Nerve Growth Factor Gradient Research Articles

The use of immobilized neurotrophins to support neuron survival and guide nerve fiber growth in compartmentalized chambers

We answered two major questions: (1) does retrograde signaling involve retrograde transport of nerve growth factor (NGF); and (2) is a gradient of immobilized NGF sufficient to promote and guide local axonal growth? To answer these questions, we developed a technique that resulted in stably immobilized NGF and combined this with compartmented chambers. NGF was photochemically-immobilized on a chitosan surface either in the cell body (CB) compartment, distal axon (DA) compartment, or both. Neuron survival and axon outgrowth were found to be insignificantly different from positive controls where soluble NGF was present. When NGF was immobilized on chitosan surfaces in the DA compartment, and in the absence of soluble NGF, neuron survival was observed, likely due to the retrograde signal of the activated TrkA receptor and NGF-induced signals, but not the retrograde signal of NGF itself. Axons were guided towards the higher end of the step concentration gradient of NGF that was photoimmobilized on the chitosan surface in the DA compartment by laser confocal patterning, demonstrating axonal guidance. These studies provide better insight into NGF signaling mechanisms which are important to both understanding developmental disorders and degenerative diseases of the nervous system, as well as improving design strategies to promote nerve regeneration after injury.

Activation of ADF/cofilin mediates attractive growth cone turning toward nerve growth factor and netrin‐1

Proper neural circuitry requires that growth cones, motile tips of extending axons, respond to molecular guidance cues expressed in the developing organism. However, it is unclear how guidance cues modify the cytoskeleton to guide growth cone pathfinding. Here, we show acute treatment with two attractive guidance cues, nerve growth factor (NGF) and netrin-1, for embryonic dorsal root ganglion and temporal retinal neurons, respectively, results in increased growth cone membrane protrusion, actin polymerization, and filamentous actin (F-actin). ADF/cofilin (AC) family proteins facilitate F-actin dynamics, and we found the inactive phosphorylated form of AC is decreased in NGF- or netrin-1-treated growth cones. Directly increasing AC activity mimics addition of NGF or netrin-1 to increase growth cone protrusion and F-actin levels. Extracellular gradients of NGF, netrin-1, and a cell-permeable AC elicit attractive growth cone turning and increased F-actin barbed ends, F-actin accumulation, and active AC in growth cone regions proximal to the gradient source. Reducing AC activity blunts turning responses to NGF and netrin. Our results suggest that gradients of NGF and netrin-1 locally activate AC to promote actin polymerization and subsequent growth cone turning toward the side containing higher AC activity.

Analyzing neurite outgrowth from explants by fitting ellipses

The establishment of appropriate connectivity in the developing nervous system depends on many molecular guidance cues. A key method for studying the response of nerve fibers to such guidance cues is to culture explants of neural tissue in three-dimensional collagen gels. However, most previous analyses of the neurite outgrowth patterns from these explants have been very simple, often measuring only one or two parameters. Here we introduce a more sophisticated method for characterizing neurite outgrowth from explants, based on fitting an ellipse to the pattern of outgrowth. This provides 5 parameters describing the outgrowth: x and y position of the center of the ellipse, the elongation, the area and the tilt. We then apply this method to a large dataset of dorsal root ganglion explants grown in the presence of precisely controlled gradients of nerve growth factor. This analysis reveals a number of new features of these data. For instance, we find that it is the position of the center of the ellipse rather than the shape of the ellipse that is correlated with the strength of the gradient. Together these results show that ellipse-fitting of explant data can give new insights into the biological processes underlying neurite guidance by molecular cues.

Magnetic alginate microspheres: system for the position controlled delivery of nerve growth factor

The use of polymeric carriers containing dispersed magnetic nanocrystalline particles for targeted delivery of drugs in clinical practice has attracted the interest of the scientific community. In this paper a system comprised of alginate microparticles with a core of magnetite and carrying nerve growth factor (NGF) is described. The magnetic properties of these microspheres, typical of superparamagnetic materials, allow precise and controlled delivery to the intended tissue environment. Experiments carried out on PC12 cells with magnetic alginate microspheres loaded with NGF have confirmed the induction of cell differentiation which is strongly dependent on the distance from the microsphere cluster. In addition, finite element modelling (FEM) of the release profile from the microspheres in culture, indicated the possibility of creating defined and predictable NGF gradients from the loaded microspheres. These observations on the carriage and release of growth factors by the proposed microparticles open new therapeutic options for both neuronal regeneration and of the development of effective neuronal interfaces.

