- Email: [email protected]
Increased expression and nuclear accumulation of basic fibroblast growth factor in primary cultured astrocytes following ischemic-like insults
Molecular Brain Research 71 Ž1999. 171–177 www.elsevier.comrlocaterbres
Research report
Increased expression and nuclear accumulation of basic fibroblast growth factor in primary cultured astrocytes following ischemic-like insults Xuan Liu, Xing-Zu Zhu
)
Department of Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 294 Tai-yuan Road Shanghai, 200031, China Accepted 18 May 1999
Abstract Basic fibroblast growth factor ŽbFGF. is a biological active polypeptide with potent trophic effects on neurons, glia and endothelial cells. In the present study, we examined the temporal expression profile of bFGF protein in cultured cortical astrocytes under ischemic-like conditions such as serum-free, glucose-free or glutamate application. A peak increase of bFGF level was observed at 24 h after the initiation of insults. A striking increase in the bFGF immunoreactivity and a moderate increase in the fibroblast growth factor receptor-1 ŽFGFR-1. immunoreactivity were also found in the astrocytes treated with serum- or glucose-deprivation or glutamate. The increased bFGF immunoreactivity and FGFR-1 immunoreactivity were mainly accumulated in the nuclei of astrocytes. The results suggest that the expression of bFGF and FGFR-1 in the astrocytes, especially in the nuclear interior, can be up-regulated under ischemic-like conditions and that the up-regulation of bFGF and FGFR-1 may play an important role in the maintenance and repair of the central nervous system ŽCNS. after ischemia. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Basic fibroblast growth factor; Fibroblast growth factor receptor; Astrocyte; Serum-free; Glucose-free; Glutamate
1. Introduction
immunoreactivities in the astrocytes was also examined by immunocytochemical staining.
Basic fibroblast growth factor ŽbFGF. is an 18 kDa polypeptide with multipotential trophic effects on neural, glia and endothelial cells. Abundant bFGF immunoreactivity has been found in the central nervous system ŽCNS. from the early development period through to the adulthood w5,10x. Several recent studies have shown that bFGF and fibroblast growth factor receptor-1 ŽFGFR-1. mRNA and protein levels were up-regulated after ischemia in the rodent brain w7,12,14,15,24,25,27,28x. The increased bFGF expression in brain was mainly associated with reactive astroglia in the regions surrounding the focal insult. In the present study, the temporal expression profile of bFGF protein in cultured astrocytes was examined under ischemic-like conditions. Three ischemic-like insults, serum-free, glucose-free and glutamate application, were preformed to compare their potentials of inducing bFGF expression. The spatial distribution of bFGF and FGFR-1
)
Corresponding author: [email protected]
Fax:
q 86-21-64370269;
E-mail:
2. Materials and methods 2.1. Enriched cortical astrocyte culture Astrocyte cultures were prepared from postnatal day 1 rat pups using the methods of Murphy w19x and Simmous and Purphy w23x with minor modifications. Briefly, cortices from a litter of pups were dissected, pooled and dissociated mechanically into cell suspensions. The cells were seeded into 162 cm2 tissue culture flasks ŽCostar. and allowed to plate for 3 days at 378C and 5% CO 2 in Dulbecco’s modified Eagle’s medium ŽDMEM.rF12 medium Ž1:1, Gibco. with 10% heat-inactivated fetal calf serum, 100 Urml penicillin and 100 mgrml streptomycin. Media were changed every 2–3 days until 10–11 days, at which time the flasks were shaken overnight at 200 rpm to remove contaminating cells, mainly microglia. The purity of the astroglial cultures was determined with glial fibril-
0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 9 . 0 0 1 8 0 - 1
X. Liu, X.-Z. Zhu r Molecular Brain Research 71 (1999) 171–177
172
added. The microplate was further incubated for 2 h and then washed to remove any unbound antibody–enzyme reagent. A substrate solution was added and incubated for 20 min at room temperature. The reaction was stopped by the addition of 2 N sulphuric acid. The absorbance of reaction product was measured at 450 nm. Both of the cell lysates and the culture medium were collected and assayed. To obtain cell lysates, astrocytic cell monolayers were scraped into PBS, pelleted and resuspended in PBS. The cells were then sonicated. The cellular debris was pelleted and the supernatant collected for analysis. Culture medium were collected and centrifuged to eliminate cell debris. 2.3. Protein assay The total protein concentration in each sample was determined using the method of Bradford w4x.
Fig. 1. Time-course of bFGF protein level of control group Ž`., serumfree group Že., glucose-free group ŽI. and 500 mM glutamate group Ž^. in cultured cortical astrocytes. Values are expressed as bFGF protein Žpg.rtotal protein Žmg., and are mean"S.E.M. U P - 0.05 compared with control Ž ns 3..
2.4. MTT assay The cell viability was evaluated with MTT 3-Ž4,5-dimethylthiazol-z-yl.-2,5-diphenyltetrazolium bromide4 assay w6x. Briefly, MTT Žsolved in PBS. was added to the culture medium to reach a final concentration of 0.5 mgrml. After the cells were incubated at 378C for 4 h, the culture medium containing MTT was removed. Dimethyl sulfoxide ŽDMSO. was added into each well and the absorbance at 540 nm was measured.
lary acidic protein ŽGFAP. staining. The cultures generated by this procedure were consistently ) 95% astrocytes. Purified astrocytes were detached from the flasks into cell suspension using 0.25% trypsin ŽGibco. and then subcultured on poly-L-lysine ŽSigma.-coated 96-well plates ŽCostar.. The density of the subcultured cells was 100,000 cellsr0.2 ml per well. After the cells were cultured for 24 h, the cells were treated with ischemic-like insult or vehicle.
2.5. Immuncytochemical staining 2.2. Enzyme-linked immunosorbent assay (ELISA) Immuncytochemical staining was performed using a rabbit polyclonal anti-bFGF antibody ŽSC-79-G, Santa Cruz Biotechnology, CA, USA, 1:50 dilution. and a mouse monoclonal anti-FGFR-1 antibody ŽGR 21, Calbiochem– Novabiochem Int., Cambridge, USA, 1:50 dilution.. In brief, the cells were washed with PBS for three times and then fixed for 30 min with 0.1 M PBS containing 4% paraformaldehyde. Following three rinses with PBS, cells were incubated for 20 min with methanol containing 0.3% H 2 O 2 to block endogenous peroxidase staining. Immunos-
The bFGF protein level was determined using ELISA kit bought from R & D System ŽMinneapolis, USA. with a detection limit of 1 pgrml bFGF protein. The assay procedure was conducted following the instruction. Briefly, samples or bFGF standards were pipetted into wells of the microplate precoated with monoclonal antibody specific for bFGF. After the microplate was incubated for 2 h at room temperature, the wells were washed and the enzyme-linked polyclonal antibody specific for bFGF was
Table 1 Induction of bFGF protein expression by glutamate at different concentrations in cultured cortical astrocytes Values are expressed as bFGF protein Žpg.rtotal protein Žmg., and are mean " S.E.M. Time Žh.
