Basic fibroblast growth factor enhances the growth of postnatal neostriatal GABAergic neurons in vitro

Basic fibroblast growth factor (bFGF) significantly enhances the short-term survival of embryonic striatal neurons in vitro but has little effect on the outgrowth of striatal cells compared to neurons from other brain regions. Studies in our laboratory have shown that bFGF protects postnatal striatal cells in vitro from NMDA receptor-induced neurotoxicity. We therefore examined the effects of bFGF on the outgrowth of GABA-containing cells taken from the postnatal (Day 1) caudate-putamen and cultured for up to 3 weeks. In control cultures GABAergic neurons formed three populations based on somatic size and developed the cytoarchitectural features characteristic of dendrites, spines, and axons. In the presence of bFGF (6 pM continuously from the day of plating), small- and medium-sized GABAergic neurons showed significant increases compared to untreated controls in axon-like growth (axon length) at 6 days in culture and in both axon- and dendrite-like neurite growth (axon length and branch order, number of primary dendrites, dendrite length, and dendritic branch order) at 13 and 17 days in culture. Large GABAergic neurons were unaffected by treatment with bFGF. Striatal GABAergic neurons exposed to nerve growth factor (10 ng/ml) were not different from untreated controls. Neuron survival was also unaffected by bFGF treatment at all days in culture examined. Other observations suggested that the neurotrophic effects of bFGF were mediated by a direct action of the growth factor on striatal neurons and not glial cells. First, glial cells (identified by the immunohistochemical localization of glial fibrillary acidic protein) were unaffected by bFGF treatment at the low concentration (6 pM) used to enhance neurite growth, but did significantly proliferate at higher concentrations of bFGF (6 nM). Second, immunoreactive bFGF receptor protein was localized predominantly to the somata and processes of striatal neurons and not to glial cells in the cultures. Finally, when neurons from control cultures were briefly exposed (1 to 4 h) to bFGF at concentrations which were neurotrophic, a marked elevation in the immediate early gene protein c-fos was observed by immunohistochemistry in the nuclei of neurons, including GABAergic cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro survival and differentiation of neurons derived from epidermal growth factor-responsive postnatal hippocampal stem cells: inducing effects of brain-derived neurotrophic factor

Neural stem cells proliferate in vitro and form neurospheres in the presence of epidermal growth factor (EGF), and are capable of differentiating into both neurons and glia when exposed to a substrate. We hypothesize that specific neurotrophic factors induce differentiation of stem cells from different central nervous system (CNS) regions into particular fates. We investigated differentiation of stem cells from the postnatal mouse hippocampus in culture using the following trophic factors (20 ng/mL): brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and glial-derived neurotrophic factor (GDNF). Without trophic factors, 32% of stem cells differentiated into neurons by 4 days in vitro (DIV), decreasing to 10% by 14 DIV. Addition of BDNF (starting at either day 0 or day 3) significantly increased neuron survival (31-43% by 14 DIV) and differentiation. Morphologically, many well-differentiated neurons resembled hippocampal pyramidal neurons. 5'-Bromodeoxyuridine labeling demonstrated that the pyramidal-like neurons originated from stem cells which had proliferated in EGF-containing cultures. However, similar application of NT-3 and GDNF did not exert such a differentiating effect. Addition of BDNF to stem cells from the postnatal cerebellum, midbrain, and striatum did not induce these neuronal phenotypes, though similar application to cortical stem cells yielded pyramidal-like neurons. Thus, BDNF supports survival of hippocampal stem cell-derived neurons and also can induce differentiation of these cells into pyramidal-like neurons. The presence of pyramidal neurons in BDNF-treated hippocampal and cortical stem cell cultures, but not in striatal, cerebellar, and midbrain stem cell cultures, suggests that stem cells from different CNS regions differentiate into region-specific phenotypic neurons when stimulated with an appropriate neurotrophic factor.     

