Basic fibroblast growth factor in neuronal cultures of human fetal brain

The presence of basic fibroblast growth factor (bFGF) was investigated in neuronal cells derived from 12 and 18 week-old human fetal brain cultures. To this purpose, the ability of bFGF to stimulate plasminogen activator (PA) production in fetal bovine aortic endothelial GM 7373 cells was used as an assay for this molecule in neuronal cell extracts. The identity of the PA-stimulating activity of neuronal cell extract with bFGF was confirmed by its high affinity for heparin and by its cross-reactivity with polyclonal antibodies to human placental bFGF. These antibodies recognized a Mr 18,000 cell-associated protein both in Western blot and in immuno-precipitation experiments. All the neurons showed bFGF immunoreactivity, as demonstrated by immunocytochemical staining, while nonneuronal cells were unstained. The data demonstrate for the first time that cultured human fetal brain neurons contain and synthesize bFGF.     

Intrathecal injection of epidermal growth factor and fibroblast growth factor 2 promotes proliferation of neural precursor cells in the spinal cords of mice with mutant human SOD1 gene

We investigated three steps of neural precursor cell activation--proliferation, migration, and differentiation--in amyotrophic lateral sclerosis spinal cord treated with intrathecal infusion of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2) into the lumbar spinal cord region of normal and symptomatic transgenic (Tg) mice with a mutant human Cu/Zn superoxide dismutase (SOD1) gene. We observed that 5-bromodeoxyuridine (BrdU) + nestin double-labeled neural precursor cells increased in the spinal cords of Tg mice compared with non-Tg mice, with a much greater increase produced by EGF and FGF2 treatment. The number of BrdU + nestin double-labeled cells was larger than that of BrdU + ionized calcium-binding adapter molecule-1 (Iba1), BrdU + glial fibrillary acidic protein (GFAP), or BrdU + highly polysialylated neural cell adhesion molecule (PSA-NCAM) double-labeled cells, but none expressed neuronal nuclear antigen (NeuN). On further analysis of the gray matter of Tg mice, the number of BrdU + nestin and BrdU + PSA-NCAM double-labeled cells increased more in the ventral horns than the dorsal horns, which was again greatly enhanced by EGF and FGF2 treatment. Because neural precursor cells reside close to the ependyma of central canal, the present study suggests that proliferation and migration of neural precursor cells to the ventral horns is greatly activated in symptomatic Tg mice and is further enhanced by EGF and FGF2 treatment and, furthermore, that the neural precursor cells preferentially differentiate into neuronal precursor cells instead of astrocytes in Tg mice with EGF and FGF2 treatment.     

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.     

Leukemia inhibitory factor promotes the neuronal development of spinal cord precursors from the neural tube.

Recent evidence from our laboratory has shown that leukemia inhibitory factor (LIF) can act early in peripheral nervous system development. We have investigated a potential role of LIF in the developing spinal cord. In explants and dissociated cultures of spinal cord primordium, LIF stimulated a profuse neurite outgrowth. To determine if these effects were related to neuronal differentiation, cells were plated at low cell density and stained for neurofilament. LIF stimulated an increase in the number of newly differentiated neurons, without inducing proliferation of the precursors. Given that LIF has previously reported effects as a cholinergic switching factor for sympathetic neurons, we investigated whether LIF had similar effects in these spinal cord cultures. LIF increased the number of cholinergic neurons in proportion to its overall effect on the stimulation of all neurofilament positive neurons in the culture. These data show that LIF stimulates the generation of spinal cord neurons from their precursors and further implicates a role for LIF in nervous system development.     

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.     

