Inhibition of neurite outgrowth on astroglial scars in vitro

Traumatic injury to the adult mammalian CNS results in the formation of an astroglial-mesenchymal scar that seals the wound site but blocks axonal regeneration in the process. The mechanism that leads to this inhibition of axon outgrowth has been proposed to be either a physical barrier blocking the advancement of the growth cone or chemical factors actively inhibiting axon outgrowth. At present, it is unknown whether one or both of these mechanisms are responsible for the inhibitory nature of the glial scar in vivo. Using a model of CNS trauma that allows for removal of an adult rat glial scar intact on a nitrocellulose support and placement in vitro with the upper surface exposed, we addressed the question of whether the inhibitory effects could be accounted for by chemical components at the scar surface. A purified population of rat hippocampal neurons was seeded onto the scar explants as well as onto explants taken from neonatal rat cerebral cortex, and the extent of neurite outgrowth was compared. We found that the glial scar, at best, stimulates only minimal neurite outgrowth over its surface when compared to the immature environment explanted in the same manner. This growth-inhibitory state cannot merely be explained by neuronotoxic factors or fibroblasts preventing astrocyte-mediated neurite outgrowth. The inhibition is more probably due to the expression of molecules on the surface of the adult scar that either directly inhibit growth cones or inhibit them indirectly by occluding neurite-promoting factors in the extracellular matrix or on the astrocyte surface.     

Basal lamina enhances hippocampal neurite outgrowth in vitro

To investigate the involvement of the extracellular matrix in central neuron neurite outgrowth, dissociated hippocampal neurons were cultured on basal lamina prepared from chick embryo retina. After 2 days in culture, the basal lamina was found to promote marked hippocampal neurite outgrowth. The average neurite length was over twice that on collagen plus laminin-treated coverslips. These results clearly indicate that central neurons as well as peripheral neurons respond to the components of basal lamina and thus extend their processes.     

Stimulation of neurite outgrowth in vitro by a glycero-ganglioside

A glycerol-containing analog of ganglioside, with sialic acid attached to a diglyceride-like structure possessing two ether-linked alkyl chains, was prepared synthetically and applied exogenously to three culture systems; neuro-2A neuroblastoma cells, PC12 cells and dorsal root ganglia. This resulted in pronounced stimulation of neurite outgrowth in all three, demonstrating that sialo-lipids(s) lacking ceramide and possessing sialic acid as the sole carbohydrate are able to promote neuritogenesis in approximately the same manner as naturally occurring gangliosides.     

A multiplexed proteomics approach to differentiate neurite outgrowth patterns

We report here a method for proteomics pattern discovery by utilizing a self-organizing map approach to analyze data obtained from a novel multiplex iTRAQ proteomics method. Through the application of this technique, we were able to delineate the early molecular events preceding dorsal root ganglia neurite outgrowth induced by either nerve growth factor (NGF) or an immunophilin ligand, JNJ460. Following pattern analysis we discovered that each neurotrophic agent promoted mostly distinct increases in protein expression with few overlapping patterns. In the NGF-treated group, proteins possessing "biosynthesis function" (p < 0.002) and "ribosome localization" (p < 0.0003) were increased, while proteins promoting "organogenesis" (p < 0.004) and related "signal transduction" (p < 0.008) functions were notably increased in the JNJ460-treated group. This study suggests that the properties of neurite outgrowth triggered by NGF and JNJ460 can be distinguished at the proteome level. Multiplexed proteomics analysis, along with pattern discovery bioinformatics tools, has the capability to differentiate subtle neuroproteomics patterns.     

A quantitative method for analysis of in vitro neurite outgrowth

The adult mammalian CNS is extremely limited in its ability to regenerate axons following injury. Glial scar, neuroinflammatory processes and molecules released from myelin impair axonal regrowth and contribute to the lack of neural regeneration. An in vitro assay that quantitates neurite outgrowth from cultured neurons as a model of neuronal regenerative potential is described. Specifically, the neurite outgrowth from primary neurons (rat cerebellar granule neurons; CGNs) and a neuronal cell line (NG108-15) were quantitatively measured after optimization of culture conditions. After cultures were fixed and immunostained to label neurons and nuclei, microscope images were captured and an image analysis algorithm was developed using Image-Pro Plus software to allow quantitative analysis. The algorithm allowed the determination of total neurite length, number of neurons, and number of neurons without neurites. The algorithm also allows for end-user control of thresholds for staining intensity and cell/nuclei size. This assay represents a useful tool for quantification of neurite outgrowth from a variety of neuronal sources with applications that include: (1) assessment of neurite outgrowth potential; (2) identification of molecules that can block or stimulate neurite outgrowth in conventional culture media; and (3) identification of agents that can overcome neurite outgrowth inhibition by inhibitory substrates.     

