically promoted this action of EGCG. GTPP also induced an activation of extracellular signal-regulated kinases (ERKs). PD98059, an inhibitor of the ERK pathway, blocked the expression of GAP-43. K252a, an inhibitor of TrkA-associated tyrosine kinase, partially blocked the expression of these genes and ERK activation. Antioxidants, catalase (cell-permeable form), and N-acetylcysteine (both L and D-forms) inhibited these events and abolished the GTPP potentiation of NGF-induced neuritogenesis. Taken together, these results show for the first time that GTPP potentiates NGF-induced neuritogenesis, likely through the involvement of sublethal levels of reactive oxygen species, and suggest that unfractionated GTPP is more effective in this respect than its fractionated polyphenols.

Nerve Growth Factor Promotes the Survival of Goat Preantral Follicles Cultured in vitro

The aim of this study was to investigate the effects of nerve growth factor (NGF) on the in vitro culture of goat preantral follicles. Ovarian cortex fragments were cultured in α-MEM+ supplemented with 0, 1, 10, 50, 100 or 200 ng/ml NGF for 1 or 7 days. Small fragments of noncultured ovarian tissue as well as those cultured for 1 or 7 days were processed for histology and transmission electron microscopy. The results showed that after 1 or 7 days of culture at all concentrations of NGF, except at 1 ng/ml after 1 day of culture, there was a significant reduction in the percentage of normal follicles compared to noncultured tissues. At higher NGF concentrations (100 and 200 ng/ml) after 7 days of culture, there was a significant reduction in the percentage of normal follicles compared to tissues cultured in α-MEM+ alone or at the other concentrations of NGF. It is important to note that ultrastructural and fluorescent analyses confirmed only the integrity of follicles cultured with 1 ng/ml of NGF after 7 days. In contrast to noncultured control tissues, the percentage of developing follicles was significantly increased at all concentrations of NGF after 1 or 7 days of culture. We observed that follicular diameter was greater at 1 and 10 ng/ml NGF after culture for 7 days than at the other concentrations but was similar to follicles cultured in α-MEM+ alone. In conclusion, NGF improved the survival of goat preantral follicles cultured in vitro in a dose-dependent manner.

The Effect of Pulse-Released Nerve Growth Factor from Genipin-Crosslinked Gelatin in Schwann Cell–Seeded Polycaprolactone Conduits on Large-Gap Peripheral Nerve Regeneration

Different lag-time of pulse-released nerve growth factor (NGF) from genipin-crosslinked gelatin within polycaprolactone (PCL) conduits was evaluated in large-gap peripheral nerve repair. In this study, 10% (w/v) gelatin was mixed with NGF, crosslinked with 0%, 0.1%, 0.5%, and 1% (w/v) genipin, and then sucked into the wall of PCL conduits. These controlled-release nerve conduits were named NCL (non-crosslink), LCL (low crosslink), MCL (medium crosslink), and HCL (high crosslink), respectively. The NGF releasing character showed four distinctive curves, including initial burst within 5 days, pulse releasing at 5-20 days, pulse releasing at 10-25 days, and steadily releasing. The bioactivity of the released NGF was shown by neurite outgrowth of PC12 cells after culturing in all groups. Finally, the controlled-release conduits were seeded with 9 x 10(3) Schwann cells. Conduits were used to bridge a 15-mm rat sciatic nerve defect, and the results were compared with the isografts (control group). Eight weeks after implantation, morphological analysis revealed that LCL, MCL, and HCL groups were similar to autograft treatment in the numbers and area of myelinated axons. The LCL group, although insignificant, showed a trend to have the highest myelinated axon counts of the conduit-treated groups. Thus, comparing the different NGF release characteristics among NCL, MCL, and LCL groups, we concluded that a high concentration of NGF at 5-10 days in LCL groups is needed in bridging a 15-mm peripheral nerve injury.

High-dose dietary supplementation of vitamin A induces brain-derived neurotrophic factor and nerve growth factor production in mice with simultaneous deficiency of vitamin A and zinc

Marginal vitamin A and zinc (Zn) deficiency often co-exist in many populations. Vitamin A plays a trophic role in brain and is important for its development. We investigated effects of dietary supplementation of vitamin A on brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) production in mice depleted for vitamin A and Zn. After 3 months' feeding with a low vitamin A and Zn (LVA-LZ) diet, mice were divided into two groups and replenished with either normal or high vitamin A with low Zn diet for an additional 2 months. Levels of BDNF and NGF were measured from extracts of hippocampus, cortex and cerebellum at the end of the third and fifth months. The LVA-LZ group tended to show decreased amounts of the BDNF and NGF, while animals supplemented with high vitamin A along with Zn deficiency had high BDNF and NGF concentrations. From these results, we conclude that vitamin A may increase BDNF and NGF levels.

