Fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor AB (PDGF AB) promote adult SVZ-derived oligodendrogenesis in vivo

The capacity of multipotential progenitor cells of the adult mammalian forebrain to generate myelin-forming oligodendrocytes was tested by grafting fragments of different regions of the subventricular zone (SVZ) of the lateral ventricle and the striatum of 6-month-old wild-type mice into the brain of neonate shiverer and wild-type mice. Without growth factor treatment, only few cells of the rostral SVZ survived and formed myelin after engraftment. Treating donors prior to transplantation with a single intraperitoneal injection of epidermal growth factor, basic fibroblast growth factor 2 (FGF-2), and platelet-derived growth factor AB (PDGF(AB)) vigorously promoted the survival, migration, and differentiation of the grafted SVZ cells into myelin-forming oligodendrocytes. In situ, both growth factors expanded the constitutively proliferative PSA-NCAM+ population and favored their differentiation toward the neuronal and oligodendroglial cell fate. The adult central nervous system thus harbors a focal reservoir of FGF-2 and PDGF(AB)-responsive cells which are able to generate substantial amounts of myelin-forming oligodendrocytes in vivo, opening a new prospective area for therapy in demyelinating diseases.     

Effects of Kir2.1 gene transfection in cochlear hair cells and application of neurotrophic factors on survival and neurite growth of co-cultured cochlear spiral ganglion neurons

Immature inner hair cells (IHCs) produce spontaneous action potentials, which may be associated with the survival of spiral ganglion neurons (SGNs) during early development. Later, this activity ceases in part by the expression of Kir channels. In the present study, SGNs were co-cultured with organ of Corti in which a Kir2.1 channel was over-expressed in an attempt to block the spontaneous activity of IHCs. The over-expression led to a reduced survival and neurite growth accompanied by increased SGN apoptosis. The enhanced activation of apoptosis was consistent with the inhibition of the survival-promoting pathway and the disruption of [Ca²⁺]_i homeostasis. Furthermore, the effect of Kir2.1 over-expression can be reversed by exogenous neurotrophic factors (NTFs). These results are consistent with the hypothesis that the earlier-than-normal expression of Kir2.1 in HCs inhibits their spontaneous activity required for SGN survival and neurite growth.     

Interactive roles of fibroblast growth factor 2 and neurotrophin 3 in the sequence of migration, process outgrowth, and axonal differentiation of mouse cochlear ganglion cells

A growth factor may have different actions depending on developmental stage. We investigated this phenomenon in the interactions of fibroblast growth factor 2 (FGF2) and neurotrophins on cochlear ganglion (CG) development. The portions of the otocyst fated to form the CG and cochlear epithelium were cocultured at embryonic day 11 (E11). Cultures were divided into groups fed with defined medium, with or without FGF2 and neurotrophin supplements, alone or in combination, for 7 days. We measured the number of migrating neuroblasts and distances migrated, neurite outgrowth, and axonlike processes. We used immunohistochemistry to locate neurotrophin 3 (NT3) and its high-affinity receptor (TrkC) in the auditory system, along with FGF2 and its R1 receptor, at comparable developmental stages in vitro and in situ from E11 until birth (P1) in the precursors of hair cells, support cells, and CG cells. Potential sites for interaction were localized to the nucleus, perikaryal cytoplasm, and cell surfaces, including processes and growth cones. Time-lapse imaging and quantitative measures support the hypothesis that FGF2 alone or combined with neurotrophins promotes migration and neurite outgrowth. Synergism or antagonism between NT3 and other factors suggest interactions at the receptor level. Formation of axons, endings, and synaptic vesicle protein 2 were increased by interactions of NT3 and FGF2. Similar experiments with a mutant overexpressor for FGF2 suggest that endogenous FGF2 supports migration and neurite outgrowth of CG neuroblasts as well as proliferation, leading to accelerated development. The findings suggest interactive and sequential roles for FGF2 and NT3.     

Fibroblast growth factor 2 (FGF-2) differentially regulates connexin (cx) 43 expression and function in astroglial cells from distinct brain regions