碱性成纤维生长因子和神经生长因子联合应用培养成年大鼠海马神经干细胞向神经元样细胞的分化

背景：影响神经干细胞向神经元分化的因素很多,各种营养因子可以不同程度地刺激神经干细胞向神经元分化,如何使神经干细胞大量分化为神经元是研究的热点问题。 目的：观察联合应用碱性成纤维生长因子和神经生长因子对成年大鼠海马神经干细胞为神经元的影响。 方法：无菌条件下分离大鼠脑海马组织,传至第4代克隆球直径约为200 μm时,滴加DMEM/F12+2% B27+20 μg/L表皮生长因子+20 μg/L碱性成纤维细胞生长因子,进行单细胞克隆培养,传代的神经干细胞分成空白对照组、碱性成纤维细胞生长因子组、神经生长因子组、碱性成纤维细胞生长因子+神经生长因子组。观察传代后的克隆球进行神经干细胞免疫细胞化学染色鉴定,计数神经元特异性烯醇化酶阳性细胞率,检测神经干细胞向神经元的分化情况。 结果与结论：①单细胞克隆培养后,克隆球细胞表达巢蛋白,诱导分化后神经元特异性烯醇化酶、胶质纤维酸性蛋白均呈阳性表达。②与空白对照组神经干细胞分化为神经元的比例比较,碱性成纤维细胞生长因子组、神经生长因子组、碱性成纤维细胞生长因子组+神经生长因子组均明显提高(P < 0.05),且碱性成纤维细胞生长因子组+神经生长因子组神经元的比例最高(P < 0.05)。提示,碱性成纤维细胞生长因子可以提高神经生长因子诱和神经生长因子均可促进神经干细胞向神经元分化,且二者联合应用效果更佳。     

Nerve growth factor (NGF) induces motoneuron apoptosis in rat embryonic spinal cord in vitro

Recent studies have demonstrated that nerve growth factor (NGF) induces apoptosis of several cell types in the central nervous system through its low-affinity p75 neurotrophin receptor (p75NTR). To test the effect of NGF on embryonic motoneuron survival, we developed an organotypic culture system which allowed the in vitro development of intact embryonic rat spinal cords. In our system, neural tubes were taken and cultured at E13, just before the onset of physiological motoneuron death. After 2 days in vitro (DIV), motoneurons underwent apoptosis over a time-course similar to that in vivo. In this system, the addition of NGF (200 ng/mL) for 2 days enhanced the number of apoptotic motoneurons by 37%. This pro-apoptotic effect was completely reversed by the blocking anti-p75NTR (REX) antibody which inhibits NGF binding to p75NTR. Other neurotrophins, e.g. brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3) and neurotrophin 4/5 (NT4/5) did not have any effect, while glial cell-derived neurotrophic factor (GDNF) promoted motoneuron survival. Anti-BDNF blocking antibodies enhanced motoneuron death indicating that endogenous BDNF promotes motoneuron survival in explants. Our results demonstrate, for the first time, that NGF can induce embryonic motoneuron apoptosis through its receptor p75NTR.     

FGF和EGF对神经干细胞增殖及分化的影响

胚胎和成年哺乳动物脑内均存在的神经干细胞,成纤维细胞生长因子和表皮生长因子对神经干细胞的增殖及分化有一定的影响,ＦＧＦ和ＥＧＦ及其受体在胚胎期和成年期表达各异。ＦＧＦ和ＥＧＦ能促进神经干细胞增殖,在不同的条件下对分化和作用不同。     

Regulation of chondroitin sulphate proteoglycan and reactive gliosis after spinal cord transection: effects of peripheral nerve graft and fibroblast growth factor 1