Glucocorticoids differentially increase nerve growth factor and basic fibroblast growth factor expression in the rat brain

Adrenocorticotropin hormone (ACTH) and adrenal steroids may influence trophic processes operative in neuronal plasticity. Because nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) participate in neuronal trophism, we have investigated whether adrenal steroids induce the expression of these two trophic factors in the rat brain. The systemic administration of dexamethasone (DEX) elicited a rapid (within 3 hr) and sustained accumulation of bFGF and NGF mRNA in the cerebral cortex and hippocampus. Regional studies showed that DEX increases bFGF but not NGF mRNA in the cerebellum, striatum, and hypothalamus. In situ hybridization studies revealed that DEX increases NGF mRNA in superficial layers of the cerebral cortex and in the dentate gyrus of the hippocampus, and bFGF mRNA throughout the brain, suggesting that DEX induces NGF mRNA in neurons and bFGF in glial cells. ACTH administered systemically elicited a temporal and regional induction in NGF and bFGF mRNA similar to that obtained with DEX. Increases in NGF and bFGF mRNAs were also observed after administration of corticosterone and, albeit to a lesser extent, aldosterone, suggesting that the pituitary-adrenocortical axis plays an important role in the regulation of NGF and bFGF expression in the brain. Our data suggest that NGF and bFGF represent a link by which the adrenal cortical system can exert trophic action on the CNS.     

Cell death of spinal motoneurons in the chick embryo following deafferentation: rescue effects of tissue extracts, soluble proteins, and neurotrophic agents

In the absence of descending spinal and supraspinal afferent inputs, neurons in the developing lumbar spinal cord of the chick embryo undergo regressive changes including cellular atrophy and degeneration between embryonic days 10 and 16. There are significant decreases in the number of motoneurons, interneurons, and sensory (dorsal root ganglion) neurons. Although there are several possible explanations for how afferents might regulate the maintenance of neuronal viability, we have focused attention on the putative role of neurotrophic agents in these events. Previous studies have shown that specific tissue extracts (e.g., muscle, brain), soluble proteins, growth factors, and trophic agents can promote the in vitro and in vivo survival of avian motoneurons during the period of natural cell death (embryonic days 6-10). Several of these agents were also effective following deafferentation. These included brain extract (BEX), muscle extract (MEX), conditioned medium from astrocyte cultures (ACM), as well as the following neurotrophic agents: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), S-100, insulin-like growth factor-I (IGF-I), ciliary neurotrophic factor (CNTF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and leukemia inhibitory factor (CDF/LIF). Both transforming growth factor-beta (TGF-beta) and acidic fibroblast growth factor (aFGF) were ineffective. Although considerable more work is needed to determine which (and how) specific CNS-derived trophic agents regulate motoneuron survival, the present results are consistent with the notion that neurotrophic agents released from or modulated by synaptic inputs to target neurons promote neuronal differentiation and survival in the CNS.     

Distinctive morphological features of a subset of cortical neurons grown in the presence of basal forebrain neurons in vitro

Basal forebrain cholinergic neurons (BFCNs) provide the major subcortical source of cholinergic input to cerebral cortex and play an important role in regulating cortical activity. The present study examined the ability of BFCNs to influence neocortical neuronal growth by examining effects of the presence of BFCNs on certain cortical neurons grown under the controlled conditions of dissociated cell culture. Initial experiments demonstrated distinctive morphological features of a population of neurons (labeled with SMI-32, a monoclonal antibody to nonphosphorylated neurofilament proteins that labels pyramidal neurons in vivo) in cocultures containing basal forebrain (BF) and cortical cells. These neurons (large neurons immunoreactive for SMI-32 [SMI-32(+) neurons]) were characterized as having extensive axons, greater soma size, and more dendritic growth than did most SMI-32(+) neurons in the cultures. Staining for SMI-32 in cocultures in which the cortical neurons were labeled with a fluorescent marker before adding the BF cells indicated that virtually all large SMI-32(+) neurons were of cortical origin. Eliminating BFCNs with the selective cholinergic immunotoxin 192 IgG-saporin resulted in a >80% decrease in the number of large SMI-32(+) neurons, although causing little damage to other cells in the treated cultures; this suggests that survival or maintenance of large SMI-32(+) neurons may depend on ongoing trophic support from BFCNs. Thus, present findings suggest that BFCNs may provide powerful growth- and/or survival-enhancing signals to a subset of cortical neurons.     

A C-terminal region of the Saccharomyces cerevisiae transcription factor ADR1 plays an important role in the regulation of peroxisome proliferation by fatty acids