Control
3 6 12 24 48
2.28 " 0.11 2.33 " 0.15 2.27 " 0.18 2.26 " 0.13 2.24 " 0.19
U
P - 0.05 compared with control Ž n s 3..
Glutamate ŽmM. 20
100
200
500
2.27 " 0.17 2.28 " 0.14 2.75 " 0.67 2.42 " 0.13 2.23 " 0.17
2.29 " 0.17 2.41 " 0.11 2.34 " 0.66 2.86 " 0.17U 2.39 " 0.13
2.28 " 0.13 2.91 " 0.38U 3.14 " 0.56U 3.05 " 0.22U 2.31 " 0.25
2.23 " 0.26 3.10 " 0.50U 3.56 " 0.55U 3.55 " 0.45U 2.29 " 0.13
X. Liu, X.-Z. Zhu r Molecular Brain Research 71 (1999) 171–177
173
Table 2 Cell viability of cultured astrocytes Žabsorbance at 540 nm. after treatment with ischemic-like insults Values are expressed as A540 and are mean " S.E.M. Group
Vehicle Serum-free Glucose-free 20 mM glutamate 100 mM glutamate 200 mM glutamate 500 mM glutamate U
Time Žh. 3
6
12
24
48
1.31 " 0.07 1.28 " 0.09 1.26 " 0.11 1.30 " 0.09 1.29 " 0.05 1.29 " 0.08 1.33 " 0.06
1.27 " 0.04 1.29 " 0.06 1.19 " 0.05U 1.30 " 0.10 1.29 " 0.05 1.24 " 0.11 1.23 " 0.14
1.29 " 0.07 1.15 " 0.08U 1.12 " 0.10U 1.28 " 0.07 1.25 " 0.09 1.24 " 0.11 1.21 " 0.10
1.24 " 0.05 1.09 " 0.08UU 1.03 " 0.07UU 1.20 " 0.08 1.18 " 0.10 1.12 " 0.09U 1.10 " 0.06UU
1.25 " 0.06 0.88 " 0.10UU 0.76 " 0.05UU 1.21 " 0.07 1.12 " 0.11U 1.03 " 0.09UU 0.96 " 0.08UU
P - 0.05 compared with vehicle-treated group Ž n s 6.. P - 0.01 compared with vehicle-treated group Ž n s 6..
UU
taining was performed as follows: cells were permeabilized by a 5 min exposure to 0.2% Triton X-100 in PBS and incubated for 24 h at 48C with the primary antibody.
After three rinses, cultures were incubated with biotinylated goat anti-rabbit IgG Žfor bFGF staining. or biotinylated goat anti-mouse IgG Žfor FGFR-1 staining. at 378C
Fig. 2. bFGF immunoreactivity in cultured astrocytes at 24 h after treated with ischemic-like insults or vehicle. ŽA. GFAP staining to show the purity of astrocytes used; ŽB. negative control; ŽC. vehicle-treated; ŽD. serum-free-treated; ŽE. glucose-free-treated; ŽF. 500 mM glutamate-treated. For ŽA., =400; for ŽB–F., =200.
174
X. Liu, X.-Z. Zhu r Molecular Brain Research 71 (1999) 171–177
for 1 h. After three rinses, cultures were incubated with avidin-linked peroxidase complex at 378C for 1 h, rinsed, and then incubated with 3,3X-diaminobenzidine ŽDAB, 0.5 mgrml. at the presence of 0.003% H 2 O 2 for 4 min. In order to verify the specificity of the chromogen reaction, some dishes were processed identically except that the cells were incubated with the primary antibody solvent rather than the primary antibody Žnegative control.. No immunostaining was generated when the primary antibody solvent was used.
mate.; or Ž4. vehicle Žchange the normal medium into fresh normal medium.. At each designed time point Ž6, 12, 24 or 48 h after the initiation of treatment., cells of insult-treated wells or vehicle-treated wells were assayed. Each set of cells from an individual cell suspension was considered a single replicate Ž n.. The Student’s t-test was used to assess the effects of ischemic-like insults on bFGF protein level and cell viability. The criterion for statistical significance was set at P - 0.05.
2.6. Experiment design and statistical analysis
3. Results
Cell cultures were randomly treated with Ž1. serum-free insult Žchange the normal medium into fresh medium without serum.; Ž2. glucose-free insult Žchange the normal medium into fresh medium without glucose.; Ž3. glutamate-application insult Žchange the normal medium into fresh medium containing different concentrations of gluta-
3.1. Effect of ischemic-like insults on bFGF leÕel An increase in the bFGF level was found in the astrocyte lysates after the astrocytes were treated with the ischemic-like insults. When astrocytes were treated with serum-free medium, bFGF level in the cells was gradually
Fig. 3. FGFR-1 immunoreactivity in cultured astrocytes at 24 h after treated with ischemic-like insults or vehicle. ŽA. GFAP staining to show the purity of astrocytes used; ŽB. negative control; ŽC. vehicle-treated; ŽD. serum-free-treated; ŽE. glucose-free-treated; ŽF. 500 mM glutamate-treated. For ŽA., =400; for ŽB–F., =100.
X. Liu, X.-Z. Zhu r Molecular Brain Research 71 (1999) 171–177
increased. The level became significantly elevated at 24 h time point and followed by a decline to normal at 48 h time point ŽFig. 1.. When astrocytes were treated with glucose-free medium, bFGF level increased quickly and the increase became significant at 6 h time point. The level peaked at 24 h time point and then declined to normal at 48 h time point ŽFig. 1.. When astrocytes were treated with glutamate, the increase of bFGF level was dose-dependent. Glutamate at 20 mM had no effect on bFGF level while 500 mM glutamate induced a significant increase in the bFGF level. The increase was found at as early as 6 h time point, reached the peak at 24 h time point then declined at 48 h time point ŽTable 1, Fig. 1.. The bFGF protein levels in the culture medium of every culture were lower than the detection limit of the bFGF-assay kit. 3.2. Effect of ischemic-like insults on cell Õiability of astrocytes The MTT assay was used to show the extents of cellular damage of astrocytes after ischemic-like insults. When astrocytes were treated with serum-free medium, the decrease of cell viability became significant at 12 h time point. When astrocytes were treated with glucose-free medium, the cell viability decreased quickly and became significant at 6 h time point. The effect of glutamate on the cell viability was dose-dependent. Glutamate at 20 mM had no effect. While 100 mM glutamate only partially decreased the number of living cells, 200 or 500 mM glutamate significantly decreased the living cell number at 24 and 48 h time points ŽTable 2.. 3.3. Effect of ischemic-like insults on spatial distribution of bFGF and FGFR-1 expression in astrocytes When the distribution of bFGF immunoreactivity in the astrocytes was observed by immunocytochemical staining, bFGF immunoreactivity was at low level in vehicle-treated cells, predominately in the cytoplasm of astrocytes ŽFig. 2C.. All three insults induced significant increase in bFGF immunoreactivity. It is noteworthy that the increased bFGF immunoreactivity appeared mainly in the nuclei of astrocytes ŽFig. 2D,E and F.. The FGFR-1 immunoreactivity in vehicle-treated astrocytes was relatively low ŽFig. 3C.. After astrocytes were treated with serum- or glucose-free medium or glutamate, FGFR-1 immunoreactivity in the cells, especially in the nuclear interior, increased ŽFig. 3D,E and F..
