Axotomy enhances the outgrowth of neurites from embryonic rat septal-basal forebrain neurons on a laminin substratum

Previous studies have suggested that embryonic (nonaxotomized) and regenerating central nervous system neurons differentially respond to the same substrata. In the present study, we have used an in vitro model to test the ability of laminin and type I collagen to promote the outgrowth of neurites from nonaxotomized and axotomized, embryonic septal-basal forebrain (SBF) neurons. Neurons within explants derived from Embryonic Day (E) 15 rats extended neurites that demonstrated similar growth characteristics on a collagen or laminin substratum. E15 neurons could be induced to extend longer neurites on laminin if they were axotomized in vitro and subsequently replated onto a laminin substratum. The carbocyanine dye DiI indicated that neurons which were axotomized could survive and regenerate processes. These regenerating neurites grew 27% longer on laminin than they did on collagen. Similarly, neurons that were axotomized in situ, i.e., E18 SBF neurons, extended neurites that were 29% longer on a laminin substratum. In contrast, E15 explants that were maintained in suspension culture prior to being plated onto a substratum exhibited similar growth on laminin or collagen. The increase in regeneration by E15 neurons on laminin was augmented, by 22%, if nerve growth factor was supplemented to the culture medium. These results demonstrate that laminin is a better substratum, as compared to collagen, for the elongation of neurites from axotomized SBF neurons. Nonaxotomized neurites, on the other hand, do not appear to prefer one substratum over the other. Furthermore, regeneration from embryonic, SBF neurons on laminin is augmented if NGF is used simultaneously.     

Secretion of nerve growth factor from septum stimulates neurite outgrowth and release of the amyloid protein precursor of Alzheimer's disease from hippocampal explants

Alzheimer's disease (AD) is characterized by the deposition of amyloid in the extracellular and intracellular compartments of the cerebral cortex. The extracellular amyloid consists of a protein (βA4) which is derived from a larger precursor, the amyloid protein precursor (APP). Several studies have implicated APP in the regulation of neurite outgrowth during development, although the precise function of APP remains unknown. To examine the role of APP in the regulation of neutrite outgrowth from hippocampal neurons, an explant culture system was developed. Explants of E18 mouse hippocampus were found to extend neurites when co-cultured with explants of E18 mouse septum. This finding demonstrated that the septum can release a neurite outgrowth-promoting factor (NOPF). As nerve growth factor (NGF) was also able to stimulate neurite outgrowth from the hippocampal explants, this suggested that the NOPF might be NGF. Immunoprecipitation of NGF from septal conditioned medium using a specific monoclonal antibody (27/21) completely blocked the neurite outgrowth-promoting effect, supporting this conclusion. Concomitant with its ability to stimulate neurite outgrowth, NGF stimulated the release of APP from the hippocampal explants. As previous studies have suggested that the binding of APP to heparan sulfate proteoglycans (HSPGs) in the extracellular matrix might be an important step in the regulation of neurite outgrowth by NGF, we examined the effect of APP on neurite outgrowth from dissociated hippocampal cells cultured on various protein substrates. When cells were cultured on a substrate of APP and HSPG, neurite outgrowth was markedly stimulated. No stimulation of neurite outgrowth was seen when neurons were cultured on substrates of either APP or HSPG alone. The results suggest that secreted forms of APP may be involved in stimulating neurite outgrowth from hippocampal neurons and that interactions between APP and HSPG may be important for a neurite outgrowth-promoting function. © 1994 Wiley-Liss, Inc.     

Effects of nerve growth factor and heart cell conditioned medium on neurite regeneration of aged sympathetic neurons in culture

The effects of nerve growth factor (NGF) and heart-cell-conditioned medium (HCM) on the neurite regeneration of aged sympathetic neurons were investigated in culture. Investigation of HCM was carried out by two different methods: one was the use of whole HCM on collagen substratum, which reflected component(s) effective in solution (HCM-S); the other was the use of polyornithine (PORN)-binding component(s) (P-HCM). Superior cervical ganglion neurons prepared from male mice from 6 to 30 months of age were cultured in MEM-10% FCS on collagen or gelatin-PORN substratum for 3 days. The number of neurons with neurites and the length of neurites were quantified as neurite production and elongation, respectively. Neuronal survival was not affected by addition of NGF, HCM-S or P-HCM. Neurite production of early adult neurons was enhanced by NGF, HCM-S or P-HCM. In contrast, neurite production of aged neurons was enhanced by only HCM-S, but not NGF or P-HCM. HCM-S did not promote neurite elongation in neurons at any age. Neurite elongation of early adult neurons was enhanced by NGF or P-HCM. Neurite elongation of aged neurons was enhanced by P-HCM. However, responsiveness of NGF for neurite elongation varied according to substrata. No age-related difference was found in neurite production and elongation in the absence of NGF, HCM-S or P-HCM. These results indicate that responsiveness of aged sympathetic neurons is various in different growth factors.     