Sulfated proteoglycans in astroglial barriers inhibit neurite outgrowth in vitro

In vivo studies of the roof plate of the spinal cord and midline optic tectum in rodent and the developing subplate in the telencephalon of the chick showed that two glycosaminoglycans, keratin sulfate and chondroitin sulfate, possibly in the proteoglycan form (KS-PG, CS-PG, or KS/CS-PG), were present at times when axons approach closely but do not invade these territories. To address the question of whether KS/CS-PG actively inhibits growth cone elongation and to determine which component(s) of the proteoglycan may be critical to this phenomenon, we used a technique employing nitrocellulose-coated petri dishes onto which stripes of various purified macromolecules were attached. Isolated E9 chick dorsal root ganglia were grown on lanes of KS/CS-PG in alteration with lanes of the growth-promoting molecule laminin (LN). Neurite outgrowth was abundant along stripes of LN. In contrast, upon encountering a stripe containing KS/CS-PG, neurites either stopped abruptly or turned and traveled along the KS/CS-PG stripe border. The effect was dependent upon the concentration of the proteoglycan with intermediate concentrations producing intermittent patterns of crossing. We mixed LN with the KS/CS-PG, where the LN was in concentrations which alone support outgrowth, and observed that the KS/CS-PG was still inhibitory when such a growth-promoting molecule was present. A 10-fold higher concentration of LN was able to overcome the inhibitory effect of the KS/CS-PG. These results suggest that the interaction of inhibitory and growth-promoting molecules can interact to produce a wide spectrum of neurite patterns ranging from complete inhibition to totally unimpeded outgrowth. Selective enzymatic removal of the KS or CS from the KS/CS-PG permitted various degrees of neurite outgrowth to occur across the previously inhibitory lanes, and digestion of both glycoaminoglycan moieties, leaving only the protein core of the molecule, resulted in a complete lack of inhibition. These assays demonstrated that KS/CS-PG is inhibitory to embryonic dorsal root ganglia neurites in vitro and that complete inhibition requires contributions from both KS and CS moieties.     

IGF-1 and BDNF promote chick bulbospinal neurite outgrowth in vitro

Injured neurons in the CNS do not experience significant functional regeneration and so spinal cord insult often results in permanently compromised locomotor ability. The capability of a severed axon to re-grow is thought to depend on numerous factors, one of which is the decreased availability of neurotrophic factors. Application of trophic factors to axotomized neurons has been shown to enhance survival and neurite outgrowth. Although brainstem-spinal connections play a pivotal role in motor dysfunction after spinal cord injury, relatively little is known about the trophic sensitivity of these populations. This study explores the response of bulbospinal populations to various trophic factors. Several growth factors were initially examined for potential trophic effects on the projection neurons of the brainstem. Brain derived neurotrophic factor (BDNF) and insulin-like growth factor (IGF-1) significantly enhance mean process length in both the vestibulospinal neurons and spinal projection neurons from the raphe nuclei. Nerve growth factor (NGF), neurotrophin-4 (NT-4) and glial derived neurotrophic factor (GDNF) did not effect process outgrowth in vestibulospinal neurons. At the developmental stages used in this study, it was determined that receptors for BDNF and IGF-1 were present both on bulbospinal neurons and on surrounding cells with a non-neuronal morphology.     

