Ciliary neurotrophic factor stimulates neurite outgrowth from spinal cord neurons

Ciliary neurotrophic factor (CNTF) has been shown to promote the survival of motoneurons, but its effects on axonal outgrowth have not been examined in detail. Since nerve growth factor (NGF) promotes the outgrowth of neurites within the same populations of neurons that depend on NGF for survival, we investigated whether CNTF would stimulate neurite outgrowth from motoneurons in addition to enhancing their survival. We found that CNTF is a powerful promoter of neurite outgrowth from cultured chick embryo ventral spinal cord neurons. An effect of CNTF on neurite outgrowth was detectable within 7 hours, and at a concentration of 10 ng/ml, CNTF enhanced neurite length by about 3- to 4-fold within 48 hours. The neurite growth-promoting effect of CNTF does not appear to be a consequence of its survival-promoting effect. To determine whether the effect of CNTF on spinal cord neurons was specific for motoneurons, we analyzed cell survival and neurite outgrowth for motoneurons labeled with diI, as well as for neurons taken from the dorsal half of the spinal cord, which lacks motoneurons. We found that the effect of CNTF was about the same for motoneurons as it was for neurons from the dorsal spinal cord. The responsiveness of a variety of spinal cord neurons to CNTF may broaden the appeal of CNTF as a candidate for the treatment of spinal cord injury or disease. © 1996 Wiley-Liss, Inc.     

Trophic effect of olmesartan,a novel AT1R antagonist,on spinal motor neurons in vitro and in vivo

Olmesartan is a novel compound which has been shown to exhibit various neuropharmacological effects. For the purpose of clarifying the effect of Olmesartan on spinal motor neurons, we studied the following tests. We studied the effect in vitro of Olmesartan on neurite outgrowth and choline acetyltransferase (ChAT) activity in primary explant cultures of ventral spinal cord (VSCC) of fetal rats. Olmesartan-treated VSCC, compared with control VSCC, had a significant neurite outgrowth and increased activity of ChAT. The effect was dose-related in neurite outgrowth. However, there was no relationship between activity of ChAT andgiven doses of Olmesartan. We examined in vivo the effect of Olmesartan on axotomized spinal motor neuron death in the rat spinal cord. After post-natal unilateral section of sciatic nerve, there was approximately a 50% survival of motor neurons in the fourth lumbar segment. In comparison with vehicle, intraperitoneal injection of Olmesartan for consecutive 14 days reduced spinal motor neuron death. There was no relationship between number of surviving neurons and doses of Olmesartan. These in vitro and in vivo studies showed that Olmesartan has a neurotrophic effect on spinal motor neurons. Our data suggest a potential therapeutic use of Olmesartan in treating diseases that involve degeneration and death of motor neurons, such as motor neuropathy and amyotrophic lateral sclerosis.     

Bone marrow stromal cells promote neurite outgrowth of spinal motor neurons by means of neurotrophic factors in vitro

Transplantation of bone marrow stromal cells (BMSCs) into spinal cord injury models has shown significant neural function recovery; however, the underlying mechanisms have not been fully understood. In the present study we examined the effect of BMSCs on neurite outgrowth of spinal motor neuron using an in vitro co-culture system. The ventral horn of the spinal grey matter was harvested from neonatal Sprague–Dawley rats, cultured with BMSCs, and immunostained for neurofilament-200 (NF-200). Neurite outgrowth of spinal motor neurons was measured using Image J software. ELISA was used to quantify neurotrophic factors such as brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) in culture media, and antibodies or exogenous neurotrophic factors were used to block or mimic the effect of BMSCs on neurite outgrowth, respectively. The results showed that neurite outgrowth significantly increased in spinal motor neurons after co-cultured with BMSCs, while the secretion level of BDNF, GDNF and NGF was dramatically elevated in co-culture. However, the neurite outgrowth-promoting effect of BMSCs was found to significantly reduced using antibodies to BDNF, GDNF and NGF. In addition, a fraction of BMSCs was found to exhibit NF-200 immunoreactivity. These results indicated that BMSCs could promote neurite outgrowth of motor neurons by means of neurotrophic factors. The findings of the present study provided new cues for the treatment of spinal cord injury.     