A conditioning lesion enhances sympathetic neurite outgrowth

Axonal regeneration can be influenced by a conditioning lesion (an axonal injury made prior to a second test lesion). Previously, sympathetic neurons in vivo were shown to respond to a conditioning lesion with decreased neurite outgrowth, in contrast to the enhanced outgrowth observed in all other peripheral neurons examined. The present experiments tested the effects of a conditioning lesion on neurite outgrowth in vitro from the superior cervical ganglion (SCG) and the impact of several factors on that response. Ganglia axotomized 1 week earlier and then explanted in Matrigel or collagen gel responded with a significant increase in neurite extension compared to sham-operated ganglia. A distal axotomy produced by unilateral removal of the salivary glands (sialectomy) caused an increase in neurite outgrowth similar to that of a proximal axotomy. These conditioning lesions induced both an increase in the rate of elongation, and, in the case of the proximally axotomized SCG, a shorter initial delay of outgrowth. The enhanced outgrowth following sialectomy was specific to the nerve containing the majority of axons projecting to the salivary glands, suggesting that the conditioning lesion effect is restricted to previously injured neurons. Deletion of the gene for leukemia inhibitory factor (LIF), a gene induced by axotomy, did not abolish the conditioning lesion effect in SCG explants or dissociated cell cultures. In conclusion, sympathetic neurons are capable of responding to a conditioning lesion with increased neurite outgrowth. The hypothesis that the neuronal cell body response to axotomy plays an important role in the conditioning lesion response is discussed.     

Serum- and substratum-dependent modulation of neuritic growth

Explants of embryonic day 8 (E8) chicken dorsal root ganglia (DRG) have been cultured with medium containing serum or the serum-free supplement N1 on one of three substrata: collagen, polyornithine (PORN), or PORN exposed to a polyornithine-binding neurite-promoting factor (PNPF-PORN). Replicate cultures were maintained with or without nerve growth factor (NGF). NGF elicited its classical neuritic outgrowth on all three substrata in serum-containing or serum-free medium. In the absence of NGF, however, a gradation of increasing neurite growth was seen with: PNPF-PORN greater than PORN greater than collagen. This response occurred in both media. In addition, the neuritic halo in each instance was markedly more developed in the absence of serum, especially on PNPF-PORN. Nonneuronal behaviors reflected both serum and substratum influences: thus, nonneuronal outgrowth consisted mainly of flat cells with serum and collagen, was nonexistent with serum and PORN or PNPF-PORN, and involved mostly Schwann-like scattered cells in the absence of serum on any one substratum. The serum-dependent behaviors of ganglionic neurites were examined further with explants from chicken E11 sympathetic ganglia. A single substratum was used (PORN), without exogenous trophic factor. Neurite outgrowth was depressed by the presence of fetal calf serum, thus supporting the generality of this phenomenon. Lastly, PC12 cells, a clonal line of rat pheochromocytoma, will grow neurites in the presence of NGF after 48 hr in serum-free, but not serum-containing media. Addition of serum to serum-free cultures at this time results in the rapid and complete retraction of neurites.     

Neurite outgrowth and GAP-43 mRNA expression in cultured adult rat dorsal root ganglion neurons: Effects of NGF or prior peripheral axotomy