Effect of lens lesion on neurite outgrowth of retinal ganglion cells in vitro

Recent studies have shown that lens lesion promotes axonal regeneration in the optic nerve of adult rats. In the present investigations, dissociated retinal ganglion cells (RGC) from intact postnatal (P) 9-11 rats showed spontaneous neurite outgrowth on laminin-1, in contrast to RGC from intact P14-adult rats. Neurite outgrowth from P9-14 RGC on laminin-1 was promoted by prior lens lesion and also during coculture with lesioned lenses. Neurite outgrowth from adult RGC following prior lens lesion, or in cocultures with lesioned lenses, required the presence of laminin-2. In media conditioned by lesioned lenses, the stimulatory effect on neurite outgrowth was still observed in the presence of K252a (trk receptor blocker) and mAb 228 (which blocks the effects of leukemia inhibitory factor and ciliary neurotrophic factor). Together, these results suggest the existence of a neuritogenic factor(s) associated with the lesioned lens that belongs to neither the neurotrophin nor the gp130 cytokine family.     

Integrin-laminin interactions controlling neurite outgrowth from adult DRG neurons in vitro

A prerequisite for axon regeneration is the interaction between the growth cone and the extracellular matrix (ECM). Laminins are prominent constituents of ECM throughout the body, known to support axon growth in vitro and in vivo. The regenerative capacity of adult neurons is greatly diminished compared to embryonic or early postnatal neurons. Since most lesions in the nervous system occur in the adult, we have examined neurite outgrowth from adult mouse DRG neurons on four laminin isoforms (laminin-1/LM-111, laminin-2/LM-211, laminin-8/LM-411 and laminin-10/LM-511) in vitro. The growth on laminin-1 and -10 was trophic factor-independent and superior to the one on laminin-2 and -8, where growth was very poor in the absence of neurotrophins. Among other ECM proteins, laminins were by far the most active molecules. Using function-blocking antibodies to laminin-binding integrins, we identified non-overlapping functions of integrins alpha3beta1, alpha7beta1 and alpha6beta1 on different laminin isoforms, in that alpha3beta1 and alpha7beta1 integrins appeared to be specific receptors for both laminin-1 and-2, whereas integrin alpha6beta1 was a receptor for laminin-8 and-10. Lastly, by use of immunohistochemistry, expression of subunits of laminin-1, -2, -8 and -10 in sensory organs in the human epidermis could be demonstrated, supporting an important role for these laminins in relation to primary sensory axons.     

Riluzole promotes cell survival and neurite outgrowth in rat sensory neurones in vitro

This study explored the effects of riluzole administration on cell survival and neurite growth in adult and neonatal rat dorsal root ganglion (DRG) neurones in vitro. Neuronal survival was assessed by comparing numbers of remaining neurones in vehicle- and riluzole-treated cultures. A single dose of 0.1 microm riluzole was sufficient to promote neuronal survival in neonatal DRG cultures, whereas repeated riluzole administration was necessary in adult cultures. However, a single administration of riluzole was sufficient to induce neuritogenesis, promote neurite branching and enhance neurite outgrowth in both neonatal and adult DRG cultures. The effects of a single dose of riluzole on adult DRG neurones after peripheral nerve or dorsal root injury were also studied in vitro at 48 h. For both types of injury, riluzole enhanced neurite outgrowth in terms of number, length and branch pattern significantly more on the injured side as compared with the contralateral side. No effect was seen on cell survival. The results suggest that, in addition to its cell survival effects, riluzole has novel growth-promoting effects on sensory neurones in vitro and that riluzole may offer a new way to promote sensory afferent regeneration following peripheral injury.     

Methyl 3,4-dihydroxybenzoate promotes neurite outgrowth of cortical neurons cultured in vitro

Cerebral cortical neurons from neonatal rats were cultured in the presence of methyl 3,4-dihydroxybenzoate (MDHB;2,4,and 8 μM).Results showed that MDHB significantly promoted neurite outgrowth and microtubule-associated protein 2 mRNA expression,and increased neuronal survival in a dose-dependent manner.Moreover,MDHB induced brain-derived neurotrophic factor expression.These findings suggest that MDHB has a neurotrophic effect,which may be due to its ability to increase brain-derived neurotrophic factor expression.     

Antisera to an axolemma-enriched fraction inhibit neurite outgrowth and destroy axons in vitro

Antisera prepared to an axolemma-enriched fraction derived from rat brain inhibited neurite outgrowth and destroyed mature axons in spinal cord-dorsal root ganglia cultures. Similar antibody-mediated anti-axon effects may be important in some diseases of the human nervous system.     