Trophic effect of Olmesartan,a novel AT1R antagonist,on spinal motor neurons in vitro and in vivo

Abstract

Olmesartan is a novel compound which has been shown to exhibit various neuropharmacological effects. For the purpose of clarifying the effect of Olmesartan on spinal motor neurons, we studied the following tests. We studied the effect in vitro of Olmesartan on neurite outgrowth and choline acetyltransferase (ChAT) activity in primary explant cultures of ventral spinal cord (VSCC) of fetal rats. Olmesartan-treated VSCC, compared with control VSCC, had a significant neurite outgrowth and increased activity of ChAT. The effect was dose-related in neurite outgrowth. However, there was no relationship between activity of ChAT and given doses of Olmesartan. We examined in vivo the effect of Olmesartan on axotomized spinal motor neuron death in the rat spinal cord. After post-natal unilateral section of sciatic nerve, there was approximately a 50% survival of motor neurons in the fourth lumbar segment. In comparison with vehicle, intraperitoneal injection of Olmesartan for consecutive 14 days reduced spinal motor neuron death. There was no relationship between number of surviving neurons and doses of Olmesartan. These in vitro and in vivo studies showed that Olmesartan has a neurotrophic effect on spinal motor neurons. Our data suggest a potential therapeutic use of Olmesartan in treating diseases that involve degeneration and death of motor neurons, such as motor neuropathy and amyotrophic lateral sclerosis. [Neurol Res 2002; 24: 468-472]     

Regulation of sympathetic trophic factors in smooth muscle

The level and nature of trophic activity present in the chicken expansor secundariorum muscle has been shown to be altered by denervation. This muscle receives a dense, sympathetic innervation and contains high concentrations of trophic factors, which were found to be immunologically and functionally distinct from mouse Nerve Growth Factor. In young birds, denervation increased the number of neurons which could be supported by muscle extract. This difference was apparent with regard to E8 to E16 sympathetic neurons. Innervated but not denervated extract was additive with NGF in promoting neurite outgrowth. In contrast, when extracts of denervated and innervated muscle from mature birds were examined, no difference was seen in the number of neurons supported by each extract. However when the denervated and innervated extracts from mature birds were combined more neurons were supported than by a saturating dose of either extract alone. Furthermore, muscle from mature birds responded to denervation only between 2 and 9 days, whereas in young birds the effect was apparent for at least 3 weeks. Analysis of intact, control muscles during the first 8 weeks posthatch demonstrated that the number of neurons that could be supported by the individual extracts varied with the age of the bird. It is concluded that denervation does not in all instances lead to an increase in trophic activity, but does produce a change in the nature of the activity present, such that a different neuronal subpopulation may be supported.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholinergic neuronotrophic factors: I. Survival, neurite outgrowth and choline acetyltransferase activity in monolayer cultures from chick embryo ciliary ganglia

Two key components of neural development and regeneration, survival of the involved neurons and elongation of neuritic elements, are likely to depend on the availability of an appropriate trophic drive to these neurons. At present, only one trophic factor, Nerve Growth Factor, is known to ensure both survival and neuritic growth for its target neurons. A search for a second such agent, a putative cholinergic neuronotrophic factor (CNTF), has been undertaken using as indicators neuronal survival, neurite outgrowth and choline acetyltransferase (CAT) activity in monolayer cell cultures. Eight-day chick embryo ciliary ganglia yielded two monolayer culture systems which appear to be well suited for a CNTF assay. Ciliary ganglionic dissociates, seeded on a highly adhesive collagen substratum, show no neuronal survival by 24 h if the medium is supplemented only with serum or chick embryo extract. However serum and embryo extract combined support survival of, and extensive neuritic outgrowth from, nearly the theoretical number of ganglionic neurons seeded. Alternatively, ciliary ganglionic neurons can be made to survive and produce a profuse neuritic outgrowth on polyornithine-coated dishes if supplied with medium conditioned over chick embryo heart muscle cultures, as already described by other laboratories. The two trophic sources differ markedly in their effects on the ganglionic neurons when tested on collagen or polyornithine substrata, and in some cases when different serum supplements are used. Neuronal survival, neurite production and, possibly, CAT activity appear to be subject to independent regulation. The culture systems used in this study can be developed into quantitative bioassays for the isolation of the different agents responsible for neuronal survival and neurite promotion, and for the investigation of their activities.     