Spatially patterned gene expression for guided neurite extension

Axon pathfinding by localized expression of guidance molecules is critical for the proper development of the nervous system. In this report, we present a well-defined spatially patterned gene expression system to investigate neurite guidance in vitro. Nonviral gene delivery was patterned by combining substrate-mediated gene delivery with soft lithography techniques, and the amount of protein produced at the region of localized expression was varied by altering the vector concentration and the width of the pattern, highlighting the flexibility of the system. A neuronal coculture model was used to investigate responses to spatial patterns of nerve growth factor (NGF) expression. The soluble NGF gradient elicited a guidance cue, and the degree of guidance was governed by the distance a neuron was cultured from the pattern and the time between accessory cell and neuron seedings. A portion of the diffusible NGF bound to the culture surface in the extracellular space, and the surface-associated NGF supported neuron survival and neurite outgrowth. However, the surface-bound NGF gradient alone did not elicit a guidance signal, and in fact masked the guidance cue by soluble NGF gradients. Mathematical modeling of NGF diffusion was used to predict the concentration gradients, and both the absolute and fractional gradients capable of guiding neurites produced by patterned gene expression differed substantially from the values obtained with existing engineered protein gradients. Spatially patterned gene expression provides a versatile tool to investigate the factors that may promote neurite guidance.

Guiding Adult Mammalian Sensory Axons During Regeneration

Misdirection of axons after nerve injury impairs successful regeneration of adult neurons. Investigations of axon guidance in development have provided an understanding of pathfinding, but their relevance to regenerating adult axons is unclear. We investigated adult mammalian axon guidance during regeneration after peripheral nerve injury and focused on the effects of the prototypic guidance molecule nerve growth factor (NGF). Adult rat sensory neurons from dorsal root ganglia that expressed the NGF receptor tropomyosin-related kinase A (trkA) were presented with a point source of NGF in vitro. Naive trkA neurons had no net turning response to NGF, but if they had been preconditioned by a peripheral nerve transection in vivo before culturing, their growth cones were attracted toward the NGF gradient. A laminin substrate was required for this behavior and an anti-trkA antibody interrupted turning. These data demonstrate that injured adult mammalian axons can be guided as they regenerate. Moreover, despite the downregulation of trkA mRNA and protein levels within the dorsal root ganglion after injury, sensory neurons retain and increase trkA protein at the injury site where the regenerating axons are found. This may enhance the axonal response to NGF and allow guidance along an NGF gradient created in vivo in the distal nerve stump.

Differences between the effect of anisotropic and isotropic laminin and nerve growth factor presenting scaffolds on nerve regeneration across long peripheral nerve gaps

Anisotropic scaffolds of agarose hydrogels containing gradients of laminin-1 (LN-1) and nerve growth factor (NGF) molecules were used to promote sciatic nerve regeneration across a challenging 20 mm nerve gap in rats. Step and continuous gradient anisotropic scaffolds were fabricated and characterized, and regeneration was compared to that in isotropic scaffolds with uniform concentrations of LN-1 and NGF and sciatic nerve grafts harvested from syngenic rats. Polysulfone tubular guidance channels were used to present the agarose-based scaffolds to the nerve stumps. Four months after implantation, regenerating axons were observed in animals implanted with anisotropic scaffolds with gradients of both LN-1 and NGF molecules and nerve grafts, but not in animals with isotropic scaffold implants. Also, the scaffolds with gradients of either LN-1 or NGF, with the other component being uniformly distributed in the scaffold, did not elicit axonal regeneration. The total number of myelinated axons was similar for the anisotropic scaffold and the nerve graft conditions, with the anisotropic scaffolds having a higher density of axons than the nerve grafts. Axonal diameter distribution was similar for the anisotropic scaffolds and the nerve grafts. The nerve grafts and anisotropic scaffolds resulted in better functional outcome compared to isotropic scaffolds as measured by the relative gastrocnemius muscle weight (RGMW). Additionally the state of neuromuscular junctions as assessed by pre- and post-synaptic staining revealed that both the anisotropic scaffolds performed as well as nerve grafts.