4. Discussion Altered levels of several growth factors have been reported to play an important role in mediating growth and repair after CNS injury w8,21x. Among these factors, bFGF was a focus of study because administration of extraneous
175
bFGF can exhibit potent protective effects against ischemic injury w2,9,13,16,20,22,29,30,32 x. Though several papers have reported the increase of bFGF after ischemia in vivo, the induction of bFGF expression in astrocytes in vitro has not been examined in detail. In the present studies we demonstrated that ischemic-like insults could considerably induce bFGF expression in cultured astrocytes. The increase of bFGF was significant at as early as 6 h after glucose-free or 500 mM glutamate treatment. The bFGF level peaked at 24 h time point and then the level decreased and returned to normal at 48 h after the initiation of insults. The results indicated that the induction of bFGF was a relatively quick but transient event. Our results further demonstrated that some differences might exist between different ischemic-like insults. The glucose-free insult significantly increased the bFGF level at as early as 6 h time point while the increase of bFGF following serum-free insult became statistically significant only after 24 h treatment. Besides, the peak increase of bFGF by the glucose-free insult Ž1.43-fold of the control. was higher than that by the serum-free insult Ž1.23-fold of the control.. Glutamate induced a dose-dependent increase in the bFGF level in astrocytes. While glutamate at low concentration Ž20 mM. had no effect on the bFGF level, glutamate at high concentration Ž500 mM. induced an significant increase in the bFGF level at 6 h time point. The peak level of bFGF was 1.57-fold of the control. It has been well known that many factors including glucose- and oxygen-deprivation and glutamate-induced excitotoxicity could be involved in the cascade of ischemic injury of CNS. Since all the ischemic-like insults tested in our study were able to induce bFGF expression in astrocytes, we proposed that the increase of bFGF in vivo following ischemia might be the result of the effects of several factors. There were also differences in the extents of damage caused by different ischemic-like insults. Glucose-free insult exhibited a higher potential in causing cell death than serum-free insult. It seemed that the bFGF expression was correlated with the extent of cellular damage. However, the results from glutamate insult were different. Though 500 mM glutamate could quickly and significantly induce the expression of bFGF in astrocytes, the extent of cellular damage caused by the same concentration of glutamate was relatively slight. While the increase of bFGF expression became significant at as early as 6 h time point, the decrease of cell viability became statistically significant at 24 h time point. It seemed that glutamate might induce the bFGF expression by a mechanism different from that of glucose-free or serum-free insult. Unlike other growth factors, bFGF is not freely secreted from cells by classical mechanism w1,31x. Accordingly, only very low level of bFGF-like activity could be detected in conditioned medium of cells that produce bFGF w18,31x. It has been proposed that bFGF might have both exocrine and intracrine Žautocrine. actions. On one hand,
176
X. Liu, X.-Z. Zhu r Molecular Brain Research 71 (1999) 171–177
the extracellular bFGF Žmainly released from dead cells. may interact with its receptor on the plasma membrane of the intact cells and undergo receptor-mediated actions. On the other hand, the intracellular bFGF may also regulate the proliferation of the cell by moving from the cytoplasm into the nuclear and changing the expression of many other growth factors w3,11,26x. Our results that bFGF could only be detected in the cells but not the medium and that the increased bFGF immunoreactivity after ischemic-like insults appeared mainly in the nuclei of astrocytes further support the proposal. In the case of ischemia in vivo, the nuclear accumulation of bFGF in astrocytes may induce the expression and release of other growth factors to protect themselves as well as surrounding neurons against the injury. To explain the effect of nuclear bFGF, the presence of nuclear FGFRs was also postulated. Evidence has shown that quiescent human astrocytes had little or no nuclear FGFRs. However, an induction of nuclear FGFRs was observed in reactive astrocytes w26x. FGFR-1 is one of the high-affinity receptors of bFGF. It is widely distributed in the intact rodent brain and becomes localized on reactive astroglia after brain injury or ischemia. It has been found that both the number of bFGF mRNA-positive cells and the number of FGFR-1-positive cells increased after ischemia. The time course of the appearance and distribution of FGFR-1 mRNA-positive cells were quite similar to those of bFGF mRNA-positive cells w7,17,24,28x. In our in vitro studies, we found that all three insults also induced a moderate increase in FGFR-1 immunoreactivity while the same treatment induced a significant increase in bFGF immunoreactivity. In summary, our results demonstrated that the three ischemic-like insults, serum-free, glucose-free or glutamate could induce an up-regulation in the bFGF and FGFR-1 expression in astrocytes in vitro. The induction of bFGF was a relatively quick but transient event. Some differences may exist when astrocytes were treated with different insults. Glutamate may induce the bFGF expression by a mechanism different from that of glucose-free or serumfree insult. Our results also suggest that the increased astrocytic bFGF levels might act on the astrocytes via autocrine mechanism and that the increased bFGF and FGFR-1 expression may play an important role in the maintenance and repair of the CNS after ischemia.
