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. 1999 Jul 15;19(14):6006-16.
doi: 10.1523/JNEUROSCI.19-14-06006.1999.

Stimulation of neonatal and adult brain neurogenesis by subcutaneous injection of basic fibroblast growth factor

J P Wagner  1 , I B Black, E DiCicco-Bloom
Affiliations

Stimulation of neonatal and adult brain neurogenesis by subcutaneous injection of basic fibroblast growth factor

J P Wagner et al. J Neurosci. .

Abstract

Mounting evidence indicates that extracellular factors exert proliferative effects on neurogenetic precursors in vivo. Recently we found that systemic levels of basic fibroblast growth factor (bFGF) regulate neurogenesis in the brain of newborn rats, with factors apparently crossing the blood-brain barrier (BBB) to stimulate mitosis. To determine whether peripheral bFGF affects proliferation during adulthood, we focused on regions in which neurogenesis persists into maturity, the hippocampus and the forebrain subventricular zone (SVZ). In postnatal day 1 (P1) rats, 8 hr after subcutaneous injection (5 ng/gm body weight), bFGF increased [(3)H]thymidine incorporation 70% in hippocampal and SVZ homogenates and elicited twofold increases in mitotic nuclei in the dentate gyrus and the dorsolateral SVZ, detected by bromodeoxyuridine immunohistochemistry. Because approximately 25% of proliferating hippocampal cells stimulated in vivo expressed neuronal traits in culture, bFGF-induced mitosis may reflect increased neurogenesis. bFGF effects were not restricted to the perinatal period; hippocampal DNA synthesis was stimulated by peripheral factor in older animals (P7-P21), indicating the persistence of bFGF-responsive cells and activity of peripheral bFGF into late development. To begin defining underlying mechanisms, pharmacokinetic studies were performed in P28 rats; bFGF transferred from plasma to CSF rapidly, levels rising in both compartments in parallel, indicating that peripheral factor crosses the BBB during maturity. Consequently, we tested bFGF in adults; peripheral bFGF increased the number of mitotic nuclei threefold in the SVZ and olfactory tract, regions exhibiting persistent neurogenesis. Our observations suggest that bFGF regulates ongoing neurogenesis via a unique, endocrine-like pathway, potentially coordinating neuron number and body growth, and potentially providing new approaches for treating damaged brain during development and adulthood.