Adult dorsal root ganglion (DRG) cells are capable of neurite outgrowth in vivo and in vitro after axotomy. We have investigated, in cultured adult rat DRG cells, the relative influence of nerve growth factor (NGF) or a prior peripheral nerve lesion on the capacity of these neurons to produce neurites. Since there is evidence suggesting that the growth-associated protein GAP-43 may play a crucial role in axon elongation during development and regeneration, we have also compared the effect of these treatments on GAP-43 mRNA expression. NGF increased the early neurite outgrowth in a subpopulation of DRG cells. This effect was substantially less, however, than that resulting from preaxotomy, which initiated an early and profuse neurite outgrowth in almost all cells. No difference in the expression of GAP-43 mRNA was found between neurons grown in the presence or absence of NGF over 1 week of culture, in spite of the increased growth produced by NGF. In contrast, cultures of neurons that had been preaxotomized showed substantial increase in GAP-43 mRNA and NGF had, as expected, a significant effect on substance P mRNA levels. Two forms of growth may be present in adult DRG neurons: an NGF-independent, peripheral nerve injury-provoked growth associated with substantial GAP-43 upregulation, and an NGF-dependent growth that may underlie branching or sprouting of NGF-sensitive neurons, but which is not associated with increased levels of GAP-43 mRNA. © 1994 Wiley-Liss, Inc.     

Extension of sympathetic neurites in vitro towards explants of embryonic and neonatal mouse heart and stomach: ontogeny of neuronotrophic factors

In order to establish when target organs first produce neuronotrophic factors, extension of neurites in vitro from sympathetic ganglia (superior cervical and coeliac) of 1-day neonatal mice towards explants of 10-, 11-, 14- and 17-day embryonic and 1-day neonatal atrium and stomach was examined in co-cultures. Longer neurites extended from ganglia towards, than away from, atrial targets at all stages examined, and was most marked towards 17-day embryonic and neonatal explants. Treatment of atrial co-cultures with antiserum to nerve growth factor (NGF) almost totally blocked preferential neurite outgrowth. Directional growth of neurites towards stomach explants in co-cultures was not as pronounced as that towards atrium; extension of neurites was most marked when stomach was provided by 11-, 14- and 17-day embryos. Such outgrowth was only partially blocked by antiserum to NGF, significant preferential extension of neurites towards stomach persisting in the presence of the antiserum. These results indicate that atrium and stomach produce neuronotrophic factors from the earliest ages studied; the evidence indicates that in the case of atrium, NGF predominates but that stomach produces NGF as well as another factor immunologically distinct from NGF. It is of interest that both types of target explanted before they receive sympathetic innervation show evidence of producing NGF in culture.     

Raphe-spinal neurons display an age-dependent differential capacity for neurite outgrowth compared to other brainstem-spinal populations

Functional regeneration of brainstem-spinal pathways occurs in the developing chick when the spinal cord is severed prior to embryonic day (E) 13. Functional spinal cord regeneration is not observed in animals injured after E13. This developmental transition from a permissive to a restrictive repair period may be due to the formation of an extrinsic inhibitory environment preventing axonal growth, and/or an intrinsic inability of mature neurons to regenerate. Here, we investigated the capacity of specific populations of brainstem-spinal projection neurons to regrow neurites in vitro from young (E8) versus mature (E17) brainstem explants. A crystal of carbocyanine dye (DiI) was implanted in ovo into the E5 cervical spinal cord to retrogradely label brainstem-spinal projection neurons. Three or 12 days later, discrete regions of the brainstem containing DiI-labeled neurons were dissected to produce explant cultures grown in serum-free media on laminin substrates. The subsequent redistribution of DiI into regenerating processes permitted the study of in vitro neurite outgrowth from identified brainstem-spinal neurons. When explanted on E8, i.e., an age when brainstem-spinal neurons are normally elongating through the spinal cord and are capable of in vivo functional regeneration, robust neurite outgrowth was observed from all brainstem populations, including rubro-, reticulo-, vestibulo-, and raphe-spinal neurons. In contrast, when explanted on E17, robust neurite outgrowth was seen only from raphe-spinal neurons. Neurite outgrowth from raphe-spinal neurons was 5-hydroxy-tryptamine immunoreactive. This study demonstrates that in growth factor-free environments with permissive growth substrates, neurite outgrowth from brainstem-spinal neurons is dependent on both neuronal age and phenotype.     

