Vasoactive intestinal peptide influences neurite outgrowth in cultured rat spinal cord neurons.

Vasoactive intestinal peptide (VIP) is a neuropeptide which has been shown to exhibit a wide range of neurotrophic effects both in vivo and in vitro. For the purpose of clarifying the effect of VIP on spinal cord neurons, we studied the effect of VIP on neurite outgrowth of fetal rat ventral and dorsal portions of spinal cord in cultures. VIP-treated ventral spinal cord cultures (VSCC), compared with control VSCC, had a significant neurite outgrowth at 10(-8), 10(-6), and 10(-4) M. The effect was considered to be concentration dependent. Morphological changes of the dorsal spinal cord cultures (DSCC) remained unchanged by VIP treatment. Because of their close sequence homology with VIP, PHI-27 (peptide, histidylisoleucine amide) and secretin were also examined with the same experimental conditions as was VIP. Both PHI-27 and secretin had neurite promoting effects in VSCC at 10(-8) and 10(-6) M, respectively. However, there were no neurite promoting effects in DSCC in both of them at any concentrations. VIP had the most potent effect on neurite outgrowth in VSCC, followed by PHI-27, and secretin in their effectiveness concentrations. Our data showing VIP, PHI-27 and secretin have neurotrophic action on VSCC and suggest that a potential therapeutic use of VIP and its related peptides in treating diseases that involve degeneration and death of spinal motor neurons, such as motor neuropathy and amyotrophic lateral sclerosis.     

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]     

Acidic and basic fibroblast growth factors enhance neurite outgrowth in cultured rat spinal cord neurons

Abstract

We have studied neurotrophic effects of acidic fibroblast growth factor (aFCF) and basic fibroblast growth factor (bFGF) on explanted ventral and dorsal spinal cord cultures from 13- and 14-day-old rat embryos. Cultures treated with aFCF and bFGF significantly enhanced neurite outgrowth with cultures of ventral spinal cord, but not with cultures of dorsal spinal cord. Our data suggest that aFCF and bFGF are potent neurotrophic factors on rat ventral spinal cord neurons in vitro. [Neurol Res 1995; 17: 70-72]     

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 mesenchymal stromal cells stimulate neurite outgrowth of spinal neurons by secreting neurotrophic factors

Abstract

It has been demonstrated that bone mesenchymal stromal cells (BMSCs) stimulate neurite outgrowth from dorsal root ganglion (DRG) neurons. The present in vitro study tested the hypothesis that BMSCs stimulate the neurite outgrowth from spinal neurons by secreting neurotrophic factors. Spinal neurons were cocultured with BMSCs, fibroblasts and control medium in a non-contact system. Neurite outgrowth of spinal neurons cocultured with BMSCs was significantly greater than the neurite outgrowth observed in neurons cultured with control medium or with fibroblasts. In addition, BMSC-conditioned medium increased the length of neurites from spinal neurons compared to those of neurons cultured in the control medium or in the fibroblasts-conditioned medium. BMSCs expressed brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). The concentrations of BDNF and GDNF in BMSC-conditioned medium were 132±12 and 70±6 pg ml^?1, respectively. The addition of anti-BDNF and anti-GDNF antibodies to BMSC-conditioned medium partially blocked the neurite-promoting effect of the BMSC-conditioned medium. In conclusion, our results demonstrate that BMSCs promote neurite outgrowth in spinal neurons by secreting soluble factors. The neurite-promoting effect of BMSCs is partially mediated by BDNF and GDNF.     

TRH analogue, TA-0910 (3-methyl-(s)-5,6-dihydroorotyl-L-histidyl-L-prolinamide) enhances neurite outgrowth in rat embryo ventral spinal cord in vitro.

We have studied effects of TRH analogue, TA-0910 (3-methyl-(s)-5,6-dihydroorotyl-L-histidyl-L-prolinamide) (from Tanabe, Osaka, Japan) on explanted ventral and dorsal spinal cord cultures from 13- and 14-day-old rat embryos. TA-0910-treated cultures had significantly increased neurite outgrowth with cultures of ventral spinal cord, but not with cultures of dorsal spinal cord. The effect was dose-dependent. A possible role for TRH in amyotrophic lateral sclerosis remains to be defined.     