M‐J. Lee, C. J. Chen, W‐C. Huang, M‐C. Huang, W‐C. Chang, H‐S. Kuo, M‐J. Tsai, Y‐L. Lin and H. Cheng (2011) Neuropathology and Applied Neurobiology 37, 585–599 Regulation of chondroitin sulphate proteoglycan and reactive gliosis after spinal cord transection: effects of peripheral nerve graft and fibroblast growth factor 1 Aims: The combined treatment of peripheral nerve (PN) graft and fibroblast growth factor (FGF)‐1 for spinal cord injury produces functional recovery, but how it affects injury events is still unknown. This project studied the effect of PN graft and FGF‐1 on white matter degeneration following spinal cord injury. Methods: Rats were divided into four groups: (i) complete spinal cord transection and T8 segment removed; the remaining three groups underwent transection followed by (ii) PN grafting; (iii) supply of exogenous FGF‐1; and (iv) PN grafting plus FGF‐1 treatment. Chondroitin sulphate proteoglycan (CSPG) deposition, astrocytes and macrophage activation, cavity size, and calcitonin gene‐related peptide and synaptophysin immunoreactivity were compared. Results: Peripheral nerve grafting increased CSPG levels compared to transection surgery alone. This CSPG was associated with the proximity to the PN graft. FGF‐1 reduced CSPG deposition in grafted animals regardless of the proximity to the graft. The CSPG reduction was accompanied by reduced GFAP expression and macrophage activation. The amount of CSPG with dissociated glycosaminoglycan did not differ between groups. FGF‐1 in Schwann cell–astrocyte coculture did not reduce CSPG deposition. Furthermore, the PN graft increased the calcitonin gene‐related peptide immunoreactivity and altered the distribution of synaptophysin‐positive axons. Conclusion: Peripheral nerve graft supported sensory re‐innervation and partial protection of the grey matter, but up‐regulated CSPG in the graft–stump junction compared to non‐grafted rats. The reduction of CSPG was caused by FGF‐1–PN synergy, and did not involve dissociation of CSPG or the suppression of a general immune response.     

Basic fibroblast growth factor attenuates the degeneration of injured spinal cord motor endplates**

The distal end of the spinal cord and neuromuscular junction may develop secondary degeneration and damage following spinal cord injury because of the loss of neural connections. In this study, a rat model of spinal cord injury, established using a modified Allen’s method, was injected with basic fibroblast growth factor solution via subarachnoid catheter. After injection, rats with spinal cord injury displayed higher scores on the Basso, Beattie and Bresnahan locomotor scale. Motor function was also well recovered and hematoxylin-eosin staining showed that spinal glial scar hyperplasia was not apparent. Additionally, anterior tibial muscle fibers slowly, but progressively, atrophied. Immunohistochemical staining showed that the absorbance values of calcitonin gene related peptide and acetylcholinesterase in anterior tibial muscle and spinal cord were similar, and injection of basic fibroblast growth factor increased this absorbance. Results showed that after spinal cord injury, the distal motor neurons and motor endplate degenerated. Changes in calcitonin gene related peptide and acetylcholinesterase in the spinal cord anterior horn motor neurons and motor endplate then occurred that were consistent with this regeneration. Our findings indicate that basic fibroblast growth factor can protect the endplate through attenuating the decreased expression of calcitonin gene related peptide and acetylcholinesterase in anterior horn motor neurons of the injured spinal cord.     

Characterization of nerve growth factor binding to embryonic rat spinal cord neurons

The binding of iodinated beta-nerve growth factor, [125I]-NGF, to embryonic (E16) rat spinal cord cells, was investigated to characterize the binding properties and cellular distribution of nerve growth factor receptors. Spinal cord cells prepared without trypsin yielded two classes of NGF binding sites with Kd's of 3 x 10(-11) M and 4 x 10(-9) M. Fractionation of the cells by discontinuous gradients composed of 8%, 12%, and 17% metrizamide was used to separate motoneurons from other cell types. The motoneuron enriched fraction (8% metrizamide) contained approximately 10% of the cells and 64% of the choline acetyltransferase (ChAT) activity. In contrast, the 12% metrizamide fraction contained most (51%) of the cells and 36% of the ChAT activity, while the 17% metrizamide fraction contained the remainder of the cells and negligible amounts of ChAT activity. Characterization of [125I]-NGF binding to each metrizamide fraction showed that the motoneuron-enriched fraction exhibited both high and low affinity binding sites, while the other metrizamide fractions exhibited only the low affinity binding sites. These findings indicate that although low affinity NGF receptors appear to be relatively evenly distributed amongst embryonic rat spinal cord cells, high affinity NGF receptors are found primarily on motoneurons.     