We examined the hypoxic tolerance phenomenon in vitro. Brief exposure to hypoxia induced the production of basic fibroblast growth factor (bFGF) mRNA and protein in rat cortical neurons and protected them from hypoxic injury. Cortical neurons were cultured from 18th-day rat embryos in a serum-free medium and subjected to brief (4 h) and/or prolonged (24 h) hypoxia. Neuronal damage was assessed by quantifying lactate dehydrogenase (LDH) activity in the medium. After brief hypoxia, LDH release was identical to that of the controls, whereas prolonged hypoxia caused a significant increase in LDH release, indicating neuronal death. However, if brief hypoxia was applied 2 days prior to the prolonged hypoxia, no increase in LDH release was observed. The bFGF mRNA expression was assessed with Northern blot and protein immunoreactivity with Western blot analysis. The brief period of hypoxia caused a 2.5-fold increase in bFGF mRNA and considerable bFGF protein expression 1 day later, but prolonged hypoxia caused increase in the expression of bFGF mRNA at 2 days and no protein expression until 3 days after the start of the hypoxia. When cells were subjected to prolonged hypoxia 2 days after brief hypoxia, however, no increase in bFGF mRNA was observed, while bFGF protein was expressed continuously. We also observed that exogenously applied bFGF reduced neuronal injury produced by prolonged hypoxia. The results obtained with this model suggest that brief hypoxia induces bFGF protein and thus tolerance to subsequent lethal hypoxia. Basic FGF might play a role as a tolerance-associated factor in this process. Thus, an in vitro model is useful for assessing the response of cortical neurons to hypoxic stress and for researching new factors related to ischemic tolerance.     

Establishment and characterization of a multipotential neural cell line that can conditionally generate neurons, astrocytes, and oligodendrocytes in vitro

In the mammalian central nervous system (CNS), multipotential neural stem cells in the neuroepithelium generate the three major types of neural cells, namely, neurons, astrocytes, and oligodendrocytes. To explore the molecular mechanisms underlying proliferation and differentiation of these neural stem cells, we established a cell line named MNS-57 from the embryonic day 12 rat neuroepithelium by introducing the mycer fusion gene, in which c-myc can be conditionally activated by adding oestrogen to the culture medium. MNS-57 cells expressed nestin, vimentin, and the RC1 antigen, which are potential markers for neural stem cells. We show that under particular culture conditions, MNS-57 cells can conditionally generate neurons, astrocytes, and oligodendrocytes in vitro, indicating that they are likely to originate from multipotential neural stem cells. Incubating MNS-57 cells with either oestrogen, which activates mycer, or growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) stimulated their growth, and the combination of oestrogen and bFGF (or EGF) had a synergistically stronger mitogenic effect than the single factors. Furthermore, both c-myc activation and bFGF appeared to be necessary for the differentiation of MNS-57 cells, and only when stimulated by both signals simultaneously, the cells committed to generating multiple neural cell types. Thus, the property of the cell line is unique in that its differentiation into neurons and glia can be conditionally manipulated in vitro in an exogenous signal-dependent manner. We propose that the cell line described here will provide an useful in vitro model to understand genetic and environmental mechanisms that control the generation of neural cell diversity in the CNS.     

Internalization of basic fibroblast growth factor (bFGF) in cultured endothelial cells: role of the low affinity heparin-like bFGF receptors

We have shown (Presta et al., Cell Regul., 2:719-726, 1991) that a long-lasting interaction of basic fibroblast growth factor (bFGF) with endothelial GM 7373 cells is required to induce cell proliferation. In the present work, we have investigated the interaction of 125I-bFGF with GM 7373 cells, its pathway of internalization, and its intracellular fate under the same experimental conditions previously utilized to assess the mitogenic activity of the growth factor. Cell cultures were incubated with 10 ng/ml 125I-bFGF for 2 h at 4 degrees C. Then, cells were shifted to 37 degrees C without changing the medium. A rapid down-regulation of high affinity sites, paralleled by a rapid internalization of 125I-bFGF, was observed during the first 1-2 h at 37 degrees C. After 6-8 h, also low affinity sites down-regulate. This was paralleled by a continuous internalization of 125I-bFGF and by a slow disappearance of the growth factor from the culture medium. This suggests that GM 7373 cells activate, when exposed to bFGF for a long period of time, a late internalization pathway mediated by low affinity sites. This hypothesis was supported by the following experimental evidence: 1) soluble heparin inhibited the prolonged internalization of 125I-bFGF and its binding to low affinity sites with the same potency; 2) treatment of GM 7373 cells with heparinase, which removes most of the low affinity sites, also inhibited the prolonged internalization of 125I-bFGF. 125I-bFGF internalized via low affinity sites was partially protected from lysosomal degradation. This was the case also when 125I-bFGF was internalized in the presence of soluble heparin, suggesting that the complexes bFGF-cell surface glycosaminoglycan and bFGF-soluble heparin are maintained during the internalization of the growth factor. Moreover, the capacity of soluble heparin to inhibit the mitogenic activity of bFGF also when added to cell cultures several hours after the growth factor indicates that the requirement for a prolonged interaction of bFGF with GM 7373 cells in order to induce cell proliferation might be related to the late internalization of the growth factor via low affinity sites.     