Trophic effect of angiotensin II, vasopressin and oxytocin on the ventral spinal cord of rat embryo

We studied trophic effects of angiotensin II, vasopressin and oxytocin on explanted ventral spinal cord cultures derived from 13 to 14-old day rat embryos. There was a significant neurite promoting effect in angiotensin II and vasopressin-treated cultures. Angiotensin II had the most potent effect at any concentrations. It became clear that minimum effective concentration was 10(-8)M in both angiotensin II and vasopressin. However, oxytocin had no neurotrophic effect at any concentrations. Our results demonstrated that angiotensin II and vasopressin have a neurotrophic effect on ventral spinal cord in cultures, and may contribute to therapeutic strategy of amyotrophic lateral sclerosis.     

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.     

Growth inhibitory effects of a mu opioid on cultured cholinergic neurons from fetal rat ventral forebrain, brainstem, and spinal cord

Cholinergic pathways play a role in respiration in the mammalian brain, and agents that affect respiratory function such as opioid peptides might have positive or negative neurotrophic effects during the development of these cholinergic connections. Rat fetal nerve cell cultures from developmental stages E14-E18 were established in 96-well plates from ventral forebrain (VFB), an area rich in cholinergic neurons, and from brainstem and rostral spinal cord, areas where respiratory control systems and cholinergic neurons co-exist. High affinity 3H-choline uptake was highest in E14 VFB cultures and decreased to 20% of this value by E16 and E18. Choline uptakes in E14 brainstem and spinal cord were only 20% and 13%, respectively, of E14 VFB uptake. A mu opioid receptor agonist, d-ala2-mePhe4-gly(ol)5]-enkephalin (DAMGO), was tested for its effect on somal area and neurite outgrowth in E16 cultures. Cholinergic neurons were identified by immunostaining with choline acetyltransferase antibody. DAMGO (10(-8) M) significantly decreased somal area in VFB cultures and spinal cord, but had no effect on somal area in brainstem. Naltrexone (10(-6) M) reversed this inhibition. Spinal cord cell neurite outgrowth was inhibited by DAMGO, and this inhibition was reversed by naltrexone. DAMGO had no significant effect on neurite length in VFB. Brainstem neurite length was paradoxically increased by both DAMGO and naltrexone. It was concluded that mu-selective opioid peptides inhibit growth of cultured cholinergic neurons in VFB and spinal cord, but not in the brainstem. There was no evidence for endogenous opioid activity in either VFB or spinal cord cultures.     

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.     

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 angiotensin II, vasopressin and other peptides on the cultured ventral spinal cord of rat embryo

We studied trophic effects of angiotensin II, vasopressin, cholecystokinin, and oxytocin on explanted ventral spinal cord cultures from 13- and 14-day-old rat embryos. There was a significant neurite promoting effect of the spinal cord cultures by using angiotensin II, vasopressin, and cholecystokinin. Cholecystokinin had the most potent effect at any concentrations. The minimum effective concentration was 10(-8) M in angiotensin II and vasopressin and 10(-12) M in cholecystokinin, respectively. The effect of angiotensin II and vasopressin was dependent on concentrations. However, the rate and grade of neurite appearance did not correlate with the concentrations of cholecystokinin. Oxytocin had no neurotrophic effect at any concentrations. Our results demonstrated that angiotensin II, vasopressin and cholecystokinin have neurotrophic effects on the ventral spinal cord in cultures, and may be candidates for therapeutic trials of amyotrophic lateral sclerosis.     

