Effect of basic fibroblast growth factor on neurons cultured from various regions of postnatal rat brain

Neurons from various brain regions of postnatal (15 days after birth) and fetal (16 days gestation) rats were cultured in the presence of basic fibroblast growth factor (bFGF). bFGF increased the survival of neurons from postnatal septum, striatum, midbrain, and hippocampus. Fetal neurons derived from cerebral cortex, septum, striatum, midbrain, thalamus, and colliculus were far more dependent on bFGF for survival in comparison with postnatal neurons. In contrast, cerebellum neurons of postnatal and fetal rat brain did not respond to bFGF. The increase of postnatal and fetal neuronal survival with bFGF treatment (0.01–10 ng/ml) was dose-dependent and reached 2–4-fold and 5–10-fold more than the control, respectively. Fetal cortical neurons showed almost complete dependence on bFGF since almost all neurons died in control cultures. Nerve growth factor was slightly effective only on postnatal septal and striatal neurons, being ineffective on the other neurons tested. These results indicate that bFGF can function as a neurotrophic factor not only on fetal but also on postnatal neurons of the central nervous system, and that bFGF has great potential for application in vivo.     

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.     

Acidic fibroblast growth factor prevents death of spinal cord motoneurons in newborn rats after nerve section

Abstract

The death of spinal axotomised motoneurons provides a useful tool for studying neurotrophic factors which could prevent motoneuron loss in vivo. Median and ulnar nerves of newborn rats were unilaterally sectioned and topically treated with either a vehicle solution or acidic fibroblast growth factor (aFCF). aFCF treatment increased the survival of the median and ulnar spinal motoneurons, after 7 days of axotomy, from 37% to 63%. These results show that aFGF is a neurotrophic factor for newborn spinal motoneuronsand suggest that this protein is a potential therapeutic agent for preventing the death of damaged motoneurons. [Neurol Res 1995; 17: 396^399]     

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

Abstract

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. [Neurol Res 2001; 23: 851-854]     

Retinoic acid responsive gene product,midkine, has neurotrophic functions for mouse spinal cord and dorsal root ganglion neurons in culture

Midkine (MK) is the product of a retinoic acid responsive gene and is a member of a new family of heparin-binding growth factors. Neurotrophic effects of MK were examined using cultured spinal cord and dorsal root ganglion (DRG) neurons derived from fetal mouse. MK, which was added to the culture medium at concentrations of 1–100 ng/ml, promoted survival of both types of neurons approximately 5-fold after 7 days in culture. For spinal cord neurons, the increased survival was reflected in an increase of choline acetyltransferase activity. MK also promoted neurite extension in spinal cord (2-fold) and DRG (1.7-fold) neurons. The survival-promoting activity of MK to these neurons was comparable to that of basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF). In spite of its significant effects on fetal neurons, MK was ineffective in sustaining survival of DRG neurons derived from postnatal mice. From these results, we conclude that MK is a neurotrophic factor to embryonic spinal cord and DRG neurons, and we propose that MK plays a significant role in embryogenesis of the nervous system. © 1993 Wiley-Liss, Inc.     

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.     

蛋白激酶C调节脊髓神经元突起的生长(英文)

目的关于蛋白激酶C(PKC)在神经元突起生长和神经再生中的作用,目前仍存有争议。本研究主要观察PKC对离体培养的脊髓神经元生长的调节作用,旨在阐明PKC对突起生长的调节作用。方法分离纯化胎龄14天(E14)的SD胎鼠的脊髓前角神经元,进行原代培养,并检测不同时相点膜/浆PKC活性(m/c-PKCactivity)的比值。结果神经元培养3-11d期间,神经元内m/c-PKC比值以及PKC-βII在突起中的表达水平均与突起生长呈显著相关关系(r=0.95,P<0.01;r=0.73,P<0.01)。此外,PKC激动剂PMA能显著提高m/c-PKC比值,且与神经突起的生长一致(r=0.99,P<0.01)。而PKC抑制剂GF109203X则能显著抑制突起生长,且不被PMA作用所逆转。结论PKC的活性在脊髓神经元突起生长调节中具有重要作用,其中βII亚型可能扮演重要角色。     

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.     