A novel neurotrophic pyrimidine compound MS-818 enhances neurotrophic effects of basic fibroblast growth factor

MS-818 (2-piperadino-6-methyl-5-oxo-5, 6-dihydro (7H) pyrrolo [2,3-d]pyrimidine maleate), a newly synthesized heterocyclic pyrimidine derivative, promotes neurite outgrowth in neuronal cell lines. The survival-promoting effect of MS-818 on cultured neurons isolated from mouse cortices was examined. MS-818 promoted neuronal survival by inhibiting apoptosis in a dose-dependent manner. MS-818 treatment also activated mitogen-activated protein kinase (MAPK) of the extracellular signal regulation kinase 2, as demonstrated by Western blot analysis. The MAPK activation level in the cultures treated with MS-818 was almost equivalent to that in cultures treated with nerve growth factor but was less than that in cultures treated with epidermal growth factor and basic fibroblast growth factor (bFGF). MAPK was activated within 3 min after the addition of MS-818, and its activity level returned to baseline within 120 min. Its activation was protein kinase C independent. We further investigated the effect of concurrent treatment with MS-818 and bFGF on neuronal survival. MS-818 enhanced the neuronal survival-promoting effect of bFGF in shifting the half-maximally effective dose from 2.1 ng/ml to 0.036 ng/ml in the sigmoidal dose effect of bFGF and permitted nearly maximum MAPK activation. The enhancement by MS-818 of the neuronal survival-promoting effect of bFGF was accompanied by sustained activation of MAPK to a degree that far exceeded, in magnitude and duration, the cooperative effect of MS-818 and bFGF. These results indicate that MS-818 promotes neuronal survival and enhances the neurotrophic actions of bFGF through stimulation of synchronous signals that may elevate MAPK levels within neurons.     

Involvement of protease-activated receptor-1 in the in vitro development of mesencephalic dopaminergic neurons

In situ hybridization studies have revealed high levels of protease (thrombin)-activated receptor-1 messenger RNA in the mesencephalon of rats, suggesting that dopaminergic neurons are a target for thrombin's actions. We have evaluated the effect of thrombin receptor activation, either by thrombin or by thrombin receptor agonist peptide, a 14 amino acid agonist of protease-activated receptor-1, on tyrosine hydroxylase-positive neurons. Pure cultures of rat mesencephalic neurons or co-cultures of mesencephalic neurons and glial cells were treated with either thrombin or thrombin receptor agonist peptide the day after plating. Tyrosine hydroxylase-positive cell counting, [3H]dopamine uptake and morphometric analysis were performed on day 5. Thrombin and thrombin receptor agonist peptide influenced neurite elongation, branching and the number of primary, secondary and tertiary neurites of tyrosine hydroxylase-positive neurons. In pure cultures, the most significant effects of thrombin and thrombin receptor agonist peptide were to delay branching and to increase the centrifugal growth of neurites without affecting the total neuritic length. Thrombin (up to 10 nM) and thrombin receptor agonist peptide did not affect the number of tyrosine hydroxylase-positive neurons or [3H]dopamine uptake. Neurotrophin-4 also influenced the morphology of tyrosine hydroxylase-positive neurons. The increase of neuritic length initiated by this neurotrophin is complementary to the radial elongation induced by protease-activated receptor-1 activation. When neurons were cultured in the presence of glial cells, the effects of thrombin and thrombin receptor agonist peptide on most of these parameters were larger than those observed with pure cultures. Thus, thrombin is able to initiate a complex remodelling of the architecture of tyrosine hydroxylase-positive neurons through the activation of protease-activated receptor-1. These results provide further support for the involvement of protease-activated receptor-1 activation in the development and differentiation of the central nervous system.     