w4x
w5x
w6x
w7x
w8x
w9x
w10x
w11x
w12x
w13x
w14x
w15x
w16x
w17x
w18x
References w19x w1x J.A. Abraham, A. Mergia, J.L. Whang, A. Tumolo, J. Friedman, K.A. Hjerrild, D. Gospodarowiez, J.C. Fiddes, Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor, Science 233 Ž1986. 545–548. w2x A. Bethel, J.R. Kirsch, R.C. Koehler, S.P. Finklestein, R.J. Traystman, Intravenous basic fibroblast growth factor decrease brain injury resulting from focal ischemia in cats, Stroke 28 Ž1997. 609–616. w3x A. Bikfalvi, S. Kleen, G. Pintucci, N. Quatro, P. Mignatti, D.B. Rifkin, Differential modulation of cell phenotype by different molec-
w20x
w21x
ular weight forms of basic fibroblast growth factor: possible intracellular signaling by the high molecular weight forms, J. Cell Biol. 129 Ž1995. 233–243. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 Ž1976. 248–254. C.G. Caday, M. Klagsburn, P.J. Fanning, A. Mirzabegian, S.P. Finklestein, Fibroblast growth factor ŽFGF. levels in the developing rat brain, Brain Res. Dev. Brain Res. 52 Ž1990. 241–246. J. Carmichael, W.G. Degraff, A.F. Gazdar, J.D. Minna, J.B. Mitchell, Evaluation of a Tetrazolium-based semi-automated colorimetric assay: assessment of chemosensitivity testing, Cancer Res. 47 Ž1987. 936–942. M. Emdoh, W.A. Pulsinelli, J.A. Wagner, Transient global ischemia induces dynamic changes in the expression of bFGF and the FGF receptor, Mol. Brain Res. 22 Ž1994. 76–88. S.P. Finklestein, C.G. Caday, M. Kano, D.J. Berlove, C.Y. Hsu, M. Moskowitz, Klagsbrun, Growth factor expression after stroke, Stroke 21 Ž1990. 122–124. M. Fisher, M.E. Meadows, T. Do, J. Weise, V. Trubetskoy, M. Charette, S.P. Finklestein, Delayed treatment with intravenous basic fibroblast growth factor reduces infarct size following permanent focal ischemia in rats, J. Cereb. Blood Flow Metab. 15 Ž1995. 953–959. A.M. Gonzalez, M. Buscaglia, M. Ong, A. Baired, Distribution of basic fibroblast growth factor in the 18-day rat fetus: localization in the basement membranes of diverse tissues, J. Cell Biol. 110 Ž1990. 753–765. A. Joy, J. Moffett, K. Neary, E. Mordechai, E.K. Stachowiak, S. Coons, J. Rankin-Shapiro, R.Z. Florkiewilz, M.K. Stachowiak, Nuclear accumulation of FGF-2 is associated with proliferation of human astrocytes and glioma cell, Oncogene 17 Ž1997. 171–183. Y. Kiyota, K. Takami, M. Iwane, A. Shino, M. Miyomoto, R. Tsukuda, A. Nagaoka, Increase in basic fibroblast growth factor-like immunoreactivity in rat brain after forebrain ischemia, Brain Res. 545 Ž1991. 322–328. N. Koketsu, D.J. Berlove, M.A. Moskowitz, N.W. Kowall, C.G. Caday, S.P. Finklestein, Pretreatment with intraventricular basic fibroblast growth factor decrease infarct size following focal cerebral ischemia in rats, Ann. Neurol. 35 Ž1994. 451–457. T.N. Lin, J. Te, M. Lee, G.Y. Sun, C.Y. Hsu, Induction of basic fibroblast growth factor ŽbFGF. expression following focal cerebral ischemia, Mol. Brain Res. 49 Ž1997. 255–265. A. Lippoldt, B. Andbjer, L. Rosen, E. Richter, D. Ganten, Y. Cao, R.F. Petterson, K. Fuxe, Photochemically induced focal cerebral ischemia in rat, time dependent and global increase in expression of bFGF mRNA, Brain Res. 625 Ž1993. 45–56. X. Liu, X.Z. Zhu, Basic fibroblast growth factor protected forebrain against ischemia-reperfusion damage in rats, Acta Pharmacol. Sin. 19 Ž1998. 527–530. A. Logan, S.A. Frautschy, A.M. Gonzalez, A. Baird, A time course for the focal elevation of synthesis of basic fibroblast growth factor and one of its high-affinity receptors Žflg. following a localized cortical brain injury, J. Neurosci. 12 Ž1992. 3827–3828. D. Moscatelli, M. Presta, J. Joseph-Silverstein, D.B. Rifkin, Both normal and tumor cells produce basic fibroblast growth factor, J. Cell Physiol. 129 Ž1986. 273–276. S. Murphy, Generation of astrocyte cultures from normal and neoplastic central nervous system, in: P.M. Coon ŽEd.., Cell Culture, Academic Press, San Diego, 1990, pp. 45–56. N. Nakata, H. Kato, K. Kogure, Protective effects of basic fibroblast growth factor against hippocampal neuronal damage following cerebral ischemia in the gerbil, Brain Res. 605 Ž1995. 354–356. M. Nieto-Sampedro, Growth factor induction and order of event in CNS repair, in: D.G. Stain, B.S. Sabei ŽEds.., Pharmacological Approaches to the Treatment of Brain and Spinal Cord Injury, Plenum, New York, 1988, pp. 301–337.
X. Liu, X.-Z. Zhu r Molecular Brain Research 71 (1999) 171–177 w22x J.M. Ren, S.P. Finklestein, Time window of infarct reduction by intravenous basic fibroblast growth factor in focal cerebral ischemia, Eur. J. Pharmacol. 327 Ž1997. 11–16. w23x M. Simmous, S. Purphy, Protein kinases in the induction of nitric oxide synthase in astrocytes, Glia 11 Ž1994. 227–234. w24x E.K. Speliotes, C.G. Caday, T. Do, J. Weise, N.W. Kowall, S.P. Finklestein, Increased expression of basic fibroblast growth factor ŽbFGF. following cerebral infarction in the rat, Mol. Brain Res. 39 Ž1996. 31–42. w25x E.K. Speliotes, N.W. Kowall, C.G. Caday, B.F. Shanti, S.P. Finklestein, Time course of changes in basic fibroblast growth factor ŽbFGF. immunoreactivity in global hippocampus following global cerebral ischemia, Ann. Neurol. 34 Ž1993. 287, ŽAbstr... w26x E.K. Stachowiak, P.A. Maher, J. Tucholiski, E. Mordechai, A. Joy, J. Moffett, S. Coons, M.K. Stachowiak, Nuclear accumulation of fibroblast growth factor receptors in human glial cells — association with cell proliferation, Oncogene 14 Ž1997. 2201–2211. w27x K. Takami, M. Iwane, Y. Kiyota, M. Miyamoto, R. Tsukuda, S. Shiosaka, Increase of basic fibroblast growth factor immunoreactivity and its mRNA level in rat brain following transient forebrain ischemia, Exp. Brain Res. 90 Ž1992. 1–10.
177
w28x K. Takami, Y. Kiyota, M. Iwane, M. Miyamoto, R. Tsukuda, K. Igarashi, A. Shino, A. Wanaka, S. Shisaka, M. Tohyama, Up-regulation of fibroblast growth factor–receptor message RNA expression in rat brain following forebrain ischemia, Exp. Brain Res. 97 Ž1993. 185–194. w29x R. Tanaka, Y. Miyasaka, K. Yada, T. Ohwada, T. Kameya, Basic fibroblast growth factor increases regional cerebral blood flow and reduces infarct size after experimental ischemia in a rat model, Stroke 26 Ž1995. 2154–2159. w30x T. Tatlisumak, K. Takano, R.A. Carano, M. Fisher, Effect of basic fibroblast growth factor on experimental focal ischemia studied by diffusion-weighted and perfusion imaging, Stroke 27 Ž1996. 2292– 2298. w31x I. Vlodavskey, R. Bar-Shavit, R. Ishai-Michaeli, P. Bashkin, Z. Fuks, Extracellular sequestion and release of fibroblast growth factor: a regulating mechanism, Trends Biochem. Sci. 16 Ž1991. 268– 271. w32x T.C. Wen, S. Matsuda, H. Yoshimura, J. Aburaya, F. Kushihata, M. Sakanaka, Protective effect of basic fibroblast growth factor-heparin and neurotoxic effect of platelet factor 4 on ischemic neuronal loss and learning disability in gerbils, Neuroscience 65 Ž1995. 513–521.