Neurotrophic effects of FPF-1070 (Cerebrolysin®) on cultured neurons from chicken embryo dorsal root ganglia, ciliary ganglia, and sympathetic trunks

We examined the effect of FPF-1070 (Cerebrolysin) on neurite outgrowth in explant cultures of dorsal root ganglia (DRG), sympathetic trunks (ST), and ciliary ganglia (CG) from 10- to 11-day chicken embryos. FPF-1070 significantly promoted neurite outgrowth in DRG and ST neurons at all concentrations examined, in comparison with phosphate buffered saline-treated negative controls; however, this effect on neurite outgrowth was not as significant as that observed for nerve growth factor-treated positive controls on DRG and ST neurons. Additionally, FPF-1070 exhibited an inverted U relationship between concentration and effectiveness in DRG and ST neurons. In contrast, FPF-1070 did not affect neurite outgrowth in CG neurons although ciliary neurotrophic factor-treated positive controls showed striking neurite outgrowth. Our results demonstrate that FPF-1070 has different neurotrophic effects depending on the subpopulation of neurons. This study clarifies a role for neurotrophic activity in the mechanism of action of FPF-1070.     

A subpopulation of embryonic telencephalic neurons survive and develop in vitro in response to factors derived from the periphery

Denervated chick muscle contains factors that enhance neurite outgrowth in cultures of embryonic chicken spinal neurons. Chromatography of muscle extract on a column of DEAE-Sepharose yielded a fraction which retained most of the starting neurite-promoting activity. This DEAE fraction was tested for its activity on neurons from other regions of the central nervous system of 5-day-old chicken embryos. Both neurite outgrowth and survival of telencephalic neurons in vitro were greatly enhanced when the DEAE fraction was added at protein concentrations around 1 microgram/ml. When cultures were prepared from embryos later than 6 days in ovo, the effects of the DEAE fraction progressively diminished with age. Neurons from the embryonic diencephalon, mesencephalon and rhombencephalon were not responsive to the DEAE fraction, although they all developed neurites on a laminin substratum. Similar neurite-promoting activities for telencephalic neurons were found in extracts of neonatal brain, liver and heart, but not lung.     

Skeletal muscle extract and nerve growth factor have developmentally regulated survival promoting effects on distinct populations of mammalian sensory neurons

Neurotrophic factors appear to be relevant to the therapy of degenerative diseases as well as neural regeneration. In this respect, we have investigated the neurotrophic effects of skeletal muscle extract on DRG neuron survival by examining the survival and neurite outgrowth promoting activity of factor(s) present in skeletal muscle extracts (SME) on dissociated cultures of embryonic or early postnatal mouse dorsal root ganglion (DRG) sensory neurons. The numbers of surviving neurons resulting from SME addition increased continuously from embryonic day 13 (15%) to birth (55%), then decreased up to 7 days after hatching (0%). Preliminary characterization of the factor(s) present in SME suggests that the active molecule is a protein different from the known neurotrophic factors NGF, BDNF, NT3, CNTF, and bFGF, and that its neurotrophic effect is not mediated by direct interaction with the substratum. © 1993 John Wiley & Sons, Inc.     