Fibroblast growth factor (FGF)-2 is a peptide growth factor that promotes the generation, differentiation, and survival of neurons and glial cells. In the CNS, astroglial cells are coupled in a region-specific manner by gap junctions consisting of connexin 43 (cx43). In the present study we have investigated effects of FGF-2 and of other growth factors on the expression and function of cx43 in astroglial cells cultured from telencephalic cortex, striatum, and mesencephalon of newborn rats. Confluent cultures were maintained for two days in low serum, and then exposed to FGF-2 (10 ng/ml) for 48 h. FGF-2 caused a reduction of cx43-protein, -mRNA, and intercellular communication revealed by dye spreading. These changes occurred in cortical and striatal cells, but not in mesencephalic astroglial cells. Effects of FGF-2 were time- and concentration-dependent, with a minimal effective dose of 1 ng/ml FGF-2, and an onset of effects after 6 h of incubation. The reduction of coupling by FGF-2 was transient, since in cortical and striatal cultures coupling recovered to control levels 48 h after removal of the growth factor. Like FGF-2, transforming growth factor-β3 (TGF-β3) decreased coupling of cortical and striatal, but not mesencephalic astroglial cells. Astroglial cells from all brain regions showed a slight FGF-mediated increase in 5-bromo-2′-desoxy-uridine (BrdU) incorporation, which was abolished upon co-treatment with TGF-β3. However, TGF-β3 did not interfere with the repression of cx43-function by FGF-2. Epidermal growth factor (EGF) that has been demonstrated to influence coupling in other cell types had no effect on dye spreading but significantly increased BrdU incorporation. Our results reveal a novel function of FGF-2 on cultured astroglial cells which may be relevant to the regulation of astroglial cell connectivity in vivo. GLIA 22:19–30, 1998. © 1998 Wiley-Liss, Inc.     

Carbon disulfide inhibits neurite outgrowth and neuronal migration of dorsal root ganglion in vitro

Carbon disulfide (CS?) is a neurotoxic industrial solvent and widely used in the vulcanization of rubber, rayon, cellophane, and adhesives. Although the neurotoxicity of CS? has been recognized for over a century, the precise mechanism of neurotoxic action of CS? remains unknown. In the present study, a embryonic rat dorsal root ganglia (DRG) explants culture model was established. Using the organotypic DRG cultures, the direct neurotoxic effects of CS? on outgrowth of neurites and migration of neurons from DRG explants were investigated. The organotypic DRG cultures were exposed to different concentrations of CS? (0.01 mmol/L, 0.1 mmol/L, 1 mmol/L). The number of nerve fiber bundles extended from DRG explants decreased significantly in the presence of CS? (0.01 mmol/L, 15.00 ± 2.61, p < .05; 0.1 mmol/L, 11.17 ± 1.47, p < .001; 1 mmol/L, 8.00 ± 1.41, p < .001) as compared with that in the absence of CS? (17.83 ± 2.48). The number of neurons migrated from DRG explants decreased significantly in the presence of CS? (0.01 mmol/L, 79.50 ± 9.40, p < .01; 0.1 mmol/L, 62.50 ± 14.15, p < .001; 1 mmol/L, 34.67 ± 7.58, p < .001) as compared with that in the absence of CS? (99.33 ± 15.16). And also, the decreases in the number of nerve fiber bundles and migrated DRG neurons were in a dose-dependent manner of CS?. These data implicated that CS? could inhibit neurite outgrowth and neuronal migration from DRG explants in vitro.     

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.     

NT3基因转染对小鼠耳蜗螺旋神经节细胞生长的影响

目的通过神经营养素-3(NT3)基因转染的方法,了解NT3对耳蜗螺旋神经节细胞(SGC)生长的影响.方法建立体外培养小鼠耳蜗螺旋神经节细胞的方法,并进行神经丝蛋白(NF)免疫组化染色鉴定及受体酪氨酸激酶C(TrKC)免疫组化染色.利用阳离子脂质体作为载体,将重组质粒pIRES2-EGFP(增强型绿色荧光蛋白)-NT-3瞬时转染耳蜗螺旋神经节细胞,观察转染后耳蜗螺旋神经节细胞的形态、数量等改变.结果成功培养了小鼠耳蜗螺旋神经节细胞.NT3基因转染48 h后,荧光显微镜下见聚合分布的、胞体及轴突发出绿色荧光的螺旋神经节细胞,与未转染细胞相比,螺旋神经节细胞形态未见明显变化.继续培养2周后,各实验组细胞的数量均减少,但NT3转染组细胞减少的数量低于空载体组及空白对照组.结论 NT3基因转染可以在一定程度上减轻耳蜗螺旋神经节细胞的退行性改变.     

Vascular endothelial cell growth factors promote the in vitro development of rat photoreceptor cells.