Cooperation between nerve growth factor and laminin or fibronectin in promoting sensory neuron survival and neurite outgrowth

Chick embryo dorsal root ganglion (DRG) neurons were purified by differential adhesion to plastic. The purified neurons were used to study the cooperation between nerve growth factor (NGF) and laminin or fibronectin in promoting neuron survival and neurite outgrowth. NGF alone supported the survival of only 20% embryonic day 10 (E10) cells, of which only 40-50% had neurites. Treatment of the substrate with fibronectin or laminin increased survival in the presence of NGF up to 80% of the seeded neurons, all of which showed extensive neurite outgrowth. Survival and neurite outgrowth were also enhanced by the combined effects of elevated potassium and laminin. In contrast to E8-10 cells, 85% of E16 neurons survived in the basal culture conditions, i.e. without additional NGF, fibronectin or laminin, although neurite outgrowth was enhanced by all 3 proteins. Antisera to NGF, laminin and fibronectin, each independently decreased survival and neurite outgrowth of DRG neurons, totally with E9 and partially with E16 cells. The results suggest that the cooperative actions of extracellular matrix proteins and NGF are essential for survival and neurite outgrowth of embryonic DRG neurons and that these neuronal requirements change during development.     

Regeneration of axons from adult human retina in vitro

In an effort to establish an in vitro model of regenerating adult human central nervous system (CNS) neurons, we have investigated the potential for neurite growth from explants prepared from normal adult human retina. Eyes (donated for corneal transplantation) were removed within 2.0 h postmortem and stored on ice for 1.5 to 7.0 days. Retinal explants (1 mm2) were prepared and cultured at 37 degrees C on cellular or acellular substrata in an oxygen-rich, humidified atmosphere. Neurite outgrowth, visualized by neurofilament immunofluorescence, was observed only in the presence of Schwann cells, after a quiescent period of approximately 6 days in vitro. Of 50 explants cultured for 7 days or more on substantia containing Schwann cells, 43 showed evidence of viability in vitro and 28 extended neurites onto Schwann cell surfaces. Estimated rates of neurite growth on Schwann cell substrata reached a maximum of 0.22 mm/day. Neurites did not grow beyond the explant border onto culture substrata composed of either polylysine, laminin, type-I collagen, or monolayers of adult human retinal glia. These results demonstrate that under selected conditions, explants prepared from adult human retina harbor viable neurons and that Schwann cells promote and support regeneration of neurites from these neurons in vitro, allowing systematic analysis of conditions favorable to axonal regeneration from adult human CNS neurons.     

Growth stimulation and chemotropic attraction of rat retinal ganglion cell axons in vitro by co-cultured optic nerves, astrocytes and astrocyte conditioned medium

The effects of explants of optic nerves of different ontogenetic ages (P0-P14, adult), and of cultured astrocytes of various ages on the neurite regeneration of rat retinal ganglion cells (RGC) were assessed in vitro, using a three-dimensional culture system which allows the co-cultivation of various explants. Both co-cultured P0-P12 optic nerves and astrocyte cultures from P2 cerebral cortex stimulated the regeneration of neurites from the retinal explants after 3 days in culture. By contrast, P14 and older explants of the optic nerve, astrocytes from P17 optic nerve and astrocytes that had previously been grown in culture for more than 6 weeks had no effect on RGC neurite outgrowth. Moreover, both the P0–P12 optic nerve explants and the astrocytes from P2 cerebral cortex also seemed to have a chemotropic effect on the regenerating neurites, because the latter were longer on the side facing the co-explantat. The absence of a cellular bridge between retinal and optic nerve explants suggests that the effects are mediated by astroglia-derived diffusible neurite growth promoting factors. Accordingly, astrocyte-conditioned medium from P2 astrocytes also stimulated the outgrowth of neurites from the retinal explants. These findings show that immature astrocytes of a limited ontogenetic period release as yet unknown diffusible neurite growth-promoting factors which stimulate the regeneration of neurites from retinal explants.     

Influence of nerve growth factor on developing dorso-medial and ventro-lateral neurons of chick and mouse trigeminal ganglia

Trigeminal ganglia have been removed from five, six, seven and eight day chick embryos and explants of the dorso-medial (DM) and ventro-lateral (VL) parts of the maxillomandibular lobe were grown in tissue culture. Quantitative methods were used to assess the influence of nerve growth factor (NGF) on fiber outgrowth from these explants. At all ages outgrowth from DM explants was significantly greater than from VL explants, the difference being most pronounced between the extreme DM and VL poles of the maxillomandibular lobe. These observations are interpreted as indicating the existence of two distinct populations of neurons in terms of their response to NGF rather than the consequence of the asynchronous differentiation and maturation of the VL and DM neurons. A similar study of 10, 11 and 12 day embryonic mouse trigeminal ganglia revealed no significant difference in neurite outgrowth between DM and VL regions grown in the presence or absence of NGF.     