Generation of spinal motor neurons from human fetal brain-derived neural stem cells: role of basic fibroblast growth factor

Neural stem cells (NSCs) have some specified properties but are generally uncommitted and so can change their fate after exposure to environmental cues. It is unclear to what extent this NSC plasticity can be modulated by extrinsic cues and what are the molecular mechanisms underlying neuronal fate determination. Basic fibroblast growth factor (bFGF) is a well-known mitogen for proliferating NSCs. However, its role in guiding stem cells for neuronal subtype specification is undefined. Here we report that in-vitro-expanded human fetal forebrain-derived NSCs can generate cholinergic neurons with spinal motor neuron properties when treated with bFGF within a specific time window. bFGF induces NSCs to express the motor neuron marker Hb9, which is blocked by specific FGF receptor inhibitors and bFGF neutralizing antibodies. This development of spinal motor neuron properties is independent of selective proliferation or survival and does not require high levels of MAPK activation. Thus our study indicates that bFGF can play an important role in modulating plasticity and neuronal fate of human NSCs and presumably has implications for exploring the full potential of brain NSCs for clinical applications, particularly in spinal motor neuron regeneration.     

外源性血管内皮生长因子对大鼠损伤脊髓神经元的作用

目的研究外源性血管内皮生长因子(VEGF)局部应用对大鼠脊髓损伤(SCI)后脊髓灰质神经元的作用。方法采用改良Allen氏重量打击法建立大鼠急性SCI模型,将72只模型大鼠随机分为VEGF治疗组〔人工基底膜(Matrigel蛋白胶)10μL+重组人VEGF1650.2μg〕、基底膜对照组(Matrigel蛋白胶10μL)和空白对照组(生理盐水10μL)3组,分别经蛛网膜下腔给药。于SCI后1、2、3、4周分别观察模型鼠脊髓横切面灰质神经元数量、神经元截面积和尼氏体密度恢复情况。结果 SCI后1~4周VEGF治疗组脊髓横切面灰质神经元数量(分别为76.34±6.25、108.25±11.74、144.33±13.22、165.96±18.11)和神经元截面积〔分别为(177.87±14.42)(、229.63±21.74)(、340.55±32.66)(、390.30±46.19)μm2〕均高于同期基底膜对照组〔神经元数量分别为40.89±7.20、50.61±13.11、59.78±9.46、80.13±10.84,神经元截面积分别为(108.98±15.03)(、148.53±18.38)、(163.22±16.77)(、208.87±29.58)μm2〕和空白对照组〔神经元数量分别为37.66±6.69、52.36±5.17、60.58±9.13、78.56±13.56,神经元截面积分别为(114.55±14.22)(、139.24±15.17)、(157.11±20.26)(、185.23±21.65)μm2〕(P<0.01,P<0.05)。SCI后1周时VEGF治疗组神经元尼氏体密度(0.2089±0.0210)与基底膜对照组(0.2012±0.0145)、空白对照组(0.1982±0.0106)比较差异均无统计学意义(P>0.05),SCI后2、3、4周,VEGF治疗组尼氏体密度(分别为0.2489±0.0217、0.2908±0.0327、0.3508±0.0362)亦大于基底膜对照组(分别为0.2198±0.0214、0.2406±0.0198、0.2605±0.0322)和空白对照组(分别为0.2044±0.0231、0.2356±0.0311、0.2582±0.0245)(均P<0.05)。两对照组各指标均无统计学差异。结论局部应用外源性VEGF可能对SCI后大鼠脊髓灰质神经元具有保护作用。     

体外培养大鼠脊髓前角神经元：脑源性神经生长因子对突触蛋白表达的影响

探讨BDNF对体外培养的大鼠脊髓前角神经元内突触素I与突触囊泡素(SYN)表达的影响。取孕14 d大鼠子宫内胎鼠的脊髓腹侧部分神经元,体外有血清培养。在培养7 d后．随机分成对照组、BDNF组和抗BDNF组。BDNF组培养液中加入BDNF(20 ng/ml),抗BDNF组培养液中加入BDNF抗体(20цg/ml),对照组加入等量Hanks液。3 d后在倒置显微镜下计数三组神经元成活数,并用NF-200、MAP-2、NSE的免疫组化反应对神经细胞进行鉴定。行突触素I与SYN免疫组化反应,对部分细胞行突触素I mRNA原位杂交反应,运用图像分析系统对突触素I与SYN免疫反应阳性产物以及突触素I原位杂交反应阳性产物作光密度分析。结果发现有血清培养时各组脊髓前角神经元的存活数差异无显著性 (P>0.05);BDNF组突触素I与SYN免疫反应阳性产物的平均光密度值高于其它两组,抗BDNF组最低（P<0.01）。BDNP组突触素I mRNA阳性产物的平均光密度值明显高于其它两组,抗BDNF组突触素I mRNA阳性产物的平均光密度值最低（P<0.01）。本研究结果提示BDNF对有血清培养时脊髓前角神经元的存活没有明显影响,但BDNF可明显上调培养的脊髓前角神经元内突触素I与SYN的表达     

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.     