Semaphorin3A regulates axon growth independently of growth cone repulsion via modulation of TrkA signaling

Regulation of axon growth is a critical event in neuronal development. Nerve growth factor (NGF) is a strong inducer of axon growth and survival in the dorsal root ganglia (DRG). Paradoxically, high concentrations of NGF are present in the target region where axon growth must slow down for axons to accurately identify their correct targets. Semaphorin3A (Sema3A), a powerful axonal repellent molecule for DRG neurons, is also situated in their target regions. NGF is a modulator of Sema3A-induced repulsion and death. We show that Sema3A is a regulator of NGF-induced neurite outgrowth via the TrkA receptor, independent of its growth cone repulsion activity. First, neurite outgrowth of DRG neurons is more sensitive to Sema3A than repulsion. Second, at concentrations sufficient to significantly inhibit Sema3A-induced repulsion, NGF has no effect on Sema3A-induced axon growth inhibition. Third, Sema3A-induced outgrowth inhibition, but not repulsion activity, is dependent on NGF stimulation. Fourth, Sema3A attenuates TrkA-mediated growth signaling, but not survival signaling, and over-expression of constitutively active TrkA blocks Sema3A-induced axon growth inhibition, suggesting that Sema3A activity is mediated via regulation of NGF/TrkA-induced growth. Finally, quantitative analysis of axon growth in vivo supports the possibility that Sema3A affects axon growth, in addition to its well-documented role in axon guidance. We suggest a model whereby NGF at high concentrations in the target region is important for survival, attraction and inhibition of Sema3A-induced repulsion, while Sema3A inhibits its growth-promoting activity. The combined and cross-modulatory effects of these two signaling molecules ensure the accuracy of the final stages in axon targeting.

Chronic exposure of sensory neurones to increased levels of nerve growth factor modulates CB1/TRPV1 receptor crosstalk

Anandamide (AEA) activates both cannabinoid CB(1) and TRPV1 receptors, which are expressed on cultured dorsal root ganglion neurones. Increased levels of nerve growth factor (NGF) are associated with chronic pain states. The aim of this study was to compare of the effects of AEA on CB(1) receptor signalling and TRPV1-CB(1) crosstalk in low and high concentrations of NGF, using voltage-clamp electrophysiology and Fura-2 calcium imaging. Chronic exposure to high NGF (200 ng ml(-1)) as compared to low NGF (20 ng ml(-1)) increases the proportion of neurones that exhibit an inward current in response to AEA (1 microM), from 7 to 29%. In contrast, inhibition of voltage-gated calcium currents by AEA is not significantly different in low NGF (33+/-9%, compared to high NGF 28+/-6%). Crosstalk between CB and TRPV1 receptors is modulated by exposure to high NGF. In low NGF, exposure to the CB(1) receptor antagonist, SR141716A, (100 nM) increases the percentage of neurones in which AEA elicits an increase in [Ca(2+)](i), from 10 to 23%. In high NGF, the antagonist does not alter the percentage of responders (33 to 30%). In low NGF, exposure to the CB receptor agonist, WIN55 (1 microM) reduces capsaicin-mediated increases in [Ca(2+)](i) to 28+/-8% of control as compared to an enhancement to 172+/-26% of control observed in high NGF. We conclude that cannabinoid-mediated modulation of TRPV1 receptor activation is altered after exposure to high NGF.

Donepezil Potentiates Nerve Growth Factor-Induced Neurite Outgrowth in PC12 Cells

Donepezil is a potent and selective acetylcholinesterase inhibitor developed for the treatment of Alzheimer’s disease. To elucidate whether donepezil causes neuronal differentiation, we examined its effect on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Donepezil (10 µM) significantly potentiated the neurite outgrowth evoked by low (1 ng/ml) and high (50 ng/ml) concentrations of NGF. The effect of donepezil (1 – 10 µM) was concentration-dependent. The enhancement of neurite outgrowth caused by donepezil was not blocked by the acetylcholine receptor (AChR) antagonists mecamylamine and scopolamine. Furthermore, the AChR agonists nicotine and carbachol did not affect the neurite outgrowth induced by NGF. Donepezil (10 µM) also significantly potentiated neurite outgrowth evoked by dibutyryl cyclic AMP. Moreover, donepezil potentiated the NGF-induced phosphorylation of extracellular signal-regulated kinase (ERK). These results suggest that donepezil potentiated neuronal differentiation by enhancing the activation of ERK.