Immunohistochemical localization of basic fibroblast growth factor in choroid plexus of the rat

Abstract

In the present study, we report that an intense bFGF-immunoreactivity has been detected in the choroid plexus of the brain ventricles of adult rats. These results suggest that epithelial choroid plexus cells may be the source of the cerebrospinal fluid bFGF. [Neurol Res 1994; 16: 310-312]     

Basic fibroblast growth factor improves recovery after chemically induced breakdown of myelin-like membranes in pure oligodendrocyte cultures

The putative role of growth factors in remyelination was investigated in pure oligodendrocyte (OL) secondary cultures derived from newborn rat brain. These cells form myelin-like membranes and were used as a model system for toxic attack. A 24 hr treatment with 2.10^–5 M lysophosphatidylcholine (LPC) induced a loss of 59% of the cells in these cultures, with a 64% reduction in [¹²⁵I]-iododeoxyuridine incroporation compared to untreated controls. An absence of processes and myelin-like sheaths was observed in the remaining cells. Numerous intractoplasmic inclusions were observed on transmission electron microscopy. Immunocytochemical studies with A2B5 monoclonal antibody (mAb), which recognizes oligodendrocyte-type 2 astrocyte (O-2A) precursors, OL-1 mAb (directed against cell surface sulfatides), and anti-myelin basic protein (anti-MBP) antibody showed that the entire OL lineage was affected at all stages of maturation. A 3 day treatment with 10 ng/ml basic fibroblast growth factor (bFGF) induced reconstruction of myelin-like membranes, albeit less compacted than in untreated controls. The doubling in number of cells and the 46% increase in [¹²⁵I]-iododeoxyuridine incorporation was due essentially to proliferation of O-2A progenitors. These results indicate that if bFGF release occurs during demyelination, it may participate in myelin repair mechanisms in the central nervous system. © 1994 Wiley-Liss, Inc.     

外源性碱性成纤维细胞生长因子对缺血大鼠脑内源性碱性成纤维细胞生长因子表达的影响

目的 研究外源性碱性成纤维细胞生长因子(bFGF)缩小局灶性脑缺血梗死灶的机制。方法 用免疫组化ABC法检测在局灶性脑缺血模型上给予生理盐水或bFGF后早期生长反应蛋白-1(Egr-1)，bFGF，碱性成纤维细胞生长因子受体(bFGFR)的动态表达。结果 给药组在3h～3d各时间段梗死灶均有不同程度的缩小。对照组和给药组Egr-1表达均表现为3～6h的增强过程，但给药组更强于对照组。对照组12h见有bFGF表达增强，而bFGFR表达3h到达高峰，6h起下降，12h时bFGFR的表达已恢复至正常水平(出现了配体和受体表达时相上不匹配)。给药组bFGF表达提前且增强，3h即见有bFGF表达增强，6h时出现第一峰，从而与bFGFR 3～6h的表达增强过程相吻合。结论 外源性bFGF能缩小梗死灶，该神经保护作用是通过Egr-1蛋白高表达使内源性bFGF的表达增高且提前，从而与bFGFR的表达增强过程重叠而实现的。     