Schwann cell surfaces but not extracellular matrix organized by Schwann cells support neurite outgrowth from embryonic rat retina

Despite evidence that glial cell surfaces and components of the extracellular matrix (ECM) support neurite outgrowth in many culture systems, the relative contributions of these factors have rarely been compared directly. Specifically, it remains to be determined which components of peripheral nerve support growth of central nerve fibers. We have directly compared neurite outgrowth from embryonic day 15 rat retinal explants placed onto beds of (1) Schwann cells without ECM, (2) Schwann cells expressing ECM (including a basal lamina), (3) cell-free ECM prepared from neuron-Schwann cell cultures, (4) nonglial cells (fibroblasts), and (5) 2 isolated ECM components, laminin and type I collagen. From the first day in culture, retinal explants extended neurites when placed on Schwann cells without ECM. Outgrowth on Schwann cells expressing ECM was also extensive, but not obviously different form that on Schwann cells alone. Ultrastructural study revealed that 95% of retinal neurites in ECM-containing cultures contacted other neurites and Schwann cell surfaces exclusively. On cell-free ECM prepared from neuron-Schwann cell cultures, neurite extension was poor to nonexistent. No neurite outgrowth occurred on fibroblasts. Retinal explants also failed to extend neurites onto purified laminin and ammoniated type I collagen substrata; however, growth was rapid and extensive on air-dried type I collagen. In cultures containing islands of air-dried type I collagen on a laminin-coated coverslip, retinal explants attached and extended neurites on collagen, but these neurites did not extend off the island onto the laminin substratum. We conclude from these experiments that neurite extension from embryonic rat retina is supported by a factor found on the surface of Schwann cells and that neither organized nor isolated ECM components provide this neurite promotion. These findings are discussed in relation to possible species differences in growth requirements for retinal ganglion cell neurites and to the specificity of response of different CNS neurites to ECM substrata.     

Lymphoid tissues induce NGF-dependent and NGF-independent neurite outgrowth from rat superior cervical ganglia explants in culture

Induction of neurite outgrowth from superior cervical ganglia (SCG) by rat lymphoid tissues was studied using a tissue culture model. Neonatal rat SCG were cultured with 6–12-week-old rat thymus, spleen, or mesenteric lymph node (MLN) explants in a Martrigel layer, in defined culture medium without exogenous nerve growth factor (NGF). SCG were also co-cultured with neonatal rat heart (as positive control) or spinal cord (SC; as negative control). To determine whether inflammation affects the ability of lymphoid tissues to induce neurite outgrowth, we also examined MLN at various times after infecting rats with Nippostrongylus brasiliensis (Nb-MLN). In one series of experiments, a single lymphoid tissue explant was surrounded by four SCG at a distance of 1 mm. The extent of neurite outgrowth was determinded by counting the number of neurites 0.5 mm away from each ganglion at several time points. Adult thymus and, to a lesser extent, spleen had strong stimulatory effects on neurite outgrowth from SCG after 12 hr or more in culture. For thymus tissue, this was similar to the positive control heart explants. MLN from normal rats had minimal effect on neurite outgrowth; however, Nb-MLN showed a time-dependent enhancement of the neurite outgrowth, maximal at 3 weeks after infection. The relative efficacy of neurite outgrowth induction (heart ≥ thymus ≥ Nb-MLN ≥ spleen ≥ MLN ≥ SC) was confirmed in a second series of experiments where one SCG was surrounded by three different tissue explants. We then examined the role of 2.5S NGF, a well-known trophic factor for sympathetic nerves, in the lymphoid tissue-induced neurite outgrowth. Anti-NGF treatment of co-cultures of SCG and heart almost completely blocked the neurite outgrowth. Anti-NGF also significantly inhibited thymus- and spleen-induced neurite outgrowth, but not as effectively as heart-induced neuritogenesis (93,80, and 77% inhibition at 24 hr; 86,70, and 68% inhibition at 48 hr for heart, thymus, and spleen, respectively). On the other hand, anti-NGF inhibited only 8% of neurite outgrowth induced by 3-week post-infection Nb-MLN at 24 hr, and 41% at 48 hr. These data show that several adult rat lymphoid tissues exert neurotrophic/tropic effects. The predominant growth factor in thymus and spleen is NGF, while Nb-MLN produces factor(s) which is (are) immunologically distinguishable from NGF. These neurotrophic/tropic factors are produced during the reactive lymphoid hyperplasia that forms part of the inflammatory response against the nematode, N. brasiliensis. This suggests the possibility that cytokines produced by lymphocytes or other inflammatory cells may stimulate sympathetic neurite outgrowth in vivo. © 1994 Wiley-Liss, Inc.     