A novel method for establishing daily in vivo concentration gradients of soluble nerve growth factor (NGF)

Despite the capacity for spontaneous axonal regeneration, recovery following injuries to the peripheral nervous system (PNS) following transection are often incomplete and limited to short distances. Nerve growth factor (NGF) has been previously shown to support neuron survival, and direct growth of both developing and regenerating nerve fibers along a concentration gradient, based largely on in vitro studies. Here, we present a novel in vivo model of administering daily concentration gradients of NGF by directly manipulating the placement of the catheter-nerve conduit junction. Our results show that a dose of 800 pg NGF can be reliably used to establish a chemotactic concentration gradient over both a transient time period, and chronically through repeated daily administrations of the drug. Results from these studies may lead to a better mechanistic understanding of how concentration gradients of soluble NGF influence in vivo peripheral nerve regeneration.

Mathematical Modeling of Guided Neurite Extension in an Engineered Conduit with Multiple Concentration Gradients of Nerve Growth Factor (NGF)

Neurotrophic factors such as nerve growth factor (NGF) provide essential cues to navigate growing axon toward their targets. Concentration and concentration gradient of NGF are key parameters affecting the growth rate and direction of neurites and axons. However, the maximum distance for guided nerve growth under stimulation of a single concentration gradient is limited and is thus unfavorable in nerve regeneration. Since the sensitivity of PC12 cells to NGF signals is restorable even after brief removal of the factors, exposure to multiple concentration gradients of the factor can achieve longer distances and greater rates of guided growth. In this study, a mathematical model simulating nerve growth in a virtually constructed nerve conduit incorporating multiple NGF concentration gradients is established. Using a genetic algorithm, optimized initial profiles of NGF able to achieve 4.5 cm of guided growth with a significantly improved growth rate has been obtained. The model also predicts an inverse relationship between the diffusion coefficient of the factor and the neurite growth rate. This model provides a useful tool for evaluating various conduit designs before fabrication and evaluation.

Immobilized Concentration Gradients of Neurotrophic Factors Guide Neurite Outgrowth of Primary Neurons in Macroporous Scaffolds

Neurotrophic factors present as concentration gradients are neurotropic cues that direct axonal growth toward their targets. Multiple factors work together in vivo to ensure axons reach the proper targets, likely interacting with one another via intracellular signalling pathways. Nerve growth factor (NGF) and neurotrophin-3 (NT-3) are neurotrophins known to guide axons as well as promote axonal growth following injury to both the spinal cord and peripheral nerve. These molecules interact with neurons through different tyrosine kinase receptors. In this study, the receptors for these growth factors were shown to be co-localized on E10 chick dorsal root ganglion (DRG) cells, providing an opportunity for synergism. Well-defined concentration gradients of NGF and NT-3 were immobilized for the first time in a cell-penetrable, cell-adhesive scaffold of poly(2-hydroxyethylmethacrylate) and poly(L-lysine). An NGF concentration gradient of 310 ng/mL/mm was required to guide chick DRG neurites. A lower concentration gradient of 200 ng/mL/mm of NGF was shown to elicit guidance when an NT-3 concentration gradient of 200 ng/mL/mm was also present, indicating a synergistic response in the DRG neurons. These gradient scaffolds may be useful for guided regeneration following injury to the spinal cord or peripheral nerve and may also elucidate the mechanism for intracellular signaling of neurotrophic factors.