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.     

Motor neuron survival and neuritic extension from spinal cord explants induced by factors released from denervated muscle

Extracts prepared from denervated adult skeletal muscle contain increased amounts of neurotrophic activity which promotes both survival of dissociated motor neurons and the outgrowth of neurites from explants of spinal cord maintained in serum-free defined media. The trophic activity is specific for motor neurons and reaches a peak within the first week post-denervation. In these most potent extracts the neurite outgrowth enhancement is a linearly increasing function of protein concentration at low concentrations; at higher concentrations the neurite activity-concentration relationship saturates and in the milligram range the relationship becomes inhibitory. When media containing active denervated muscle extract was preincubated over polycationic substrata, it lost the ability to promote neuritic growth; this could be restored if fresh extract was added to the cultures. Thus it was demonstrated that within the denervated muscle extract there are physically separable agents responsible for neuron survival and neurite expression. It is possible that the release of neurotrophic factors may be in part responsible for the in vivo phenomenon of nerve sprouting.     

Acidic and basic fibroblast growth factor levels in spinal cord cultures are not regulated by alterations in heparan sulfate proteoglycan expression

The present study was undertaken to assess both the levels of acidic and basic fibroblast growth factors in spinal cord cultures and to determine how they were presented to responsive cells. Western blots detected a single acidic fibroblast growth factor-like protein (17 kDa) and two (18 kDa, 24 kDa) basic fibroblast growth factor-immunoreactive proteins, the levels of which varied with the antibody used. Levels of all three proteins were unaltered in cultures grown in the presence of a mitotic inhibitor, which greatly reduced the number of astrocytes. Cell blots showed increased survival of spinal cord neurons at M_r that corresponded with the three proteins detected immunologically. Solubilized cultures separated on a P100 column showed mitogenic activity for NIH3T3 cells from 17–18 and 24 kDa fractions. Treatment of the cultures with heparitinase did not decrease the levels of acidic and basic fibroblast growth factors detected by Western blots, suggesting that these proteins were not associated with extracellular membrane heparan sulfate proteoglycans. The major fraction of both proteins appeared to be intracellular with a minor amount complexed with extracellular matrix proteins. An inhibitor of xylose-linked proteoglycan synthesis significantly altered heparan sulfate proteoglycan deposition into extracellular matrix, but did not alter the levels of acidic or basic fibroblast growth factors detected by Western blots, or the levels of choline acetyltransferase, glutamic acid decarboxylase, or aspartate aminotransferase activities. These results indicate that both acidic and basic fibroblast growth factors are stored predominantly intracellularly, with only a minor fraction complexed with extracellular proteins. We suggest that these intracellular proteins may be released following injury in the CNS and mediate a cascade of neuroprotective events.     

Acidic fibroblast growth factor (aFGF) is expressed in the neuronal and glial spinal cord cells of adult mice.

Fibroblast growth factors (FGFs) are known to be synthesized in the central nervous system (CNS) and to act on CNS cells in vitro, but less is known about their synthesis, expression, and role in vivo. In this work, using specific anti-acidic fibroblast growth factor (aFGF) antibodies, we have shown for the first time, by immunohistochemistry, that aFGF is expressed in spinal cord cells of young adult normal mice. This expression is predominant in the cell nucleus. Using immunohistochemical double staining procedures, we identified the cell type expressing aFGF as neurons, astrocytes, and oligodendrocytes, but for each type, cells were not all positively immunostained.     

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.     

Electrophysiological effects of angiotensin II on cultured mouse spinal cord neurons

The effects of angiotensin II (AII) on the membrane properties of cultured spinal neurons were investigated using electrophysiological methods. In 26% of neurons tested AII induced changes in membrane potential and input resistance which varied according to the concentration of applied peptide. At low concentrations (10(-6) M), AII increased input resistance by an ionic mechanism which appears to involve a reduction in Cl- conductance. At higher concentration (10(-4) M), AII evoked depolarization associated with a decrease in input resistance. This response appears to depend on an increase in Na+ conductance. Our observations indicate that AII can have multiple effects on neuronal membrane properties dependent on the concentration of applied peptide.