Axotomized frog sciatic nerve releases diffusible neurite-promoting factors.

Using the bullfrog (Rana catesbeiana) dorsal root ganglia (DRG) and its sciatic nerve (ScN) as a model system, we have previously described neuronal and non-neuronal molecular changes associated with the early regenerative response of DRG neurons to axotomy. Since diffusible molecular factors, released by axotomized ScN, might function to stimulate axon regrowth, we have assayed the ability of ScN-conditioned bath to promote in vitro neurite outgrowth from PC-12 cells. Diffusible ScN proteins were collected by incubating segments of normal or axotomized ScN in a small volume of RPMI media for 4 h (nerve bath). The nerve baths, supplemented with serum, were then added to PC-12 cell cultures to assay for the presence of neurite growth factors released by ScN. Results showed that nerve baths, collected from sham-operated or axotomized ScN, could not induce the differentiation of PC-12 into neurite-bearing cells. Therefore, in all subsequent neurite growth assay experiments, an exogenous source of nerve growth factor (NGF) (50 ng/ml) was added to the nerve baths or unconditioned media to generate and maintain PC-12 neuritic structure. We found that nerve baths, collected from previously axotomized (at least 3 days post-injury) nerve, contained diffusible factors which enhanced PC-12 neurite growth, relative to unconditioned media. No neurite growth factors were observed to be released by sham-operated ScN or 1-day post-axotomized ScN. Further experiments were conducted to identify the diffusible neurite growth factors released from axotomized ScN. We showed that the release (if any) of endogenous diffusible NGF or laminin from axotomized nerve could not have accounted for the facilitation of neurite growth. Analysis of radiolabelled ScN proteins by two-dimensional polyacrylamide gel could not have accounted for the facilitation of neurite growth. Analysis of radiolabelled ScN proteins by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) showed that the relative abundance of two diffusible proteins (M(r) approximately 35 and 70 kDa) in the nerve bath was directly correlated with the ability of the nerve bath to facilitate PC-12 neurite growth.     

Neurotrophic stimulation of fetal rat retinal explant neurite outgrowth and cell survival: age-dependent relationships

Serum-free tissue culture conditions have been defined where stimulation of neurite outgrowth from fetal rat retinal explants occurred only in the presence of an active fraction (BE) prepared from a pig brain extract purification procedure. Under these conditions, 18-20-day fetal retinal explants survived and continued to extend long radial neurites for at least 3 weeks in the presence of BE. However, if fibronectin was not equilibrated onto the basic collagen/poly-L-lysine substrate the neurite outgrowth was restricted to a short halo about the circumference of the explant. In addition, a dose-response relationship was demonstrated in the presence of increasing concentrations of BE with respect to the neurite growth index. The half-maximal response for BE was estimated to be between 5 and 10 micrograms/ml. In addition a number of important age-dependent relationships were observed with respect to BE stimulation of retinal neurite outgrowth and cell survival. An inverse relationship was demonstrated between increased developmental age and responsiveness to BE. After 1 week in culture, there was a 3-fold reduction in retinal neurite length measured from the 2-day neonatal explant when compared to that of the 18-day fetus. There was also a significant inverse relationship demonstrated between the length of time before BE was added to the culture medium and the ability of 20-day fetal explants to extend neurites onto the culture substrate. If BE was added as late as 2 weeks after initial explant culture, the various neurite outgrowth indices were significantly lower than in those situations where BE was added at the time of culture or 1 week later. These results imply that BE not only is required for stimulating neurite outgrowth from fetal rat retinal explants, but may be important in survival and maturation of developing retinal neurons. This hypothesis was confirmed when morphometric analysis was performed on 16- and 20-day explants cultured for a week in the presence or absence of BE. The number of necrotic cells in the developing retinal ganglion plexiform-cell layer of 20-day fetal explants was significantly lower when treated with BE. Conversely, the density of identifiable differentiating retinal ganglion-like cells was significantly greater in response to BE treatment in both 16- and 20-day retinal explants.     