Reduction of fetal rat spinal cord volume following maternal morphine injection

Previous studies have shown behavioral changes in pre- and postnatal rats exposed to morphine before and during gestation. The present study has attempted to discover morphological correlates of these changes in the fetal rat spinal cord. Pregnant Wistar rats were injected subcutaneously on days 12–18 of gestation with 5 mg/kg of morphine 4 times daily. Non-injected, saline-injected and pair-fed controls were prepared for comparison. On day 18 of gestation the fetuses were perfused and the volume of the first thoracic spinal cord segment was measured using planimetric measurements of histological sections. The following volumes were measured within the segment: hemisection, gray matter, white matter, dorsal horn, ventral horn and length. All volumes were reduced by 20% in morphine-exposed fetuses and 10% in pair-fed fetuses. The length of the spinal cord segment was not different from controls. In addition, body weights were not reduced in either the morphine-exposed or pair-fed fetuses. This is the first study of rats exposed prenatally to morphine, exhibiting a decrease in nervous system volume without an accompanying decrease in body weight.     

Nerve growth factor stimulates development of substance P in the embryonic spinal cord

Development of the putative neurotransmitter, substance P (SP), in the embryonic rat dorsal root ganglion (DRG) and spinal cord was defined in vivo. SP was not detectable by radioimmunoassay before day 17 of gestation (E17). On E17, cervical sensory ganglia contained 4 pg SP/ganglion, rising to 49 pg/ganglion at birth. The dorsal cervical spinal cord contained 0.75 ng SP/mg protein on E17, rising to 6 ng SP/mg protein on postnatal day 3. The ventral spinal cord contained approximately 20% of the SP content in the dorsal cord at each gestational age. Intrauterine forelimb amputation partially prevented the normal development increase of SP in sensory ganglia destined to innervate that limb, suggesting that target structures regulate the development of peptidergic neruons. Conversely, treatment with nerve growth factor (NGF) stimulated development of SP in the DRG. Moreover, NGF treatment increased SP in the dorsal spinal cord, suggesting that NGF can modulate development within the CNS, as well as peripheral structures. It is likely that the CNS effect reflects NGF peptidergic neruons. Conversely, treatment with nerve growth factor (NGF) stimulated development of SP in the DRG. Moreover, NGF treatment increased SP in the dorsal spinal cord, suggesting that NGF can modulate development within the CNS, as well as peripheral structures. It is likely that the CNS effect reflects NGF peptidergic neruons. Conversely, treatment with nerve growth factor (NGF) stimulated development of SP in the DRG. Moreover, NGF treatment increased SP in the dorsal spinal cord, suggesting that NGF can modulate development within the CNS, as well as peripheral structures. It is likely that the CNS effect reflects NGF action on peripheral ganglia, but a direct effect on the spinal cord has not been excluded. However, treatment with antiserum to NGF failed to significantly inhibit development of ganglion SP. The system of SP-containing neurons in the DRG may provide a convenient model for defining events regulating peptidergic maturation.     

Epidermal growth factor and fibroblast growth factor 2 cause proliferation of ependymal precursor cells in the adult rat spinal cord in vivo

We investigated the mitogenic effect of continuous intrathecal infusion of epidermal growth factor (EGF) or fibroblast growth factor 2 (FGF2) on ependymal precursor cells of the adult rat spinal cord in vivo. Either EGF, FGF2, EGF plus FGF2, or artificial cerebrospinal fluid (aCSF) was infused at a flow rate of 0.5 microl/h (15 ng/h of EGF or FGF2) for 3 or 14 days into the intrathecal space at T1 through a catheter attached to an osmotic minipump. To assess proliferation, the bromodeoxyuridine (BrdU) labeling index (LI) in the ependyma at T1 was calculated at 3 or 14 days. At 3 days there was no statistical difference in LI between these groups, but at 14 days the LI was significantly higher in the EGF plus FGF2 group (27.2% = 16.0%) than in the aCSF group (5.4% +/- 4.7%; p < 0.05). With EGF alone or FGF2 alone, the LI increases were not significantly different from the aCSF group. With EGF plus FGF2 for 14 days, some BrdU-positive cells in the ependyma also expressed nestin. These results suggest that the intrathecal infusion of EGF plus FGF2 has a mitogenic effect on precursor cells in the ependyma of the adult rat spinal cord.     

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]