Nerve Growth Factor and Tyrosine Kinase A in Human Salivary Adenoid Cystic Carcinoma: Expression Patterns and Effects on In Vitro Invasive Behavior

Perineural invasion is a frequent occurrence in salivary adenoid cystic carcinoma (ACC) and may prevent complete surgical resection. Studies have indicated that nerve growth factor (NGF) and its high-affinity receptor tyrosine kinase A (TrkA) may play a role in perineural invasion in several malignancies in which perineural invasion is observed. The present study was conducted to investigate the expression of NGF and TrkA in salivary ACC and to examine the effects of NGF on adhesion, migration and invasion capacities of a salivary ACC cell line (SACC-83) in vitro. Expression of NGF and TrkA was explored using immunohistochemistry in paraffin-embedded tissues of 32 cases of salivary ACC. The effects of NGF on in vitro adhesion, migration, and invasion capacities of the SACC-83 cell line were examined using an MTT assay and a modified Boyden chamber assay respectively. In ACC specimens, 31 (96.9%) and 32 (100%) tumors showed immunoreactivity for NGF and TrkA respectively. Significant correlations were found between NGF/TrkA expression levels and perineural invasion (P < .05). In cell adhesion assay, the percent adherences of SACC-83 cells co-cultured with 25 ng/ml NGF at 1.5 hours and 5, 25 ng/ml NGF at 6 hours were significantly higher than that co-cultured with 0 ng/ml NGF (P < .05). However, high concentration of NGF (500 ng/ml) resulted in a significant inhibition of invasion (P < .05). Overexpression of NGF and TrkA in human salivary ACC tissues may constitute a reason for perineural invasion in salivary ACC.

Suitable dose of nerve growth factor in the poly d, l-lactic acid/nerve growth factor compound conduit

目的 探讨外消旋聚乳酸/神经生长因子(PDLLA/NGF)复合导管中促进大白鼠神经再生的神经生长因子的合适含量.方法 Wistar雌性大白鼠40只,随机分为5组,每组8只.各组采用PDLLA/NGF复合导管中的NGF含量分别为100u、250u、400u、500u和800u.将每组动物的右侧坐骨神经造成10mm缺损的间隙,分别以不同NGF含量的PDLLA/NGF复合导管桥接缺损.术后6个月检测比较各组坐骨神经的再生情况.结果 NGF含量为400u和500u的PDLLA/NGF复合导管桥接组的神经再生情况显著优于其它3组(P＜0.01或P＜0.05),而该2组间的差异无统计学意义(P＞0.05).其余3组中,NGF含量为800u组的神经再生情况最差.结论 PDLLA/NGF复合导管中NGF的含量为400～500 u左右,对促进大白鼠神经再生最为合适.NGF含量过高反而不利于神经再生。

Threshold levels of ERK activation for chemotactic migration differ for NGF and EGF in rat pheochromocytoma PC12 cells

In a previous study, we show that stimulation of chemotaxis in rat pheochromocytoma PC12 cells by nerve growth factor (NGF) and epidermal growth factor (EGF) requires activation of the RAS-ERK signaling pathway. In this study, we compared the threshold levels of ERK activation required for EGF and NGF-stimulated chemotaxis in PC12 cells. The threshold ERK activity required for NGF to stimulate chemotaxis was approximately 30% lower than that for EGF. PD98059 treatment inhibited EGF stimulation of growth and chemotaxis; however, stimulation of chemotaxis required an EGF concentration approximately 10 times higher than for stimulation of PC12 cell growth. Thus, ERK-dependent cellular functions can be differentially elicited by the concentration of EGF. Also, treatment of PC12 cells with the PI3-K inhibitor LY294002 reduced ERK activation by NGF; thus, higher NGF concentrations were required to initiate chemotaxis and to achieve the same maximal chemotactic response seen in untreated PC12 cells. Therefore, the threshold NGF concentration to stimulate chemotaxis could be adjusted by the crosstalk between the ERK and PI3-K pathways, and the contributions of PI3-K and ERK to signal chemotaxis varied with the concentrations of NGF used. In comparison, LY294002 treatment had no effect on ERK activation by EGF, but the chemotactic response was reduced at all the concentrations of EGF tested indicating that NGF and EGF differed in the utilization of ERK and PI3-K to signal chemotaxis in PC12 cells.