Enteric glia mediate neuronal outgrowth through release of neurotrophic factors

Previous studies have shown that transplanted enteric glia enhance axonal regeneration, reduce tissue damage, and promote functional recovery following spinal cord injury. However, the mechanisms by which enteric glia mediate these beneficial effects are unknown. Neurotrophic factors can promote neuronal differentiation, survival and neurite extension. We hypothesized that enteric glia may exert their protective effects against spinal cord injury partially through the secretion of neurotrophic factors. In the present study, we demonstrated that primary enteric glia cells release nerve growth factor, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor over time with their concentrations reaching approximately 250, 100 and 50 pg/mL of culture medium respectively after 48 hours. The biological relevance of this secretion was assessed by incubating dissociated dorsal root ganglion neuronal cultures in enteric glia-conditioned medium with and/or without neutralizing antibodies to each of these proteins and evaluating the differences in neurite growth. We discovered that conditioned medium enhances neurite outgrowth in dorsal root ganglion neurons. Even though there was no detectable amount of neurotrophin-3 secretion using ELISA analysis, the neurite outgrowth effect can be attenuated by the antibody-mediated neutralization of each of the aforementioned neurotrophic factors. Therefore, enteric glia secrete nerve growth factor, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor and neurotrophin-3 into their surrounding environment in concentrations that can cause a biological effect.     

Schwann cell-conditioned medium supports neurite outgrowth and survival of spinal cord neurons in culture

The effect of Schwann cell-conditioned medium (SCM) on the development in vitro of spinal cord neurons was studied. Spinal cord neurons from 18-day-old rat embryos were cultured in serum-free conditioned medium obtained from confluent rat Schwann cells. In cultures fed SCM, the cells developed typical neuronal morphology and were identified by indirect immunofluorescence using a monoclonal antibody to neurofilament protein. SCM stimulated neurite outgrowth and supported survival of spinal cord neurons. Preliminary characterization suggests that the neurotrophic factor in SCM appears to be a protein with a molecular weight greater than 8000 daltons.     

Neurotrophic effect of angiotensin II, vasopressin and oxytocin on the ventral spinal cord of rat embryo

We studied trophic effects of angiotensin II, vasopressin and oxytocin on explanted ventral spinal cord cultures from 13-14-old day rat embryos. There was a significant neurite promoting effect in angiotensin II and vasopressin-treated cultures. Angiotensin II had the most potent effect at any concentrations. It became clear that minimum effective concentration was 10(-8) M in angiotensin II and vasopressin respectively. Effect of these two neuropeptides was concentration-dependent. However, oxytocin had no neurotrophic effect at any concentrations. Our results demonstrated that angiotensin II and vasopressin have a neurotrophic effect on ventral spinal cord in cultures, and may contribute to therapeutic strategy of amyotrophic lateral sclerosis.     

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.     

Olfactory ensheathing cells represent an optimal substrate for hippocampal neurons: an in vitro study

Olfactory ensheathing cells (OECs) are cells that display Schwann cell or astrocyte-like properties. They are a source of growth factors and adhesion molecules which play a very important role as neuronal support enhancing cellular survival. Over the past 10 years, OECs have emerged as a leading reparative candidate, when transplanted into the injured spinal cord, having shown significant promise in the regeneration of spinal cord lesions. In this study we assessed the efficacy of OECs on the survival and neurite outgrowth of hippocampal neurons in vitro. Co-cultures of OECs and hippocampal of postnatal rats were successfully established and cells were immunocytochemically characterized. Some hippocampal cultures were added with growth factors, as bFGF, NGF and GDNF. Furthermore, conditioned medium from OECs cultures was used to feed some hippocampal neurons coverslips. Our results show that in co-cultures of hippocampal neurons and OECs the number of neurons and their neurite outgrowth were significantly increased in comparison with controls. Moreover, we showed that NGF and GDNF promoted a more positive effect in both neuronal survival and neurite outgrowth than bFGF. OEC-conditioned media stimulated both the neuronal survival and dense neurite outgrowth. These data indicate that OECs, as a source of growth factors, can promote the survival and the neurite outgrowth of hippocampal neurons in vitro and that bFGF, NGF and GDNF support them differently. Therefore, as OECs and their secreted growth factors appear to exert a neuroprotective effect for functional restoration and for neural plasticity in neurodegenerative disorders, they might be considered an approach for functional recovery.     

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.