Research report
Increased expression and nuclear accumulation of basic fibroblast growth factor in primary cultured astrocytes following ischemic-like insults Xuan Liu, Xing-Zu Zhu
)
Department of Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 294 Tai-yuan Road Shanghai, 200031, China Accepted 18 May 1999
Abstract Basic fibroblast growth factor ŽbFGF. is a biological active polypeptide with potent trophic effects on neurons, glia and endothelial cells. In the present study, we examined the temporal expression profile of bFGF protein in cultured cortical astrocytes under ischemic-like conditions such as serum-free, glucose-free or glutamate application. A peak increase of bFGF level was observed at 24 h after the initiation of insults. A striking increase in the bFGF immunoreactivity and a moderate increase in the fibroblast growth factor receptor-1 ŽFGFR-1. immunoreactivity were also found in the astrocytes treated with serum- or glucose-deprivation or glutamate. The increased bFGF immunoreactivity and FGFR-1 immunoreactivity were mainly accumulated in the nuclei of astrocytes. The results suggest that the expression of bFGF and FGFR-1 in the astrocytes, especially in the nuclear interior, can be up-regulated under ischemic-like conditions and that the up-regulation of bFGF and FGFR-1 may play an important role in the maintenance and repair of the central nervous system ŽCNS. after ischemia. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Basic fibroblast growth factor; Fibroblast growth factor receptor; Astrocyte; Serum-free; Glucose-free; Glutamate
1. Introduction
immunoreactivities in the astrocytes was also examined by immunocytochemical staining.
Basic fibroblast growth factor ŽbFGF. is an 18 kDa polypeptide with multipotential trophic effects on neural, glia and endothelial cells. Abundant bFGF immunoreactivity has been found in the central nervous system ŽCNS. from the early development period through to the adulthood w5,10x. Several recent studies have shown that bFGF and fibroblast growth factor receptor-1 ŽFGFR-1. mRNA and protein levels were up-regulated after ischemia in the rodent brain w7,12,14,15,24,25,27,28x. The increased bFGF expression in brain was mainly associated with reactive astroglia in the regions surrounding the focal insult. In the present study, the temporal expression profile of bFGF protein in cultured astrocytes was examined under ischemic-like conditions. Three ischemic-like insults, serum-free, glucose-free and glutamate application, were preformed to compare their potentials of inducing bFGF expression. The spatial distribution of bFGF and FGFR-1
)
Corresponding author: [email protected]
Fax:
q 86-21-64370269;
E-mail:
2. Materials and methods 2.1. Enriched cortical astrocyte culture Astrocyte cultures were prepared from postnatal day 1 rat pups using the methods of Murphy w19x and Simmous and Purphy w23x with minor modifications. Briefly, cortices from a litter of pups were dissected, pooled and dissociated mechanically into cell suspensions. The cells were seeded into 162 cm2 tissue culture flasks ŽCostar. and allowed to plate for 3 days at 378C and 5% CO 2 in Dulbecco’s modified Eagle’s medium ŽDMEM.rF12 medium Ž1:1, Gibco. with 10% heat-inactivated fetal calf serum, 100 Urml penicillin and 100 mgrml streptomycin. Media were changed every 2–3 days until 10–11 days, at which time the flasks were shaken overnight at 200 rpm to remove contaminating cells, mainly microglia. The purity of the astroglial cultures was determined with glial fibril-
0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 9 . 0 0 1 8 0 - 1
X. Liu, X.-Z. Zhu r Molecular Brain Research 71 (1999) 171–177
172
added. The microplate was further incubated for 2 h and then washed to remove any unbound antibody–enzyme reagent. A substrate solution was added and incubated for 20 min at room temperature. The reaction was stopped by the addition of 2 N sulphuric acid. The absorbance of reaction product was measured at 450 nm. Both of the cell lysates and the culture medium were collected and assayed. To obtain cell lysates, astrocytic cell monolayers were scraped into PBS, pelleted and resuspended in PBS. The cells were then sonicated. The cellular debris was pelleted and the supernatant collected for analysis. Culture medium were collected and centrifuged to eliminate cell debris. 2.3. Protein assay The total protein concentration in each sample was determined using the method of Bradford w4x.
Fig. 1. Time-course of bFGF protein level of control group Ž`., serumfree group Že., glucose-free group ŽI. and 500 mM glutamate group Ž^. in cultured cortical astrocytes. Values are expressed as bFGF protein Žpg.rtotal protein Žmg., and are mean"S.E.M. U P - 0.05 compared with control Ž ns 3..
2.4. MTT assay The cell viability was evaluated with MTT 3-Ž4,5-dimethylthiazol-z-yl.-2,5-diphenyltetrazolium bromide4 assay w6x. Briefly, MTT Žsolved in PBS. was added to the culture medium to reach a final concentration of 0.5 mgrml. After the cells were incubated at 378C for 4 h, the culture medium containing MTT was removed. Dimethyl sulfoxide ŽDMSO. was added into each well and the absorbance at 540 nm was measured.
lary acidic protein ŽGFAP. staining. The cultures generated by this procedure were consistently ) 95% astrocytes. Purified astrocytes were detached from the flasks into cell suspension using 0.25% trypsin ŽGibco. and then subcultured on poly-L-lysine ŽSigma.-coated 96-well plates ŽCostar.. The density of the subcultured cells was 100,000 cellsr0.2 ml per well. After the cells were cultured for 24 h, the cells were treated with ischemic-like insult or vehicle.
2.5. Immuncytochemical staining 2.2. Enzyme-linked immunosorbent assay (ELISA) Immuncytochemical staining was performed using a rabbit polyclonal anti-bFGF antibody ŽSC-79-G, Santa Cruz Biotechnology, CA, USA, 1:50 dilution. and a mouse monoclonal anti-FGFR-1 antibody ŽGR 21, Calbiochem– Novabiochem Int., Cambridge, USA, 1:50 dilution.. In brief, the cells were washed with PBS for three times and then fixed for 30 min with 0.1 M PBS containing 4% paraformaldehyde. Following three rinses with PBS, cells were incubated for 20 min with methanol containing 0.3% H 2 O 2 to block endogenous peroxidase staining. Immunos-
The bFGF protein level was determined using ELISA kit bought from R & D System ŽMinneapolis, USA. with a detection limit of 1 pgrml bFGF protein. The assay procedure was conducted following the instruction. Briefly, samples or bFGF standards were pipetted into wells of the microplate precoated with monoclonal antibody specific for bFGF. After the microplate was incubated for 2 h at room temperature, the wells were washed and the enzyme-linked polyclonal antibody specific for bFGF was
Table 1 Induction of bFGF protein expression by glutamate at different concentrations in cultured cortical astrocytes Values are expressed as bFGF protein Žpg.rtotal protein Žmg., and are mean " S.E.M. Time Žh.