We have identified and characterized a novel trophic effect of vascular endothelial cell growth factor (VEGF) on photoreceptor cells. Treatment of retinal cultures, derived from postnatal day 1 (P1) rats, with VEGF-2 resulted in a dose- and time-dependent increase in the level of rhodopsin protein, as determined by ELISA assay. After 7-9 d of treatment the VEGF-1 or VEGF-2, at a concentration of 10 ng/ml, induced a 200-300% increase in rhodopsin protein and a 220% increase in the number of rhodopsin-immunopositive cells. Treatment with VEGF-2 induced a 250% increase in the number of syntaxin-immunopositive cells and a 67% increase in high-affinity GABA uptake, both markers for amacrine cells. In contrast, there was no increase in the non-neuronal cell populations. VEGF-2 induced an approximately 300% increase in the number of bromodeoxyuridine-labeled (BrdU) retinal cells within 48 hr of treatment. After 3 d in culture both the basal and stimulated levels of BrdU incorporation were reduced, suggesting that the proliferative effect of VEGF was restricted developmentally. Furthermore, there was a developmentally dependent increase in the mitogenic response to VEGF-2, with retinal cultures derived from E15, E20, or P1 animals demonstrating a 50, 100, and 300% increase in thymidine incorporation, respectively. However, VEGF treatment resulted in an increase in the number of rhodopsin-immunopositive cells only when the cultures were derived from P1 animals. Therefore, retinal progenitor cells appear to be targets for VEGF, and thus VEGF may be involved in the regulation of the early developmental program of retinal neurogenesis.     

胰岛素样生长因子1与转化生长因子β2促进组织工程软骨构建的体外实验

背景：软骨组织的再生能力差,软骨组织工程能利用较少的细胞、支架材料和细胞因子对缺损进行修复。 目的：观察胰岛素样生长因子1与转化生长因子β2联合应用对组织工程软骨形成的影响。 方法：用酶消化法获取人软骨细胞,将培养的细胞以4×109 L-1的细胞浓度接种在藻酸钙凝珠支架上,分别加入200 μg/L胰岛素样生长因子1和(或)1 μg/L转化生长因子β2进行立体培养。于培养的第3,5,7,9,11,13天,进行细胞计数,观察软骨细胞的增殖情况。培养2周后,进行大体形态观察和阿尔新蓝-过碘酸雪夫氏染色 (AB-PAS)及抗Ⅱ型胶原免疫组织化学染色。 结果与结论：细胞计数及免疫组织化学染色显示,胰岛素样生长因子1和转化生长因子β2均能促进软骨细胞增殖和软骨相关基质黏多糖及Ⅱ型胶原的分泌,其中胰岛素样生长因子1的作用主要体现在促细胞增殖方面,而转化生长因子β2的作用主要体现在促进软骨相关基质形成方面,二者联合应用具有促进组织工程软骨形成的协同作用。     

IGF-I and FGF-2 coordinately enhance cyclin D1 and cyclin E-cdk2 association and activity to promote G1 progression in oligodendrocyte progenitor cells

A critical question in developmental neurobiology is how stem and progenitor cells interpret multiple signals to decide whether to proliferate or exit the cell cycle. Insulin-like growth factor (IGF)-I and fibroblast growth factor (FGF)-2 have known functions individually in development of neural stem cells as well as more restricted neuronal and glial progenitor cells. The goal of this study was to elucidate how IGF-I and FGF-2 coordinately regulate the cell cycle machinery in primary oligodendrocyte progenitors (OPs). IGF-I/FGF-2 synergistically increased the numbers of OP cells recruited into S phase. IGF-I enhanced FGF-2 induction of cyclin D1, activation of G(1) cyclin-cyclin-dependent kinase (cdk) complexes, and hyperphosphorylation of retinoblastoma protein (pRb). Moreover, IGF-I was required for G(2)/M progression. In contrast, FGF-2 decreased levels of the cdk inhibitor p27(Kip1) associated with cyclin E-cdk2. These studies provide a mechanistic basis for coordinate regulation of cell cycle progression in progenitor cells by multiple growth factors.     

Coordination of fibroblast growth factor receptor 1 (FGFR1) and fibroblast growth factor-2 (FGF-2) trafficking to nuclei of reactive astrocytes around cerebral lesions in adult rats

Traumatic injury to the adult central nervous system initiates a cascade of cellular and trophic events, culminating in the formation of a reactive gliotic scar through which transected axons fail to regenerate. Levels of fibroblast growth factor-2 (FGF-2), a potent gliogenic and neurotrophic factor, together with its full-length receptor, FGF receptor 1 (FGFR1) are coordinately and significantly increased postinjury in both nuclear and cytoplasmic fractions of extracted cerebral cortex biopsies after a penetrant injury. FGFR1 is colocalized with FGF-2 in the nuclei of reactive astrocytes, and here FGF-2 is associated with nuclear euchromatin. This study unequivocally demonstrates coordinate up-regulation and trafficking of FGF-2 and full-length FGFR1 to the nucleus of reactive astrocytes in an in vivo model of brain injury, thereby implicating a role in nuclear activity for these molecules. However, the precise contribution of nuclear FGF-2/FGFR1 to the pathophysiological response of astrocytes after injury is undetermined.     