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Figures

Fig. 1.
Fig. 1.
Effects of peripheral treatment with bFGF on [3H]thymidine incorporation in the hippocampal region and the forebrain SVZ on P1. After subcutaneous injection of bFGF (5 ng/gm body weight) at zero time and [3H]thymidine (5 μCi/gm) at 6 hr, brain regions were analyzed at 8 hr for thymidine incorporation. Data represent mean incorporation (cpm) + SEM in tissues obtained from three to four animals in each of two experiments. Results of two-way ANOVA (factors of treatment and region) indicate a global 90% elevation in DNA synthesis in response to bFGF (*p < 0.0002); this increase was equivalent in both regions (i.e., no treatment × region interaction).
Fig. 2.
Fig. 2.
Top. Effects of peripheral treatment with bFGF on mitotic nuclei in the hippocampal region and SVZ on P1. Representative photographs of tissue sections from the hippocampal region (A, B) or forebrain subventricular zone (C, D) taken from animals treated with vehicle (A, C) or bFGF (B, D) at zero time and BrdU at 6 hr. At 8 hr the whole brains were fixed, sectioned (5 μm), and stained immunohistochemically for BrdU incorporation; blacknuclei are positive, and pink nuclei are negative. bFGF treatment increased the number of BrdU-labeled cells in the hilus of the hippocampal region dentate gyrus (B) and the forebrain SVZ (D). Scale bars: B(A), D (C), 100 μm.
Fig. 4.
Fig. 4.
Bottom. Potential phenotype of hippocampal cells mitotically stimulated by peripheral bFGF in P1 rats. Afterin vivo treatment with bFGF and BrdU, cells from the hippocampal region were isolated in culture, fixed after 48 hr, and processed for dual-fluorescent immunocytochemistry. Cells are viewed under phase (top) or dark field (bottom); cells positive for BrdU appearyellowish-green (FITC), whereas phenotypic markers are red-orange (Texas Red). After bFGF treatment, BrdU-positive nuclei were found in cells expressing the neuronal markers TuJ1 and MAP-2 and the astrocytic marker GFAP. Scale bar, 100 μm.
Fig. 3.
Fig. 3.
Quantitation of peripheral bFGF effects on mitotic nuclei in the hippocampal region and SVZ on P1. A,B, The total number of BrdU-positive nuclei were counted in two regions of the hippocampal formation (A) and in the dorsolateral corner of the forebrain SVZ, depicted in one hemisphere (B). C,D, In the hippocampal formation, bFGF treatment significantly increased mitotic nuclei in both the hilus (C; region 1 in A) and stratum radiatum (D; region 2 inA); the magnitude of the effect was dependent on anatomical position in both regions. The global effect of bFGF in the neurogenetic hilus (67%) was significantly greater than the effect in the stratum radiatum (22%), as determined by three-way ANOVA [significant interaction of treatment × region;p < 0.0001;F(1,14) = 113.1]. E, In the forebrain SVZ, bFGF globally stimulated mitosis in the dorsolateral region 54%, with a similar dependence of effect on anatomical gradient. Data represent mean ± SEM from three to five sections from each of three to five animals at each region, anatomical zone, and treatment group; asterisks denote individual anatomical zones where bFGF treatment produced significant increases (attributed at p < 0.05) in the number of mitotic nuclei within a given region, determined post hoc only when significant effects of “region” and “anatomical zone” were observed (this occurred in all 3 areas).
Fig. 5.
Fig. 5.
Effects of peripheral bFGF in vivoon the proportion and phenotypic composition of responsive cells measured in vitro. A, Peripheral bFGF treatment in vivo more than doubled the proportion of BrdU-positive cells observed in culture without affecting total cell number. B, Within the BrdU-positive population, however, no change in the relative proportion of neurons and astrocytes was observed. Data represent mean + SEM of at least 100 cells counted in each of four dishes in each treatment group obtained from two experiments. There was no difference in total cell numbers among groups at 48 hr (data not shown). *p < 0.0001, one-way ANOVA.
Fig. 6.
Fig. 6.
Effects of peripheral bFGF on hippocampal region DNA synthesis in older rats. A, Rats received bFGF (5 ng/gm) and [3H]thymidine (5 μCi/gm) at each age. bFGF treatment increased the percent [3H]thymidine incorporation at each age [global effect of bFGF treatment by 2-way ANOVA, *p < 0.002;F(1,3) = 39.4]; the increase observed on P1 (65%) was significantly greater than the 25–30% increase measured at later ages (interaction of treatment × age, !p < 0.04). B, Dose–effect curve for peripheral bFGF in the P14 hippocampal region is shown. Although each dose tested significantly increased [3H]thymidine incorporation (global effect of bFGF independent of dose, *p < 0.03), the increase produced by 100 ng/gm bFGF (65%) was greater than the increase produced by the lower doses [determined post hoc after significant regression of log(dose bFGF), !p < 0.02]. Data represent mean + SEM of values obtained from three to nine animals in each treatment and age or dose group.
Fig. 7.
Fig. 7.
Distribution of bFGF after subcutaneous injection.A, B, P28 rats received 500 ng/gm peripheral bFGF, and at given times, CSF (A) and plasma (B) were collected, and bFGF was quantitated by radioimmunoassay. Significant levels were detected 2 and 4 hr after treatment in both compartments, whereas in the CSF, significant bFGF levels persisted for 24 hr. Data represent mean ± SEM from four to six animals at each time (p < 0.0001 by 1-way ANOVA).C, Similar kinetics was observed 4 hr after injection of a 25-fold lower dose of 125I-bFGF [% = (cpm/ml sample)/(total cpm of 125I − bFGF injected/gm)].
Fig. 8.
Fig. 8.
Effects of peripheral bFGF on DNA synthesis in multiple regions of the adult rat brain. P60 rats received bFGF (100 ng/gm) at zero time and [3H]thymidine (5 μCi/gm) and BrdU (100 μg/gm; Sigma) at 6 hr and were processed at 8 hr. bFGF treatment increased the percent [3H]thymidine incorporation in homogenates of the olfactory bulb/tract (OB/OT) and the forebrain SVZ (FB-SVZ); no effect was observed in the hippocampal region (HIPP) or the cerebellum (CB). Data represent mean + SEM from three animals in each treatment group (*p < 0.0001 by 1-way ANOVA).
Fig. 9.
Fig. 9.
Effect of peripheral bFGF on mitotic nuclei in neurogenetic regions of the adult rat brain. Representative photographs of tissue sections from the anterior olfactory nucleus (A, B) and the forebrain SVZ (C, D) taken from adult animals treated with vehicle (A, C) or with bFGF (100 ng/gm body weight; B, D) followed by BrdU as described in Figure 8. bFGF treatment increased the number of BrdU-labeled cells in the anterior olfactory nucleus (B) and the forebrain SVZ (D). Scale bars: B(A), D (C), 100 μm.
Fig. 10.
Fig. 10.
Quantitation of peripheral bFGF effects on mitotic nuclei within the anterior olfactory nucleus (AON) and the forebrain SVZ (FB-SVZ) in adult rats. Peripheral bFGF more than doubled the number of mitotic nuclei within both regions; this effect was equivalent in both regions, and no anatomical gradient in effect was observed (3-way ANOVA; no significant interactions of region and/or anatomical zone with treatment). Data represent counts taken from at least five sections from each of three rats within each treatment group and region [*p < 0.0001;F(1,5) = 35.7].
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