Control
3 6 12 24 48
2.28 " 0.11 2.33 " 0.15 2.27 " 0.18 2.26 " 0.13 2.24 " 0.19
U
P - 0.05 compared with control Ž n s 3..
Glutamate ŽmM. 20
100
200
500
2.27 " 0.17 2.28 " 0.14 2.75 " 0.67 2.42 " 0.13 2.23 " 0.17
2.29 " 0.17 2.41 " 0.11 2.34 " 0.66 2.86 " 0.17U 2.39 " 0.13
2.28 " 0.13 2.91 " 0.38U 3.14 " 0.56U 3.05 " 0.22U 2.31 " 0.25
2.23 " 0.26 3.10 " 0.50U 3.56 " 0.55U 3.55 " 0.45U 2.29 " 0.13
X. Liu, X.-Z. Zhu r Molecular Brain Research 71 (1999) 171–177
173
Table 2 Cell viability of cultured astrocytes Žabsorbance at 540 nm. after treatment with ischemic-like insults Values are expressed as A540 and are mean " S.E.M. Group
Vehicle Serum-free Glucose-free 20 mM glutamate 100 mM glutamate 200 mM glutamate 500 mM glutamate U
Time Žh. 3
6
12
24
48
1.31 " 0.07 1.28 " 0.09 1.26 " 0.11 1.30 " 0.09 1.29 " 0.05 1.29 " 0.08 1.33 " 0.06
1.27 " 0.04 1.29 " 0.06 1.19 " 0.05U 1.30 " 0.10 1.29 " 0.05 1.24 " 0.11 1.23 " 0.14
1.29 " 0.07 1.15 " 0.08U 1.12 " 0.10U 1.28 " 0.07 1.25 " 0.09 1.24 " 0.11 1.21 " 0.10
1.24 " 0.05 1.09 " 0.08UU 1.03 " 0.07UU 1.20 " 0.08 1.18 " 0.10 1.12 " 0.09U 1.10 " 0.06UU
1.25 " 0.06 0.88 " 0.10UU 0.76 " 0.05UU 1.21 " 0.07 1.12 " 0.11U 1.03 " 0.09UU 0.96 " 0.08UU
P - 0.05 compared with vehicle-treated group Ž n s 6.. P - 0.01 compared with vehicle-treated group Ž n s 6..
UU
taining was performed as follows: cells were permeabilized by a 5 min exposure to 0.2% Triton X-100 in PBS and incubated for 24 h at 48C with the primary antibody.
After three rinses, cultures were incubated with biotinylated goat anti-rabbit IgG Žfor bFGF staining. or biotinylated goat anti-mouse IgG Žfor FGFR-1 staining. at 378C
Fig. 2. bFGF immunoreactivity in cultured astrocytes at 24 h after treated with ischemic-like insults or vehicle. ŽA. GFAP staining to show the purity of astrocytes used; ŽB. negative control; ŽC. vehicle-treated; ŽD. serum-free-treated; ŽE. glucose-free-treated; ŽF. 500 mM glutamate-treated. For ŽA., =400; for ŽB–F., =200.
174
X. Liu, X.-Z. Zhu r Molecular Brain Research 71 (1999) 171–177
for 1 h. After three rinses, cultures were incubated with avidin-linked peroxidase complex at 378C for 1 h, rinsed, and then incubated with 3,3X-diaminobenzidine ŽDAB, 0.5 mgrml. at the presence of 0.003% H 2 O 2 for 4 min. In order to verify the specificity of the chromogen reaction, some dishes were processed identically except that the cells were incubated with the primary antibody solvent rather than the primary antibody Žnegative control.. No immunostaining was generated when the primary antibody solvent was used.
mate.; or Ž4. vehicle Žchange the normal medium into fresh normal medium.. At each designed time point Ž6, 12, 24 or 48 h after the initiation of treatment., cells of insult-treated wells or vehicle-treated wells were assayed. Each set of cells from an individual cell suspension was considered a single replicate Ž n.. The Student’s t-test was used to assess the effects of ischemic-like insults on bFGF protein level and cell viability. The criterion for statistical significance was set at P - 0.05.
2.6. Experiment design and statistical analysis
3. Results
Cell cultures were randomly treated with Ž1. serum-free insult Žchange the normal medium into fresh medium without serum.; Ž2. glucose-free insult Žchange the normal medium into fresh medium without glucose.; Ž3. glutamate-application insult Žchange the normal medium into fresh medium containing different concentrations of gluta-
3.1. Effect of ischemic-like insults on bFGF leÕel An increase in the bFGF level was found in the astrocyte lysates after the astrocytes were treated with the ischemic-like insults. When astrocytes were treated with serum-free medium, bFGF level in the cells was gradually
Fig. 3. FGFR-1 immunoreactivity in cultured astrocytes at 24 h after treated with ischemic-like insults or vehicle. ŽA. GFAP staining to show the purity of astrocytes used; ŽB. negative control; ŽC. vehicle-treated; ŽD. serum-free-treated; ŽE. glucose-free-treated; ŽF. 500 mM glutamate-treated. For ŽA., =400; for ŽB–F., =100.
X. Liu, X.-Z. Zhu r Molecular Brain Research 71 (1999) 171–177
increased. The level became significantly elevated at 24 h time point and followed by a decline to normal at 48 h time point ŽFig. 1.. When astrocytes were treated with glucose-free medium, bFGF level increased quickly and the increase became significant at 6 h time point. The level peaked at 24 h time point and then declined to normal at 48 h time point ŽFig. 1.. When astrocytes were treated with glutamate, the increase of bFGF level was dose-dependent. Glutamate at 20 mM had no effect on bFGF level while 500 mM glutamate induced a significant increase in the bFGF level. The increase was found at as early as 6 h time point, reached the peak at 24 h time point then declined at 48 h time point ŽTable 1, Fig. 1.. The bFGF protein levels in the culture medium of every culture were lower than the detection limit of the bFGF-assay kit. 3.2. Effect of ischemic-like insults on cell Õiability of astrocytes The MTT assay was used to show the extents of cellular damage of astrocytes after ischemic-like insults. When astrocytes were treated with serum-free medium, the decrease of cell viability became significant at 12 h time point. When astrocytes were treated with glucose-free medium, the cell viability decreased quickly and became significant at 6 h time point. The effect of glutamate on the cell viability was dose-dependent. Glutamate at 20 mM had no effect. While 100 mM glutamate only partially decreased the number of living cells, 200 or 500 mM glutamate significantly decreased the living cell number at 24 and 48 h time points ŽTable 2.. 3.3. Effect of ischemic-like insults on spatial distribution of bFGF and FGFR-1 expression in astrocytes When the distribution of bFGF immunoreactivity in the astrocytes was observed by immunocytochemical staining, bFGF immunoreactivity was at low level in vehicle-treated cells, predominately in the cytoplasm of astrocytes ŽFig. 2C.. All three insults induced significant increase in bFGF immunoreactivity. It is noteworthy that the increased bFGF immunoreactivity appeared mainly in the nuclei of astrocytes ŽFig. 2D,E and F.. The FGFR-1 immunoreactivity in vehicle-treated astrocytes was relatively low ŽFig. 3C.. After astrocytes were treated with serum- or glucose-free medium or glutamate, FGFR-1 immunoreactivity in the cells, especially in the nuclear interior, increased ŽFig. 3D,E and F..