Developmental patterns of neurite outgrowth from chick embryo spinal cord and retinal neurons on laminin substrates

It has been previously found that neurite outgrowth on collagen substrates decreases with increasing gestational age of chick embryo spinal cord and retinal neurons in tissue culture. In the current study, laminin, polylysine and collagen were compared in their efficacy in promoting neurite extension from chick embryo spinal cord neurons aged 6-16 days or retinal neurons aged 8-16 days in ovo. The percentage of neurons with neurites and the length of the neurites were determined at 1 and 3 days in culture. There was a significant increase in neuritogenesis by laminin and polylysine compared to collagen for both spinal cord and retinal neurons. Further, in spinal cord cultures grown on a laminin substrate, there was no decline in neurite outgrowth with increasing developmental age of the neurons as was seen on collagen and polylysine. Neurite length measurements also demonstrated a significant stimulation of neuritogenesis for spinal cord, but not retinal, neurons by laminin compared to polylysine or collagen in 1-day cultures. The results demonstrate tissue-specific differences in the developmental patterns of neurite outgrowth. Retinal neurons appear to have intrinsic changes in their ability to respond to extracellular promoting factors or substrates, while spinal cord neurite outgrowth can be regulated by these extrinsic factors.     

Carbon disulfide inhibits neurite outgrowth and neuronal migration of dorsal root ganglion in vitro

Carbon disulfide (CS?) is a neurotoxic industrial solvent and widely used in the vulcanization of rubber, rayon, cellophane, and adhesives. Although the neurotoxicity of CS? has been recognized for over a century, the precise mechanism of neurotoxic action of CS? remains unknown. In the present study, a embryonic rat dorsal root ganglia (DRG) explants culture model was established. Using the organotypic DRG cultures, the direct neurotoxic effects of CS? on outgrowth of neurites and migration of neurons from DRG explants were investigated. The organotypic DRG cultures were exposed to different concentrations of CS? (0.01 mmol/L, 0.1 mmol/L, 1 mmol/L). The number of nerve fiber bundles extended from DRG explants decreased significantly in the presence of CS? (0.01 mmol/L, 15.00 ± 2.61, p < .05; 0.1 mmol/L, 11.17 ± 1.47, p < .001; 1 mmol/L, 8.00 ± 1.41, p < .001) as compared with that in the absence of CS? (17.83 ± 2.48). The number of neurons migrated from DRG explants decreased significantly in the presence of CS? (0.01 mmol/L, 79.50 ± 9.40, p < .01; 0.1 mmol/L, 62.50 ± 14.15, p < .001; 1 mmol/L, 34.67 ± 7.58, p < .001) as compared with that in the absence of CS? (99.33 ± 15.16). And also, the decreases in the number of nerve fiber bundles and migrated DRG neurons were in a dose-dependent manner of CS?. These data implicated that CS? could inhibit neurite outgrowth and neuronal migration from DRG explants in vitro.     

Tetrodotoxin enhances initial neurite outgrowth from fetal rat cerebral cortex cells in vitro

The effect of two different concentrations of the sodium channel blocker tetrodotoxin on neurite outgrowth from fetal rat cerebral cortex neurons was studied in vitro. A concentration of 10(-6) M tetrodotoxin enhanced after two days in vitro: the percentage of isolated cells and reaggregates which formed neurites, the number of neurites per cell soma, and neurite elongation and subsequent branching. The effects observed after treatment with 10(-7) M tetrodotoxin were on the whole intermediate between control and 10(-6) M tetrodotoxin.     

Age-dependent patterns and rates of neurite outgrowth from CNS neurons on Schwann cell-derived basal lamina and laminin substrata