Enhancement of neurotrophic activity in cholinergic cells by hippocampal extract prepared from colchicine-lesioned rats

The effect of hippocampal extract on neurotrophic activity for cultured cholinergic cells was determined. Extract prepared from lesioned hippocampus caused by intrahippocampal infusion of colchicine promoted neuron survival of chick ciliary ganglion cells. The maximal level of survival-promoting activity was reached at 12 days postlesion. The biochemical differentiation of NG108-15 cells was assessed by measuring the activity of choline acetyltransferase (ChAT). After 3 days in culture, hippocampal extract was found to stimulate ChAT activity in a concentration-dependent manner. Hippocampal extract prepared after colchicine lesions elicited a large increase in ChAT-enhancing activity. The effect of hippocampal extract was additive to that of dibutyryl cyclic AMP (db-cAMP). The neurite outgrowth from NG108-15 cells was also potentiated when cells were cultured with hippocampal extract plus db-cAMP. The results suggest that one or several factors in colchicine-lesioned hippocampus promote neurotrophic activity, and the enhancing effect of hippocampal extract on cellular differentiation may act, at least in part, through a mechanism distinct from that for db-cAMP.     

Sprouting by isolated Helisoma neurons: enhancement by glutamate

We tested glutamate for its ability to modulate neurite outgrowth from isolated neurons of the adult snail, Helisoma trivolvis. Although glutamate did not induce neurite outgrowth from neurons maintained in defined medium, nevertheless it showed a dose-dependent ability to enhance the activity of conditioned medium. We concluded that glutamate can enhance the release and/or activity of CNS derived sprouting factor(s) present in conditioned medium. The general conclusion to be drawn from this study is that the ability of a neurotrophic factor(s) to promote neurite outgrowth can be regulated by a neurotransmitter. This mechanism may be important in the regulation of trophic factors in the adult nervous system.     

Neuroprotective utility and neurotrophic action of neurturin in postnatal motor neurons: comparison with GDNF and persephin.

Neurturin and persephin are recently discovered homologs of glial cell line-derived neurotrophic factor (GDNF). Here, we report that neurturin, like GDNF, increases the choline acetyltransferase activity of normal postnatal motor neurons, induces neurite outgrowth in spinal cord, and potently protects motor neurons from chronic glutamate-mediated degeneration. Persephin, in contrast, does not appear to have neurotrophic or neurite-promoting effects on mature motor neurons and may instead worsen the glutamate injury of motor neurons. This pattern in the TGF-beta family suggests certain receptor specificities, requiring at least the Ret/GFRalpha-1 receptor complex. The results predict potential benefit of neurturin, but not persephin, in the treatment of motor neuron disorders and spinal cord diseases.     

Motoneurone survival and neuritic outgrowth promoted by different cell types in embryonic muscle

Different cell types within developing chick skeletal muscle were assayed for their ability to release factors into culture media which could affect the survival and neuritic development of labelled motoneurones and lateral motor column explants. Enriched cultures of myotubes, myoblasts, fibroblasts and mesenchyme were prepared by selective preplating and trypsinisation techniques. Degrees of enrichment were assessed immunofluorescently and morphologically; fibroblasts were the main contaminating cell type. Medium conditioned over each cell type was then tested in dose-response assay against both explants and dissociated motoneurones. In both cases the myotube conditioned medium (MCM) promoted the greatest levels of both survival and neuritic outgrowth, and had the greatest relative potency of all of the cell types. When MCM was preincubated over polycationic substrata, it lost the ability to promote neuritic growth; this could be restored if fresh conditioned medium (CM) was added to the cultures. Thus it was demonstrated that within the MCM there are physically separable agents responsible for neurone survival and neurite expression. The neurite-promoting factor (NPF) within the MCM was stable to collagenase, deoxyribonuclease, neuraminidase and chondroitinase ABC, but was destroyed by trypsin and heparinase. These results imply that a heparan sulfate proteoglycan is essential for the activity of the factor.     

Pigment epithelium-derived factor promotes the survival and differentiation of developing spinal motor neurons.