Trophic effects of platelets on cultured endothelial cells are mediated by platelet-associated fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF)

In addition to their role in primary hemostasis, platelets serve to support and maintain the vascular endothelium. Platelets contain numerous growth factors including the potent angiogenic inducers VEGF and FGF-2. To characterize the function of these two platelet-associated growth factors, the effects of the addition of purified platelets to cultured endothelial cells were examined. The survival and proliferation of endothelial cells were markedly stimulated (2-3-fold and 5-15-fold respectively) by the addition of gel-filtered platelets. Acetylsalicylic acid-treated or lyophilized fixed-platelets were ineffective in supporting endothelial cell proliferation. In Transwell assays, the stimulatory effect of platelets on endothelial cells was preserved, consistent with an effect mediated by secreted factors. The combined inhibition of VEGF and FGF-2 by neutralizing antibodies, in contrast to inhibition of either alone, abrogated both platelet-induced endothelial cell survival and proliferation. FGF-2 isoforms were detected in platelet lysates, as well as in the releases of agonist-stimulated platelets. Megakaryocytes generated by ex vivo expansion of hematopoietic progenitor cells with kit ligand and thrombopoietin were analyzed for expression of FGF-2. Punctate cytoplasmic staining but no nuclear staining was observed by immunocytochemistry consistent with possible localization of the growth factor to cytoplasmic granules. The addition of platelets to cultured endothelial cells activated extracellular signal-regulated kinase (ERK) in a dose and time-dependent manner. This effect was abrogated by both anti-FGF-2 and anti-VEGF antibody. Since FGF-2 and VEGF are potent angiogenic factors and known endothelial cell survival factors, their release by platelets provides a plausible mechanism for the platelet support of vascular endothelium.     

Estrogen and insulin synergism in neurite growth enhancement in vitro: mediation of steroid effects by interactions with growth factors?

Addition of estradiol to organotypic cultures of the fetal murine hypothalamus, preoptic area and cerebral cortex has been shown to elicit a striking enhancement of neurite growth which appears restricted to estrogen receptor-containing explant regions. The mechanisms underlying this response are unknown. An important question is whether the neurite enhancement which follows exposure to estradiol is due directly to the interaction of estrogen with the cell that was stimulated (the receptor-containing cell) or whether intermediate steps involving the possible interaction of estrogen and the endogenous polypeptide neurite-promoting growth factors or their receptors may play an important role. Recent findings in the cultures suggest that the effect of estrogen on neurite growth may involve synergistic interactions between estradiol and insulin-related peptides and may be important in regulating estrogen-responsive neurite growth in the central nervous system. Concurrent addition of estradiol and high levels of insulin (10 micrograms/ml or 50 micrograms/ml) to cultures of the olfactory bulb, hypothalamus, preoptic area and cerebral cortex of the fetal rat and mouse results in a dramatic acceleration and increase of neurite outgrowth which appears localized to estrogen receptor-containing explant regions. The supraphysiological concentrations of insulin required to elicit this response suggest that the factor(s) involved is unlikely to be insulin per se. Insulin may activate the receptor of different but closely related molecules such as the insulin-like growth factors (IGF)-I or -II to which it exhibits a relatively low affinity. Interactions between hormones and endogenous growth factors have been implicated in the modulation or mediation of an increasing number of endocrine-dependent, differentiative processes in vivo and in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dibutyryl cAMP,interleukin-1β, and macrophage conditioned medium enhance the ability of astrocytes to promote neurite growth

Astrocytes purified from the neonatal rat brain were cultured for up to 3 weeks prior to being treated with agents that can induce reactive changes in astrocytes. These agents included dibutyryl cAMP, interleukin-1β, and macrophage conditioned media. After treating astrocytes for 3 days, the agents were removed and the ability of the astrocytes to support neurite growth was assessed by plating neonatal rat cerebellar cortical neurons. All the agents increased the ability of astrocytes to support the growth of long neurites. This was particularly evident with astrocytes treated with dibutyryl cAMP and LPS macrophageconditioned medium. These results provide direct evidence that the reactive changes induced in astrocytes in this in vitro model might aid the growth of neurites. Similar changes may underlie the injury-induced axonal sprouting that occurs in vivo. © 1994 Wiley-Liss, Inc.     