NT-4/5 and LIF, but not NT-3 and BDNF, promote NPY mRNA expression in cortical neurons in the absence of spontaneous bioelectrical activity

Epigenetic factors are known to influence the differentiation of neocortical neurons. The present study analyses the role of spontaneous bioelectrical activity (SBA) and neurotrophic factors on the expression of neuropeptide Y (NPY) in rat visual cortical neurons using organotypic monocultures prepared from newborn animals and in situ hybridization to detect the NPY messenger ribonucleic acid (mRNA). Spontaneously active cortex cultures display NPY mRNA expression in about 7% of all cortical neurons from 10 days in vitro (DIV) on. Blocking the SBA by chronic application of 10 mM Mg2+ for 3-30 DIV reduces the percentage of NPY neurons to about 2%. Allowing an initial phase of SBA (1-20 DIV) followed by an SBA blockade (for 21-50 DIV) results in 2% labelled neurons, indicating a dramatic reduction of NPY mRNA expression in the absence of SBA. Surprisingly, the reverse experiment (a period of SBA blockade for 1-20 DIV followed by a period of SBA recovery for 21-40 DIV) does not cause an upregulation of NPY mRNA expression. However, allowing cultures to differentiate as spontaneously active cultures, then applying a transient period of SBA blockade which is followed by a second period of SBA, does rescue the NPY mRNA expression in 7% of the cortical neurons. We conclude that SBA is a main trigger for NPY mRNA expression and it is particularly important during an early postnatal period of differentiation. We then analysed whether neurotrophic factors known to modulate cortical neuropeptide expression are able to do so in the absence of SBA. Supplementing chronically blocked cultures with the neurotrophins, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5) and the cytokine, leukaemia inhibitory factor (LIF), reveals that BDNF and NT-3 are unable to increase the percentage of NPY neurons. In contrast, LIF and NT-4/5 increase the percentage of NPY neurons to 4 and 6-7%, respectively. Moreover, neurons treated with NT-4/5 display a very high level of NPY mRNA expression in somata and in the dendritic trees. The data suggest a complex interplay and a hierarchy of epigenetic factors in regulating the neurochemical architecture of the developing neocortex.     

Proliferation, differentiation, and long-term culture of primary hippocampal neurons

Primary embryonic hippocampal neurons can develop morphologically and functionally in culture but do not survive more than a few weeks. It has been reported that basic fibroblast growth factor (bFGF) promotes the survival of and neurite elongation from fetal hippocampal neurons. We report that bFGF, in a dose-dependent manner, can induce the survival (50 pg to 1 ng/ml) and proliferation (10-20 ng/ml) of embryonic hippocampal progenitor neurons in vitro. In serum-free medium containing high concentrations of bFGF, neurons not only proliferated (4-day doubling time) and differentiated morphologically but also could be passaged and grown as continuous cell lines. The neuronal nature of the proliferating cells was positively established by immunostaining with several different neuron-specific markers and by detailed ultrastructural analyses. The proliferative effect of bFGF was used to generate nearly pure neuronal cell cultures that can be passaged, frozen, thawed, and cultured again. Neurons have been maintained > 5 months in culture. The ability to establish long-term primary neuronal cultures offers the possibility that clonal lines of distinct neuronal cell types may be isolated from specific areas of the central nervous system. Such long-term neuronal cultures should prove valuable in studying neurons at the individual cell level and also in exploring interactions between neurons in vitro. The observed dose dependence raises the possibility that cell survival and proliferation in vivo may be influenced by different levels of bFGF.     

Increased immunoreactivities for the basic fibroblast growth factor and its receptor in astrocytes at the site of cerebral lesions and oedematous change in SHR

1. To obtain information about changes of basic fibroblast growth factor (bFGF) in the brain in chronic hypertension, we immunohistochemically studied the distribution and level of bFGF and its receptor in the brain of stroke-prone spontaneously hypertensive rats (SHRSP). 2. In the control normotensive rats, immunoreactivity for bFGF was demonstrated in nerve cells, while there was almost no reactivity in astrocytes. 3. In SHRSP, there was a marked immunoreactivity in the densely accumulated reactive cells, particularly astrocytes, in and around cerebral cortical lesions. Slightly increased reaction for bFGF was found in the nerve cells around lesions. Astrocytes in the subcortical white matter on both ipsi- and contralateral sides of the cortical lesion also showed immunoreactivity for bFGF. The location of increased bFGF expression in SHRSP corresponded very well with the site of extravasated plasma fluid demonstrated by anti-fibrinogen antibody. Electron microscopically, bFGF was shown in astrocytes along the rough endoplasmic reticulum suggesting the growth factor to be produced in the cells and not to be taken up from the surroundings. Expression of FGF-receptor was also demonstrated in reactive astrocytes in the oedematous cortical portion around lesion and in the oedematous subcortical white matter. 4. These findings indicate the possibility that oedema and the simultaneously generated free radicals or some extravasated plasma components express bFGF in astrocytes and probably in nerve cells as well as FGF-receptor in astrocytes, and that the thus expressed bFGF and its receptor play some role in the sequence of developmental events of hypertensive cerebral lesions.     