Pigment epithelium-derived factor (PEDF) is a member of the serine protease inhibitor (serpin) superfamily that has been shown previously to promote the survival and/or differentiation of rat cerebellar granule neurons and human retinoblastoma cells in vitro. However, in contrast to most serpins, PEDF has no inhibitory activity against any known proteases, and its described biological activities do not appear to require the serpin-reactive loop located toward the carboxy end of the polypeptide. Because another serpin, protease nexin-1, has been shown to promote the in vivo survival and growth of motor neurons, the authors investigated the potential neurotrophic effects of PEDF on spinal cord motor neurons in highly enriched cultures and in vivo after injury. Here, it is shown that native bovine and recombinant human PEDF promoted the survival and differentiation (neurite outgrowth) of embryonic chick spinal cord motor neurons in vitro in a dose-dependent manner. A truncated form of PEDF that lacks approximately 62% of the carboxy end of the polypeptide comprising the homologous serpin-reactive loop also exhibited neurotrophic activities similar to those of the full-length protein. Furthermore, the data here showed that PEDF was transported retrogradely and prevented the death and atrophy of spinal motor neurons in the developing neonatal mouse after axotomy. These results indicate that PEDF exerts trophic effects on motor neurons, and, together with previous reports, these findings suggest that this protein may be useful as a pharmacologic agent to promote the development and maintenance of motor neurons. J. Comp. Neurol. 412:506-514, 1999. Published 1999 Wiley-Liss, Inc.     

Choroid coat extract and ciliary neurotrophic factor strongly promote neurite outgrowth in the embryonic chick retina

Previous studies have shown that extracts from the target optic tectum stimulate neurite outgrowth from retinal explants. The present study indicates that the choroid coat is an even richer source of retinotrophic activity. We thus studied the effects of recombinant rat ciliary neurotrophic factor (CNTF) on primary cultures of dissociated chick ciliary ganglion neurons and retinal explants for a comparison with choroid coat extract from the E 18 chick. For our assays, E9 ciliary neurons were incubated in collagen gels and retinal explants were cultured on collagen gels with the addition of the trophic factors and maintained for two or four days. Survival of ciliary neurons per area as well as maximal neurite length in retinal cultures were determined. Growth responses occurred in a dose-dependent manner both to CNTF and choroid extract. Immunofluorescence examination of cells and developing processes showed 200 kdal neurofilament positivity demonstrating that the cells studied were neurons with neurites. It is concluded that a trophic activity of the choroid as well as the recombinant CNTF stimulate retinal neuron survival and neurite extension. The results suggest that CNTF may have developmental functions in the establishment of the visual pathways.     

Screening for neurotrophic disturbances in amyotrophic lateral sclerosis

Neurotrophic activities in human serum and post-mortem muscle and spinal cord of possible relevance to pathophysiological mechanisms in amyotrophic lateral sclerosis (ALS) were studied. Tests included in vitro assays for nerve fibre outgrowth from sympathetic ganglia and for survival promotion of dissociated ciliary neurons, both types of neurons, of chicken embryo origin. Extracts of postmortem biceps muscle promoted survival of ciliary neurons in a dose-dependent manner. Half-maximum effect was found at a protein concentration of about 450 micrograms/ml for both ALS and control muscle. Ventral horn extracts were about 5 times as efficient as muscle in promoting neuron survival, again with no differences seen between control and ALS samples. Sera from patients suffering from ALS as well as normal sera did not enhance survival of ciliary neurons to any considerable extent, nor did they induce fibre outgrowth from sympathetic ganglia. Both groups of sera, if present above 5% in the medium, suppressed fibre outgrowth induced by added nerve growth factor (NGF). Sera from some of the ALS patients impaired survival in dissociated ciliary neurons supported by a trophic activity in choroid extract. The results do not indicate major neurotrophic deficits as the cause of ALS disease but suggest that a neurotoxic mechanism may be involved.     