Laminin supports neurite outgrowth from explants of axotomized adult rat retinal neurons

The influence of laminin on neurite outgrowth from explants of adult rat retina and its distribution in normal and lesioned rat optic nerves were examined. Neurite outgrowth required the presence of laminin in the substratum, and as with a goldfish retinal explant system, was markedly stimulated by prior axotomy. Except for blood vessels and the nerve sheath, normal rat optic nerve was devoid of laminin immunoreactivity. Unlike results seen in the goldfish optic nerve, injury to the rat optic nerve induced no observable increase in laminin content or change in its distribution. The differences in the in vivo regenerative capacities of these two species may in part be related to the differences in their abilities to provide a proper substratum for axon regrowth.     

Retinal ganglion cell response to axotomy and nerve growth factor antiserum treatment in the regenerating visual system of the goldfish (Carassius auratus): An in vivo and in vitro analysis

In vitro nerve growth factor (NGF) antiserum (anti-NGF) treatment was found to significantly depress retinal ganglion cell neurite outgrowth in goldfish explant culture. Goldfish retinas, conditioned by a 14-day prior optic nerve crush, demonstrated a significant dose response inhibition of neurite outgrowth if incubated with various concentrations of the antiserum (i.e. concentrations from full strength to 1:100) before explanation for tissue culture. NGF added to the incubation medium containing antiserum partially eliminated the inhibition of neurite outgrowth during the first 4 days of explant culture. Antiserum treatment at the higher concentrations (i.e. full strength and 1:1.5 dilution) caused a cessation of nerve growth from explants between culture days 3 and 4. However, controls at this time still exhibited vigorous neurite outgrowth.In vivo treatment with anti-NGF administered intraocularly at 7 days after optic nerve crush (i.e. 7 DPA) was found to significantly reduce the size and complexity of retinal ganglion cell nucleoli when analyzed morphometrically at 14 DPA. No other cell parameters measured (i.e. cell size, nuclear size, cell/nuclear ratios and mitochondrial, Golgi and RER densities) were found to be affected by the single antiserum treatment.     

Neurotrophic effect of hepatocyte growth factor on central nervous system neurons in vitro

Although the expression of hepatocyte growth factor (HGF) and its receptor, proto-oncogene c-met, has been demonstrated in the central nervous system (CNS), the function of HGF in the CNS was not fully understood. In the present studies, we determined the effects of HGF on neuronal development in neocortical explant and mesencephalic neurons obtained from embryonic rat brain. HGF clearly enhanced neurite outgrowth in neocortical explants. In the mesencephalic culture, the number of tyrosine hydroxylase (TH)-positive neurons was significantly higher in the HGF-treated wells and the neurites of the TH-positive neurons appear to be more developed. Moreover, the dopamine uptake into mesencephalic neurons was also enhanced by HGF treatment, indicating that HGF promotes the survival and/or maturation of mesencephalic dopaminergic neurons. In both neocortical explants and mesencephalic neurons, c-met autophosphorylation was induced by HGF and MAP kinase activation was also detected in the neocortical explant. Furthermore, Western blot analysis of the cultured CNS cells revealed that HGF was expressed mainly in microglia. These results suggest that HGF from microglia has neurotrophic activity on the CNS neurons and plays significant roles in the development of the CNS. © 1996 Wiley-Liss, Inc.     

Proteasome inhibition arrests neurite outgrowth and causes "dying-back" degeneration in primary culture

Proteasome inhibitors such as lactacystin were first isolated when assaying their ability to stimulate neurite outgrowth in neuronal-like cell lines; however, their effect on neurites in primary culture has been largely neglected. We report here that lactacystin causes immediate arrest of nerve growth factor (NGF)-stimulated neurite outgrowth in sympathetic and sensory explant cultures. This is followed by neurite degeneration that in sympathetic cultures has a distinctive "dying-back" morphology. Remarkably, this occurs even at concentrations below that required to induce neurite outgrowth in PC12 cells. Thus, lactacystin opposes rather than potentiates the effect of NGF on sympathetic neurite outgrowth and the role of the ubiquitin proteasome pathway in growth and long-term maintenance of axons and dendrites differs from that in neuritogenesis in neuronal-like cell lines. Retrograde degeneration caused by blocking of the ubiquitin proteasome pathway may mimic some aspects of gracile axonal dystrophy, a dying-back axonopathy in mice caused by ubiquitin hydrolase (Uch-l1) deficiency, and may be relevant to human neurodegenerative diseases involving ubiquitination or proteasome abnormalities.