Morphological and morphometric analysis of serotonin-containing neurons in primary dissociated cultures of human rhombencephalon: a study of development

Primary dissociated cultures of rhombencephalon were prepared from 5-9-week-old human fetuses. Half of some cultures were treated by two non-competitive N-methyl-D-aspartate antagonists, namely 1-(2-thienyl)cyclohexylpiperidine (TCP) and cis-Pip/Mel-[1-(2-thienyl)-2-methyl-cyclohexyl]piperidine (GK11) in negative enantiomeric form, which enhance the survival of human fetal central nervous system cells in culture. At different days in vitro, the treated and the control cultures were processed for immunocytochemical detection of serotonin-containing neurons which were studied by morphological and morphometric analysis. Statistical analysis showed that the surface of the stained neurons increased as a function of two parameters of time, the gestational age of the cells and the duration of the cultures. The complexity of the shape of the serotonin neurons characterized by the shape factor, the number of bifurcations and the morphological feature (bipolar or multipolar) was found to increase with the gestational age. It appears that the in vitro development of the embryonic cells which represents stages of maturation and differentiation can be specifically evaluated. Such an analysis of fetal central nervous system cells improves the knowledge of factors important in grafting experiments. We verified that the two drugs do not appreciably alter the in vitro development of the treated cells; thus they may be considered as promising drugs for human neuroprotection.     

Messenger RNA and protein expression of basic fibroblast growth factor receptor after cortical ablation

In a previous report we demonstrated that basic fibroblast growth factor (bFGF), as a multipotent neurotrophic factor, could prevent retrograde degeneration of the thalamic neurons after ablation of the somatosensory cortex. To elucidate the mechanism of this bFGF action, we examined changes in FGF receptor (FGFR) mRNA (flg) expression with in situ hybridization. The FGF receptor protein was detected with the immunoblotting method. The FGFR mRNA expression was found to be diffusely increased in the affected cortex. Microscopic observation indicated that FGFR mRNA was expressed in several types of cortical cells including neurons and non-neuronal cells. This increase could be observed as early as 6 hours after surgery and lasted for 48 hours. In the thalamus, however no change in FGFR mRNA signals was observed. Western blotting detected a protein immunoreactive to anti-FGFR antibody. Samples from the periablated cortex showed an increase in FGFR protein. Samples from the thalamus, however, showed no difference in FGFR protein level between the lesion side and the contralateral side. Application of exogenous bFGF in Gelfoam to the cortical ablation cavity did not show any effect on the gene expression or protein level of FGFR. These results suggest that FGFR is diffusely induced throughout the injured cortex in the early phase after injury and that bFGF may play an important role after injury. Topically applied bFGF might thus modulate cellular responses in the cortex and have a neurotrophic effect on the affected thalamic neurons.     

Effects of basic fibroblast growth factor on hippocampal neurons after axonal injury

OBJECTIVE: Axons of adult central nervous system neurons fail to regenerate after diffuse axonal injury in head trauma. Basic fibroblast growth factor (bFGF) has been reported to enhance neuritic extensions after neuronal injury in immature nerve cells. To investigate the effects of bFGF on adult neurons and axonal reoutgrowth, differentiated nerve cells were axonally transected and bFGF was applied. DESIGN: Cell culture study with primary rat hippocampal neurons. MATERIALS AND METHODS: After axotomy, hippocampal cultures were maintained untreated or in the presence of 0.5, 1, 10, or 20 ng/mL bFGF and evaluated over a 7-day period after injury. MEASUREMENTS AND MAIN RESULTS: Seven days after injury, axotomy decreased cell survival to 65%, increased [3H]arachidonic acid release 1.8-fold from prelabeled cells, and showed negligible effects on neuronal dendrites. bFGF reduced this neurodegeneration at all doses applied. bFGF at 10 ng/mL most efficiently increased live cells to 85% and decreased [3H]arachidonic acid release from prelabeled cells to control values (p < 0.01, vs. damaged cells). Furthermore, 10 ng/mL bFGF induced axonal branching and the longest axonal re-extensions from 60 +/- 8 to 377 +/- 10 microns 7 days after injury (p < 0.01, vs. damaged cells). CONCLUSIONS: bFGF increased cell survival and supported axonal re-elongations in adult hippocampal neurons in vitro when applied after axotomy. bFGF may play a role in new therapeutic concepts for the management of axonal injury after head trauma.