In the present study, we have examined the growth characteristics of CNS neurons on type I collagen, detergent-treated collagen (dColl), Schwann cell-derived basal lamina (SC-BL), and purified laminin substrata. Neurons from the cerebral cortex, septal basal forebrain, and lumbosacral spinal cord were obtained from embryonic age (E) 15 and E18 rats and grown in vitro as explants on the test substrata. Neurons from either embryonic age displayed radial neurite outgrowth on collagen and dColl substrata. However, pretreatment of collagen with detergents slightly diminished its ability to support neurite outgrowth, as evidence by the 20-40% decrease in the rate of neurite growth on dColl versus the rate calculated for neurons on collagen. In contrast to the similar growth characteristics of E15 and E18 neurons on collagen and dColl, the pattern of neurite outgrowth for CNS neurons on SC-BL and laminin substrata was age dependent. Most E15 neurons grown on SC-BL extended neurites that grew identically to those observed on dColl; these 'non-orienting' neurites maintained a radial orientation to their outgrowth despite encountering interposing channels of SC-BL and grew at rates equal to that calculated for neurons on dColl. E15 neurons placed on laminin substrata showed similar growth patterns and rates equal to that calculated for neurons on dColl. E15 neurons placed on laminin substrata showed similar growth patterns and rates to neurons on collagen. In contrast, neurons from E18 rats exhibited neurites that preferentially grew in intimate association with SC-BL channels once contact with the channels was established. These 'orienting' neurites faithfully elongated within the SC-BL and demonstrated a 1.4- to 2.0-fold increase in growth rate compared with the sister cultures of neurons grown on dColl. Furthermore, E18 neurons exhibited a 1.4-fold increase in growth on laminin compared with E18 neurons grown on collagen. A minor population of neurites exhibiting similar characteristics to orienting neurites was also observed in E15 cultures. It is hypothesized that orienting and non-orienting neurites reflect the outgrowth of 'regenerating' and 'developing' neurons, respectively, and may indicate an inherent difference in the ability of regenerating and developing neurons to recognize and respond to the same guidance signals.     

Adipose-derived stem cells differentiate into a Schwann cell phenotype and promote neurite outgrowth in vitro

Experimentally, peripheral nerve repair can be enhanced by Schwann cell transplantation but the clinical application is limited by donor site morbidity and the inability to generate a sufficient number of cells quickly. We have investigated whether adult stem cells, isolated from adipose tissue, can be differentiated into functional Schwann cells. Rat visceral fat was enzymatically digested to yield rapidly proliferating fibroblast-like cells, a proportion of which expressed the mesenchymal stem cell marker, stro-1, and nestin, a neural progenitor protein. Cells treated with a mixture of glial growth factors (GGF-2, bFGF, PDGF and forskolin) adopted a spindle-like morphology similar to Schwann cells. Immunocytochemical staining and western blotting indicated that the treated cells expressed the glial markers, GFAP, S100 and p75, indicative of differentiation. When co-cultured with NG108-15 motor neuron-like cells, the differentiated stem cells enhanced the number of NG108-15 cells expressing neurites, the number of neurites per cell and the mean length of the longest neurite extended. Schwann cells evoked a similar response whilst undifferentiated stem cells had no effect. These results indicate adipose tissue contains a pool of regenerative stem cells which can be differentiated to a Schwann cell phenotype and may be of benefit for treatment of peripheral nerve injuries.     

ROCK inhibition enhances neurite outgrowth in neural stem cells by upregulating YAP expressionin vitro

Spontaneous axonal regeneration of neurons does not occur after spinal cord injury because of inhibition by myelin and other inhibitory factors. Studies have demonstrated that blocking the Rho/Rho-kinase (ROCK) pathway can promote neurite outgrowth in spinal cord injury models. In the present study, we investigated neurite outgrowth and neuronal differentiation in neural stem cells from the mouse subventricular zone after inhibition of ROCK in vitro. Inhibition of ROCK with Y-27632 increased neurite length, enhanced neuronal differentiation, and upregulated the expression of two major signaling pathway effectors, phospho-Akt and phospho-mitogen-activated protein kinase, and the Hippo pathway effector YAP. These results suggest that inhibition of ROCK mediates neurite outgrowth in neural stem cells by activating the Hippo signaling pathway.     

Effects of calcium ion on neurite outgrowth of rat spinal cord neurons in vitro: the role of non-neuronal cells in regulating neurite sprouting

The interactions of nerve cells with their environment and other cells are specific to different stages of cellular differentiation. Neurite outgrowth was measured from cultured spinal cord neurons under the influence of different Ca²⁺ concentrations. We used fluorodeoxyuridine (FuDr), an antimitotic agent which reduces significantly the proportion of non-neuronal cells in spinal cord cell cultures, to examine the effects of non-neuronal cells on neurite outgrowth. Spinal cord neurons responded to changes in their environment by means of two types of neurite outgrowth: sprouting and elongation. The concurrent presence of non-neuronal cells led to increased sprouting of neurites in certain ionic environments, thus lending support to the idea that non-neuronal cells release diffusible factors which influence sprouting and guide neurite outgrowth.