Nerve growth factor activates aorta endothelial cells causing PI3K/Akt- and ERK-dependent migration

Nerve growth factor (NGF) is a well-known neurotrophin. We determined whether NGF can activate endothelial cell migration and signalling that underlie angiogenic processes. We showed that aorta endothelial cells express mRNA for both the receptor tyrosine kinase TrkA and the p75 neurotrophin receptor (p75NTR) that associates with TrkA when signalling occurs. Pig aortic endothelial cells migrated when exposed to an NGF gradient, due to the simultaneous activation of the phosphatidylinositol 3-kinase and extracellular signal-regulated kinase signalling pathways. Furthermore, morphological changes were found in migrating cells: they appear with elongated structures with a smaller cell volume than control cells. Our data show that NGF is an activator of endothelial cells and suggest that NGF plays a role in mediating angiogenesis.

Immobilized concentration gradients of nerve growth factor guide neurite outgrowth.

Axons are guided to their targets by a combination of haptotactic and chemotactic cues. We previously demonstrated that soluble neurotrophic factor concentration gradients guide axons in a model system. In an attempt to translate this model system to a device for implantation, our goal was to immobilize a stable neurotrophic concentration gradient for axonal (or neurite) guidance. Nerve growth factor (NGF) was immobilized within poly(2-hydroxyethylmethacrylate) [p(HEMA)] microporous gels using a gradient maker. The NGF was stably immobilized, with only approximately 0.05% of the amount originally incorporated into the gel released over an 8-day period. Immobilized NGF was bioactive: the percent of PC12 cells extending neurites on NGF-immobilized p(HEMA) gels was 16 +/- 2%, which was statistically the same as those exposed to soluble NGF (22 +/- 6%). We were able to predict and reproducibly create stable NGF concentration gradients in the gel. At an NGF concentration gradient of 357 ng/mL/mm, PC12 cell neurites were guided up the gradient. The facile, flexible, and reproducible nature of this method allowed us to translate soluble growth factor gradient models to stable growth factor gradient devices that may ultimately enhance axonal guidance and regeneration in vivo.

Defining the concentration gradient of nerve growth factor for guided neurite outgrowth.

The developing axon is believed to navigate towards its target tissue in response to a concentration gradient of neurotrophic factors, among other diffusible and surface-bound stimuli. However, the minimum concentration gradient required for guidance over the maximum distance is still unknown, largely because well-defined systems have not been utilized to address this question. In this study, a linear concentration gradient of nerve growth factor was achieved across a 5-mm agarose membrane that separated a nerve growth factor source compartment from a sink compartment. The concentrations in both compartments were maintained constant (and different). Both concentration and concentration gradient were well defined across the membrane, allowing us to study the relative importance of concentration gradient vs concentration for neurite guidance. The orientation of PC12 cell neurites was studied in response to a series of nerve growth factor concentration gradients in vitro. For effective guidance of PC12 cell neurite outgrowth, a minimum concentration gradient of 133ng/ml per mm was required, below which guidance was ineffective. Higher gradients were effective for guidance yet were limited by the concentration of nerve growth factor in the source compartment. At a nerve growth factor concentration of 995ng/ml, the PC12 cells' receptors were saturated, thereby limiting the maximum effective distance for guidance to less than 7.5mm in response to a diffusible nerve growth factor cue. This distance exceeds the 0.5-2mm distance observed by others for effective neurite guidance. Using this model system, we propose that the minimum concentration gradient can be defined for other cells and growth factors. Ultimately, it is anticipated that such concentration gradients could be included in a device to promote regeneration.

Phospholipase C-gamma and phosphoinositide 3-kinase mediate cytoplasmic signaling in nerve growth cone guidance.

Expression of rat TrkA in Xenopus spinal neurons confers responsiveness of these neurons to nerve growth factor (NGF) in assays of neuronal survival and growth cone chemotropism. Mutational analysis indicates that coactivation of phospholipase C-gamma (PLC-gamma) and phosphoinositide 3-kinase (PI3-kinase) by specific cytoplasmic domains of TrkA is essential for triggering chemoattraction of the growth cone in an NGF gradient. Uniform exposure of TrkA-expressing neurons to NGF resulted in a cross-desensitization of turning responses induced by a gradient of netrin-1, brain-derived neurotrophic factor (BDNF), or myelin-associated glycoprotein (MAG) but not by a gradient of collapsin-1/semaphorin III/D or neurotrophin-3 (NT-3). These results, together with the effects of pharmacological inhibitors, support the notion that there are common cytosolic signaling pathways for two separate groups of guidance cues, one of which requires coactivation of PLC-gamma and PI3-kinase pathways.