The role of neurotrophins in brain metastasis of malignant melanoma cells

We have examined the role of neurotrophin receptors and neurotrophins in brain invasion and colonization of malignant melanoma cells. Using mouse and human melanoma variant cell sublines that have the capacity to form brain tumor colonies in nude mice, we studied the effects of neurotrophins and growth factors on malignant properties. The high brain‐colonizing melanoma lines were characterized by high expression of the low‐affinity nerve growth factor (NGF) receptor p75NTR, which forms transmembrane complexes containing the neurotrophin and its receptors, but they expressed very low amounts if any of the high‐affinity neurotrophin receptor trkA. In the presence of brain endothelial cell‐derived motility factors, NGF and other neurotrophins, such as neurotrophin‐3 (NT‐3), stimulate release of basement membrane degradative enzymes and significantly increase the invasion of endothelial cell extracellular matrix and Matigel‐coated filters. Enzymes such as the collagenase‐degrading enzyme MMP‐2 and heparan sulfate‐degrading enzyme heparanase are increased in their synthesis and release from the high brain‐colonizing but not by other melanoma variant lines by NGF or NT‐3. The increase in heparanase can be blocked by antisense resulting in decreased invasion and capacity to colonize brain. Neurotrophins also stimulate the synthesis and release of autocrine growth factors by brain‐colonizing melanoma cells. Brain‐colonizing melanoma cells also respond to paracrine growth factors in the brain, and one of the important paracrine growth factors in brain metastasis has been identified as a transferrin. Examination of the invasion front in brains colonized by the brain‐colonizing melanoma cells revealed high concentrations of NGF and other neurotrophins (NT‐3) at the interface between melanoma cells and adjoining normal brain tissue (with extensive gliosis) that gradually diminished with distance from the invasion front. Using immunohistochemical techniques to detect neurotrophins, uninvolved brain tissue (adult animals) possessed very low or undetectable concentrations of these neurotrophins. Trophic factors, autocrine factors, paracrine growth factors and other factors may determine whether metastatic melanoma cells can successfully invade, colonize and grow in the CNS.

Simultaneous delivery of an active protein and neutralizing antibody: creation of separated regions of biological activity

Spatial control over the biological activity of nerve growth factor (NGF) via a novel type of controlled-release device was demonstrated in an in vitro system. Two-layer polymer matrices that simultaneously released NGF and a neutralizing antibody (anti-NGF) from opposite faces were placed in PC12 cell-populated collagen gels. Biological activity in the gels was assessed over the course of 10 days by direct observation of the cells, which extend neuronal processes in the presence of NGF in a dose-dependent manner. The concentrations of both proteins in the gels were determined by ELISA as a function of distance from the polymer matrices at various time points. A boundary in biological activity was established within a few days of the initiation of the cultures; this boundary persisted and became more pronounced throughout the duration of the experiment. ELISA analysis revealed regions of high concentration of both NGF and anti-NGF on their respective sides of the polymer matrix early in the experiment. The theoretical amount of active NGF in the gel sections was calculated on the basis of these ELISA results; the concentration of active NGF in the region adjacent to the polymer correlated with the observed degree of biological response. These experiments suggest that spatial control over the biological activity of a potent agent can be obtained by an appropriately designed controlled-release device.

Tooth pulp tissue promotes neurite outgrowth from rat trigeminal ganglia in vitro.

The mammalian tooth pulp becomes innervated by nociceptive and sympathetic axons relatively late during development, when part of the root has formed. In the adult, regenerating axons from an injured tooth nerve or sprouting axons from uninjured nerves in the vicinity rapidly reinnervate denervated tooth pulps. These observations indicate that tooth pulp tissue can use molecular factors to attract pulpal axons from local nerve trunks. The present study examines the hypothesis that these factors include nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and glial cell line derived neurotrophic factor (GDNF). Explants of trigeminal ganglia from neonatal rat pups showed a distinct neurite outgrowth when co-cultured with pulpal explants collected from molar teeth of 12-day old pups, or after application of a pulpal extract. Control cultures, containing single ganglionic explants, or explants co-cultured with heat-treated pulpal tissue, exhibited a sparse neurite outgrowth. Exogenous NGF and/or GDNF, but not exogenous BDNF, stimulated neurite outgrowth from ganglionic explants. Unexpectedly, application of antibodies against NGF, BDNF and/or GDNF to co-cultures of ganglionic and pulpal explants did not inhibit neuritogenesis. Control experiments showed that IgG molecules readily penetrate the gel used for culture and that even very high concentrations of NGF and GDNF antibodies in combination failed to block neurite growth. On the basis of these data we suggest that other as yet unknown neurite-promoting factors might be present and active in TG/pulpal co-cultures.

Denervation, but not decentralization, reduces nerve growth factor content of the mesenteric artery.

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.