Fibroblast growth factor stimulates the proliferation and differentiation of neural precursor cells in vitro

We have developed an in vitro culture system to study the regulation of proliferation and differentiation of neural precursor cells contained within the neuroepithelium of embryonic day 10 mice. A number of soluble growth factors have been tested for their ability to regulate these early events and, of these factors, we have found that the fibroblast growth factors [FGFs] can directly stimulate the proliferation and survival of the neuroepithelial cells. At least 50% of the neuroepithelial cells divide in the presence of FGF whereas in the absence of FGF all of the cells die within 6 days of culture. At higher concentrations of FGF, the cells change from being nonadherent round cells in tight clusters into a more flattened cell type which adheres to the substratum. This morphological change is accompanied by the expression of both neurofilament and GFAP, which are definitive markers of the two major cell types in the central nervous system: neurons and glia. In addition a neuroepithelial cell line, which does not rely on FGF for survival or proliferation, expresses both of these markers in response to FGF. These results indicate that FGF is stimulating the differentiation of the neuroepithelial cells into mature neurons and glia.     

Fibroblast growth factor 1 (FGFR1) modulation regulates repair capacity of oligodendrocyte progenitor cells following chronic demyelination

The adult mammalian brain contains multiple populations of endogenous progenitor cell types. However, following CNS trauma or disease, the regenerative capacity of progenitor populations is typically insufficient and may actually be limited by non-permissive or inhibitory signals in the damaged parenchyma. Remyelination is the most effective and simplest regenerative process in the adult CNS yet is still insufficient following repeated or chronic demyelination. Our previous in vitro studies demonstrated that fibroblast growth factor receptor 1 (FGFR1) signaling inhibited oligodendrocyte progenitor (OP) differentiation into mature oligodendrocytes. Therefore, we questioned whether FGFR1 signaling may inhibit the capacity of OP cells to generate oligodendrocytes in a demyelinating disease model and whether genetically reducing FGFR1 signaling in oligodendrocyte lineage cells could enhance the capacity for remyelination. FGFR1 was found to be upregulated in the corpus callosum during cuprizone mediated demyelination and expressed on OP cells just prior to remyelination. Plp/CreER^T:Fgfr1^fl/fl mice were administered tamoxifen to induce conditional Fgfr1 deletion in oligodendrocyte lineage cells. Tamoxifen administration during chronic demyelination resulted in reduced FGFR1 expression in OP cells. OP proliferation and population size were not altered one week after tamoxifen treatment. Tamoxifen was then administered during chronic demyelination and mice were given a six week recovery period without cuprizone in the chow. After the recovery period, OP numbers were reduced and the number of mature oligodendrocytes was increased, indicating an effect of FGFR1 reduction on OP differentiation. Importantly, tamoxifen administration in Plp/CreER^T:Fgfr1^fl/fl mice significantly promoted remyelination and axon integrity. These results demonstrate a direct effect of FGFR1 signaling in oligodendrocyte lineage cells as inhibiting the repair capacity of OP cells following chronic demyelination in the adult CNS.     

Amygdala neurons in vitro: neurite growth and effects of estradiol.

Dissociated cell cultures from embryonic rat medial amygdala were studied using sequential photography and immunocytochemical staining for cytoskeletal proteins and substance P (sP). Cultures were seeded with cells taken from fetuses grouped by sex; experimental cultures were raised in medium containing 17-beta-estradiol (E2). Forty-eight hours after plating a few neurons begin to define their morphological polarity by the differentiation of an axon-like process; at 5 days in vitro (DIV) almost all neurons had developed an axon. Tapering, daughter branch ratio and branch power coefficient coincided with identification of dendrites which could be confirmed by retrospective analysis of immunocytochemically stained cultures: at 5 DIV MAP-2 was restricted to dendrites whereas Tau immunoreactivity was differentially localized with a clear predominance in the axon. At 21 DIV neuronal shape parameters were strikingly like those of amygdaloid neurons in vivo. It was demonstrated in living neurons that E2 increased total dendritic length and that this is due to increased ramification of third or higher order dendritic segments whose individual lengths are not different from controls. Densitometric measurement of MAP-2 stained neurons showed a highly significant increase of immunoreactive material in cells grown in the presence of E2; readings for alpha-tubulin were not different between controls and E2 treated cultures. The effect of E2 on dendritic length was just as manifest in sP-positive as in sP-negative neurons. No sexual differences in morphological parameters, growth characteristics or effects of E2 were found in neurons taken from female fetuses versus neurons from male fetuses. The significance of these results for the generation of sexual differences in the amygdala in vivo is discussed and contrasted with reported results on the effects of E2 in cultures of different neural regions.