Correlation of gp140 trk expression and NGF-induced neuroblast chemotaxis in the embryonic rat spinal cord

During rat embryogenesis, fibers containing nerve growth factor (NGF) are present near the target destinations of migratory spinal neuroblasts, suggesting that diffusible gradients of NGF provide signals to newly generated neurons in the developing cord. In vitro, pM concentrations of NGF induce neuroblast chemotaxis (directed migration along a chemical gradient), indicating evoked motility is mediated by high-affinity receptors. Binding of 125I-labelled NGF to fetal cord cells provides additional evidence that rat spinal neuroblasts express the high-affinity receptors; however, their presence has not been directly demonstrated. In the present study, we used immunocytochemistry to show that the high-affinity NGF receptor protein, gp140 trk ( trk) is detectable in embryonic spinal tissue sections and in cord dissociates. Correlation of trk expression with NGF-induced chemotaxis revealed that both the receptor protein expression and functional responses to NGF develop along a ventro-dorsal gradient that parallels the in vivo pattern of neurogenesis and migration. Analysis of the temporal changes in trk immunoreactivity demonstrated that expression of gp140 trk is bimodal, possibly reflecting multiple effects of NGF during development. Chemotaxis to NGF was blocked by nM concentrations of the kinase inhibitor, K252a, suggesting that NGF stimulates motility via high-affinity receptors coupled to kinase activity. Elevated 3′,5′-cyclic adenosine monophosphate (cAMP) also attenuated NGF-induced chemotaxis, presenting preliminary evidence that protein kinase A (PKA) may regulate motility responses to NGF.

Local sprouting of neurites from cultured PC12D cells in response to a concentration gradient of nerve growth factor

PC12D cells, a subline of PC12 cells, extend neurites very rapidly in response to NGF, even when RNA synthesis is blocked. Several minutes after the initiation of a concentration gradient of NGF from a micropipette in the vicinity of PC12D cells, clear projections emerged from cells on the side facing the micropipette while no significant changes in morphology were observed on the other side of cells. A control solution administered from a micropipette did not produce any changes in morphology. Longer exposure to the gradient of NGF of aggregates of PC12D cells increased the length of neurites extending toward the source of NGF. The observations indicate that the sprouting of neurites occurs locally in regions of PC12D cells that are exposed to an elevated concentration of NGF.

Cellular Localization of NGF and NT-3 mRNAs in Postnatal Rat Forebrain

The presence of transiently elevated levels of mRNA for nerve growth factor (NGF) and neurotrophin-3 (NT-3) in postnatal development of several brain areas suggests that these factors may be expressed by a greater number of cell types in the immature than in the adult brain. To evaluate this possibility, in situ hybridization was used to determine the cellular localization of NGF mRNA and NT-3 mRNA in hippocampus, cingulate cortex, posterolateral neocortex, thalamus, and cerebellum of post natal rat. In areas expressing both neurotrophins (i.e., hippocampus, cingulate cortex, and anteroventral thalamus), NT-3 mRNA was detected at earlier ages than NGF mRNA. Patterns of hybridization in hippocampus and cerebellum indicate that NT-3 is expressed by neurons soon after leaving the mitotic cycle whereas NGF expression is a feature of more mature neurons. The exception to this pattern was NGF expression in the lateral geniculate nuclei which was present by Postnatal Day 1 and retained in the adult. Both neurotrophins were transiently expressed in several brain areas. The loss of expression with age was most striking in thalamus with transient expression of NT-3 mRNA by the majority of dorsal thalamic relay nuclei and of NGF mRNA by fewer nuclei including the posterior, anteroventral, ventrolateral, and ventromedial nuclei. NT-3 expression also was transient in caudal cingulate/retrosplenial cortex, hippocampal CA3 stratum pyramidale, and the granule cells of archicerebellum. In early postnatal cingulate and retrosplenial cortices there were reciprocal rostrocaudal gradients of NGF and NT-3 expression. These results suggest both distinct and overlapping functions for NT-3 and NGF in early developmental processes including involvement of NT-3 in cerebellar development and of NGF in the development and maintenance of visual afferents to thalamus. Patterns of neurotrophin expression in medial limbic cortex may establish trophic gradients which influence the topography of thalamic innervation.