4. Discussion Altered levels of several growth factors have been reported to play an important role in mediating growth and repair after CNS injury w8,21x. Among these factors, bFGF was a focus of study because administration of extraneous
175
bFGF can exhibit potent protective effects against ischemic injury w2,9,13,16,20,22,29,30,32 x. Though several papers have reported the increase of bFGF after ischemia in vivo, the induction of bFGF expression in astrocytes in vitro has not been examined in detail. In the present studies we demonstrated that ischemic-like insults could considerably induce bFGF expression in cultured astrocytes. The increase of bFGF was significant at as early as 6 h after glucose-free or 500 mM glutamate treatment. The bFGF level peaked at 24 h time point and then the level decreased and returned to normal at 48 h after the initiation of insults. The results indicated that the induction of bFGF was a relatively quick but transient event. Our results further demonstrated that some differences might exist between different ischemic-like insults. The glucose-free insult significantly increased the bFGF level at as early as 6 h time point while the increase of bFGF following serum-free insult became statistically significant only after 24 h treatment. Besides, the peak increase of bFGF by the glucose-free insult Ž1.43-fold of the control. was higher than that by the serum-free insult Ž1.23-fold of the control.. Glutamate induced a dose-dependent increase in the bFGF level in astrocytes. While glutamate at low concentration Ž20 mM. had no effect on the bFGF level, glutamate at high concentration Ž500 mM. induced an significant increase in the bFGF level at 6 h time point. The peak level of bFGF was 1.57-fold of the control. It has been well known that many factors including glucose- and oxygen-deprivation and glutamate-induced excitotoxicity could be involved in the cascade of ischemic injury of CNS. Since all the ischemic-like insults tested in our study were able to induce bFGF expression in astrocytes, we proposed that the increase of bFGF in vivo following ischemia might be the result of the effects of several factors. There were also differences in the extents of damage caused by different ischemic-like insults. Glucose-free insult exhibited a higher potential in causing cell death than serum-free insult. It seemed that the bFGF expression was correlated with the extent of cellular damage. However, the results from glutamate insult were different. Though 500 mM glutamate could quickly and significantly induce the expression of bFGF in astrocytes, the extent of cellular damage caused by the same concentration of glutamate was relatively slight. While the increase of bFGF expression became significant at as early as 6 h time point, the decrease of cell viability became statistically significant at 24 h time point. It seemed that glutamate might induce the bFGF expression by a mechanism different from that of glucose-free or serum-free insult. Unlike other growth factors, bFGF is not freely secreted from cells by classical mechanism w1,31x. Accordingly, only very low level of bFGF-like activity could be detected in conditioned medium of cells that produce bFGF w18,31x. It has been proposed that bFGF might have both exocrine and intracrine Žautocrine. actions. On one hand,
176
X. Liu, X.-Z. Zhu r Molecular Brain Research 71 (1999) 171–177
the extracellular bFGF Žmainly released from dead cells. may interact with its receptor on the plasma membrane of the intact cells and undergo receptor-mediated actions. On the other hand, the intracellular bFGF may also regulate the proliferation of the cell by moving from the cytoplasm into the nuclear and changing the expression of many other growth factors w3,11,26x. Our results that bFGF could only be detected in the cells but not the medium and that the increased bFGF immunoreactivity after ischemic-like insults appeared mainly in the nuclei of astrocytes further support the proposal. In the case of ischemia in vivo, the nuclear accumulation of bFGF in astrocytes may induce the expression and release of other growth factors to protect themselves as well as surrounding neurons against the injury. To explain the effect of nuclear bFGF, the presence of nuclear FGFRs was also postulated. Evidence has shown that quiescent human astrocytes had little or no nuclear FGFRs. However, an induction of nuclear FGFRs was observed in reactive astrocytes w26x. FGFR-1 is one of the high-affinity receptors of bFGF. It is widely distributed in the intact rodent brain and becomes localized on reactive astroglia after brain injury or ischemia. It has been found that both the number of bFGF mRNA-positive cells and the number of FGFR-1-positive cells increased after ischemia. The time course of the appearance and distribution of FGFR-1 mRNA-positive cells were quite similar to those of bFGF mRNA-positive cells w7,17,24,28x. In our in vitro studies, we found that all three insults also induced a moderate increase in FGFR-1 immunoreactivity while the same treatment induced a significant increase in bFGF immunoreactivity. In summary, our results demonstrated that the three ischemic-like insults, serum-free, glucose-free or glutamate could induce an up-regulation in the bFGF and FGFR-1 expression in astrocytes in vitro. The induction of bFGF was a relatively quick but transient event. Some differences may exist when astrocytes were treated with different insults. Glutamate may induce the bFGF expression by a mechanism different from that of glucose-free or serumfree insult. Our results also suggest that the increased astrocytic bFGF levels might act on the astrocytes via autocrine mechanism and that the increased bFGF and FGFR-1 expression may play an important role in the maintenance and repair of the CNS after ischemia.