The L2/HNK-1 Carbohydrate Epitope is Involved in the Preferential Outgrowth of Motor Neurons on Ventral Roots and Motor Nerves

Based on the observation that in adult mice the carbohydrate epitope L2/HNK-1 is detectable on Schwann cells in ventral spinal roots, but only scarcely in dorsal roots (Martini et al., Dev. Biol., 129, 330 - 338, 1988), the possibility was investigated that the carbohydrate is involved in the outgrowth of regenerating motor neuron axons on peripheral nerve substrates expressing the epitope. To monitor whether the L2 carbohydrate remains present during the time periods in which regenerating axons penetrate the denervated distal nerve stumps, the expression of L2 in motor and sensory branches of the femoral nerve was investigated in normal animals and after a crush lesion. During the first two postoperative weeks, L2 immunoreactivity remained high in the myelinating Schwann cells of the motor branch, whereas L2 immunoreactivity was virtually absent in the sensory branch. In a first experimental approach, cryosections of ventral and dorsal spinal roots and of motor and sensory nerves of adult rats and mice were used as substrates for neurite outgrowth. Neurites of motor neurons from chicken embryos were approximately 35% longer after 30 h of maintenance on ventral roots than on dorsal roots. Neurites from sensory neurons had the same length on dorsal as on ventral motors and were as long as neurites from motor neurons grown on dorsal roots. L2 antibodies reduced neurite outgrowth of motor neurons on ventral roots but not on dorsal roots. Neurite outgrowth of sensory neurons on both roots was not altered by the antibodies. Neurite outgrowth of motor neurons on a mixture of the extracellular matrix glycoprotein laminin and the L2 carbohydrate-carrying glycolipid was significantly higher than on the laminin substrate mixture with GD1b ganglioside or sulphatide. L2 antibodies reduced neurite outgrowth of motor neurons by 50% on the L2 glycolipid, but not on GD1b or sulphatide. These observations indicate that the L2 carbohydrate promotes neurite outgrowth of motor neurons in vitro and may thus contribute to the preferential reinnervation of motor nerves by regenerating motor axons in vivo.     

Neurotrophic activity of S-100 beta in cultures of dorsal root ganglia from embryonic chick and fetal rat

We report here that S-100 beta, a protein with neurotrophic activity on central nervous system neurons, stimulates neuritic outgrowth from cultures of dorsal root ganglia (DRG). S-100 beta elicited neurites from explant and dissociated cell cultures of embryonic chick DRG, and the extent of the response varied with the age of the embryo. Specificity was demonstrated by the observation that incubation of S-100 beta with antibodies directed against S-100 beta reduced the neurite outgrowth, whereas incubation of S-100 beta with normal rabbit serum had little effect. S-100 beta also stimulated the area of neuritic outgrowth from organotypic cultures of fetal rat DRG, showing that the activity of the protein is not restricted to a particular species or culture condition. A mutant S-100 beta lacking neurotrophic activity on cerebral cortex neurons was unable to effectively stimulate neurite outgrowth from DRG cultures. These studies suggest that S-100 beta may play a role in neuronal growth and/or maintenance in the peripheral nervous system.     

Factors affecting denervation-like changes at the neuromuscular junction during aging

The ability to sustain synaptic transmission diminishes with aging. To determine whether this is accompanied by alterations in the structure of the synapse, end-plate architecture was examined in EDL muscles from 10- to 25-month rats. There was a significant age-related increase in end-plate area and a decrease in the number of nerve terminals per end plate. Furthermore, the percentage of end plates with ultraterminal sprouts increased dramatically with age. To compare the morphological changes associated with aging to the changes in response to denervation, EDL muscles from 10- and 25-month animals were partially denervated. Both end-plate area and ultraterminal sprouting increased, while terminal number decreased following denervation in the 10-month muscles. To determine whether the age- and denervation-associated changes were accompanied by alterations in muscle-derived nerve-outgrowth factors, in vitro assays were performed. Neurite outgrowth was quantified from embryonic motoneurons incubated with muscle extract, or grown on cryostat-cut muscle cross sections from 10- and 25-month innervated and denervated EDL muscles. Both aged and denervated muscles induced greater degrees of neurite outgrowth compared with younger innervated muscles. Innervation to the EDL was then examined, and signs of axonal degeneration were observed. It is suggested that aging is associated with alterations in the motor axon to the EDL muscle. These changes are manifest at the neuromuscular junction. In turn, the muscle responds as if it were in a state of functional denervation.