Dynamic plasticity of dental sensory nerve structure and cytochemistry

Hypersensitive dentine responds to normal changes in touch or temperature with abnormal pain sensations. This paper reviews studies that have shown dynamic changes in sensory nerve structure, cytochemistry and location after tooth injury, suggesting that those changes contribute to dentine hypersensitivity. Nerve fibres containing calcitonin gene-related peptide (CGRP) are the main type of sensory fibre to innervate dentine. Evidence that many of those dentinal nerve endings originate from small myelinated fibres is presented here. The location of CGRP nerve terminals correlates with the pulpal gradients of nerve growth factor that have been demonstrated in normal teeth by in situ hybridization histochemistry. When shallow cavities are drilled into the outer dentine of rat molars a five-to-eight-fold increase in pulpal nerve growth factor precedes the extensive structural changes in the sensory nerve fibres in pulp and dentine near the injury. Those nerve growth factor and sensory nerve reactions eventually subside if healing occurs, but both continue if inflammation continues. Evidence correlating pulpal inflammation with long-term changes in central trigeminal pain pathways is reviewed. There can be extensive neuroplasticity after tooth injury, both within dental pain fibres and in central pain pathways. The timing of those alterations of nerve structure, location, and cytochemistry is consistent with their involvement in mechanisms of dentine hypersensitivity.

Expression of NGF in sympathetic neurons leads to excessive axon outgrowth from ganglia but decreased terminal innervation within tissues

The effects of nerve growth factor (NGF) on sympathetic axon growth were investigated by generating transgenic mice in which the β subunit of NGF was expressed in sympathetic neurons using the human dopamine β-hydroxylase (DBH) promoter. In DBH-NGF mice, the sympathetic trunk and nerves growing to peripheral tissues were enlarged and contained an increased number of sympathetic fibers. Although sympathetic axons reached peripheral tissues, terminal sympathetic innervation within tissues was decreased in DBH-NGF mice. This effect could be reversed in the pancreas by overexpression of NGF in pancreatic islets. The observations are consistent with a model in which NGF gradients are not required to guide sympathetic axons to their targets, but are required for the establishment of the normal density and pattern of sympathetic innervation within target tissues.

Transforming growth factor beta as a neuronoglial signal during peripheral nervous system response to injury.

In contrast to the central nervous system (CNS), the peripheral nervous system (PNS) displays an important regenerative ability which is dependent, at least in part, on Schwann cell properties. The mechanisms which stimulate Schwann cells to adapt their behavior after a lesion to generate adequate conditions for PNS regeneration remain unknown. In this work, we report that adult rat dorsal root ganglion (DRG) neurons are able, after a lesion performed in vivo or when they are dissociated and cultured in vitro, to synthesize transforming growth factor beta (TGF beta), a pleiotropic growth factor implicated in wound healing processes and in carcinogenesis. This TGF beta is tentatively identified as the beta-1 isoform. Adult rat DRG neurons release a biologically active form of TGF beta which is able to elicit multiple Schwann cell responses including a stimulation to proliferate. Moreover, purified TGF beta-1 produces a Schwann cell morphology alteration and decreases the secretion of tissue-type plasminogen activator (tPA) and enhances the secretion of plasminogen activator inhibitor (PAI) by Schwann cells. This generates conditions which are thought to favor a successful neuritic regrowth. Furthermore, purified TGF beta-1 stimulates type IV collagen mRNA expression in Schwann cells. This subtype of collagen is associated with the process of myelinization. Finally, TGF beta-1 decreases nerve growth factor (NGF) mRNA expression by Schwann cells, an effect which could participate in the maintenance of a distoproximal NGF gradient during nerve regeneration. We propose that neuronal TGF beta plays an essential role as a neuronoglial signal that modulates the response of Schwann cells to injury and participates in the successful regeneration processes observed in the PNS.