w4x
w5x
w6x
w7x
w8x
w9x
w10x
w11x
w12x
w13x
w14x
w15x
w16x
w17x
w18x
References w19x w1x J.A. Abraham, A. Mergia, J.L. Whang, A. Tumolo, J. Friedman, K.A. Hjerrild, D. Gospodarowiez, J.C. Fiddes, Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor, Science 233 Ž1986. 545–548. w2x A. Bethel, J.R. Kirsch, R.C. Koehler, S.P. Finklestein, R.J. Traystman, Intravenous basic fibroblast growth factor decrease brain injury resulting from focal ischemia in cats, Stroke 28 Ž1997. 609–616. w3x A. Bikfalvi, S. Kleen, G. Pintucci, N. Quatro, P. Mignatti, D.B. Rifkin, Differential modulation of cell phenotype by different molec-
w20x
w21x
ular weight forms of basic fibroblast growth factor: possible intracellular signaling by the high molecular weight forms, J. Cell Biol. 129 Ž1995. 233–243. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 Ž1976. 248–254. C.G. Caday, M. Klagsburn, P.J. Fanning, A. Mirzabegian, S.P. Finklestein, Fibroblast growth factor ŽFGF. levels in the developing rat brain, Brain Res. Dev. Brain Res. 52 Ž1990. 241–246. J. Carmichael, W.G. Degraff, A.F. Gazdar, J.D. Minna, J.B. Mitchell, Evaluation of a Tetrazolium-based semi-automated colorimetric assay: assessment of chemosensitivity testing, Cancer Res. 47 Ž1987. 936–942. M. Emdoh, W.A. Pulsinelli, J.A. Wagner, Transient global ischemia induces dynamic changes in the expression of bFGF and the FGF receptor, Mol. Brain Res. 22 Ž1994. 76–88. S.P. Finklestein, C.G. Caday, M. Kano, D.J. Berlove, C.Y. Hsu, M. Moskowitz, Klagsbrun, Growth factor expression after stroke, Stroke 21 Ž1990. 122–124. M. Fisher, M.E. Meadows, T. Do, J. Weise, V. Trubetskoy, M. Charette, S.P. Finklestein, Delayed treatment with intravenous basic fibroblast growth factor reduces infarct size following permanent focal ischemia in rats, J. Cereb. Blood Flow Metab. 15 Ž1995. 953–959. A.M. Gonzalez, M. Buscaglia, M. Ong, A. Baired, Distribution of basic fibroblast growth factor in the 18-day rat fetus: localization in the basement membranes of diverse tissues, J. Cell Biol. 110 Ž1990. 753–765. A. Joy, J. Moffett, K. Neary, E. Mordechai, E.K. Stachowiak, S. Coons, J. Rankin-Shapiro, R.Z. Florkiewilz, M.K. Stachowiak, Nuclear accumulation of FGF-2 is associated with proliferation of human astrocytes and glioma cell, Oncogene 17 Ž1997. 171–183. Y. Kiyota, K. Takami, M. Iwane, A. Shino, M. Miyomoto, R. Tsukuda, A. Nagaoka, Increase in basic fibroblast growth factor-like immunoreactivity in rat brain after forebrain ischemia, Brain Res. 545 Ž1991. 322–328. N. Koketsu, D.J. Berlove, M.A. Moskowitz, N.W. Kowall, C.G. Caday, S.P. Finklestein, Pretreatment with intraventricular basic fibroblast growth factor decrease infarct size following focal cerebral ischemia in rats, Ann. Neurol. 35 Ž1994. 451–457. T.N. Lin, J. Te, M. Lee, G.Y. Sun, C.Y. Hsu, Induction of basic fibroblast growth factor ŽbFGF. expression following focal cerebral ischemia, Mol. Brain Res. 49 Ž1997. 255–265. A. Lippoldt, B. Andbjer, L. Rosen, E. Richter, D. Ganten, Y. Cao, R.F. Petterson, K. Fuxe, Photochemically induced focal cerebral ischemia in rat, time dependent and global increase in expression of bFGF mRNA, Brain Res. 625 Ž1993. 45–56. X. Liu, X.Z. Zhu, Basic fibroblast growth factor protected forebrain against ischemia-reperfusion damage in rats, Acta Pharmacol. Sin. 19 Ž1998. 527–530. A. Logan, S.A. Frautschy, A.M. Gonzalez, A. Baird, A time course for the focal elevation of synthesis of basic fibroblast growth factor and one of its high-affinity receptors Žflg. following a localized cortical brain injury, J. Neurosci. 12 Ž1992. 3827–3828. D. Moscatelli, M. Presta, J. Joseph-Silverstein, D.B. Rifkin, Both normal and tumor cells produce basic fibroblast growth factor, J. Cell Physiol. 129 Ž1986. 273–276. S. Murphy, Generation of astrocyte cultures from normal and neoplastic central nervous system, in: P.M. Coon ŽEd.., Cell Culture, Academic Press, San Diego, 1990, pp. 45–56. N. Nakata, H. Kato, K. Kogure, Protective effects of basic fibroblast growth factor against hippocampal neuronal damage following cerebral ischemia in the gerbil, Brain Res. 605 Ž1995. 354–356. M. Nieto-Sampedro, Growth factor induction and order of event in CNS repair, in: D.G. Stain, B.S. Sabei ŽEds.., Pharmacological Approaches to the Treatment of Brain and Spinal Cord Injury, Plenum, New York, 1988, pp. 301–337.
X. Liu, X.-Z. Zhu r Molecular Brain Research 71 (1999) 171–177 w22x J.M. Ren, S.P. Finklestein, Time window of infarct reduction by intravenous basic fibroblast growth factor in focal cerebral ischemia, Eur. J. Pharmacol. 327 Ž1997. 11–16. w23x M. Simmous, S. Purphy, Protein kinases in the induction of nitric oxide synthase in astrocytes, Glia 11 Ž1994. 227–234. w24x E.K. Speliotes, C.G. Caday, T. Do, J. Weise, N.W. Kowall, S.P. Finklestein, Increased expression of basic fibroblast growth factor ŽbFGF. following cerebral infarction in the rat, Mol. Brain Res. 39 Ž1996. 31–42. w25x E.K. Speliotes, N.W. Kowall, C.G. Caday, B.F. Shanti, S.P. Finklestein, Time course of changes in basic fibroblast growth factor ŽbFGF. immunoreactivity in global hippocampus following global cerebral ischemia, Ann. Neurol. 34 Ž1993. 287, ŽAbstr... w26x E.K. Stachowiak, P.A. Maher, J. Tucholiski, E. Mordechai, A. Joy, J. Moffett, S. Coons, M.K. Stachowiak, Nuclear accumulation of fibroblast growth factor receptors in human glial cells — association with cell proliferation, Oncogene 14 Ž1997. 2201–2211. w27x K. Takami, M. Iwane, Y. Kiyota, M. Miyamoto, R. Tsukuda, S. Shiosaka, Increase of basic fibroblast growth factor immunoreactivity and its mRNA level in rat brain following transient forebrain ischemia, Exp. Brain Res. 90 Ž1992. 1–10.
177
w28x K. Takami, Y. Kiyota, M. Iwane, M. Miyamoto, R. Tsukuda, K. Igarashi, A. Shino, A. Wanaka, S. Shisaka, M. Tohyama, Up-regulation of fibroblast growth factor–receptor message RNA expression in rat brain following forebrain ischemia, Exp. Brain Res. 97 Ž1993. 185–194. w29x R. Tanaka, Y. Miyasaka, K. Yada, T. Ohwada, T. Kameya, Basic fibroblast growth factor increases regional cerebral blood flow and reduces infarct size after experimental ischemia in a rat model, Stroke 26 Ž1995. 2154–2159. w30x T. Tatlisumak, K. Takano, R.A. Carano, M. Fisher, Effect of basic fibroblast growth factor on experimental focal ischemia studied by diffusion-weighted and perfusion imaging, Stroke 27 Ž1996. 2292– 2298. w31x I. Vlodavskey, R. Bar-Shavit, R. Ishai-Michaeli, P. Bashkin, Z. Fuks, Extracellular sequestion and release of fibroblast growth factor: a regulating mechanism, Trends Biochem. Sci. 16 Ž1991. 268– 271. w32x T.C. Wen, S. Matsuda, H. Yoshimura, J. Aburaya, F. Kushihata, M. Sakanaka, Protective effect of basic fibroblast growth factor-heparin and neurotoxic effect of platelet factor 4 on ischemic neuronal loss and learning disability in gerbils, Neuroscience 65 Ž1995. 513–521.