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Increase in basic fibroblast growth factor-like immunoreactivity in rat brain after forebrain ischemia
Brain Research, 545 (1991) 322-328 ~) 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0006-8993/91/$03.50 ADONIS 000689939124598Y
322
BRES 24598
Increase in basic fibroblast growth factor-like immunoreactivity in rat brain after forebrain ischemia Yoshihiro Kiyota 1, Kenji Takami 1, Makoto Iwane 2, Akio Shino 1, Masaomi Miyamoto 1, Ryoichi Tsukuda I and A k i n o b u Nagaoka 1 Biology Research Laboratories and 2Biotechnology Research Laboratories, Takeda Chemical Ind. Ltd., Osaka 532 (Japan)
(Accepted 8 January 1991) Key words: Basic fibroblast growth factor; Immunohistochemistry; Forebrain ischemia; Hippocampus; Caudate putamen; Rat
Using immunohistochemical techniques, a study was conducted to determine whether basic fibroblast growth factor (bFGF) is generated as one of the 'self-repair' responses in rat brain following transient forebrain ischemia. In normal brain, slight bFGF-like immunoreaetivity was observed. However, in rats exposed to 20 min of forebrain ischemia, intense bFGF-like immunoreactivity was observed in the CA1 subfield of the hippocampus and the eaudate putamen, and marked activity was evident in the temporal cortex, corpus callosum and the CA4 subfield of the hippocampus. Marked neuronal degeneration was also observed in these brain regions following forebrain ischemia. These results suggest that induction of bFGF-like immunoreactivity may be related to the healing which follows brain ischemia. Several 'trophic factors' are known to regulate the survival and growth of neurons in the brain and peripheral tissues. Fibroblast growth factor (FGF), which can exist in both acidic and basic forms, has been purified and sequenced as one these trophic factors 1'5'21. It has been demonstrated that basic F G F (bFGF) promotes the fibroblast and capillary proliferation that accompanies the healing of wounds in peripheral tissues 3. b F G F also has potent effects on the survival and outgrowth of neurons from various brain regions 13'23"24 as well as on the survival and proliferation of endothelial cells 7'8. Furthermore, b F G F has been demonstrated to protect neurons from retrograde degeneration induced by axonal transection 2"14. These findings suggest that b F G F has neurotrophic effects on brain neurons and may function in their normal maintenance. Thus, b F G F may play an important role in the 'self-repair' responses that follow injury such as trauma and brain ischemia. Recently, it has been demonstrated that bFGF-like immunoreactivity increases at the site of focal brain wounds 6, suggesting that b F G F may contribute to the repair of damaged tissue. In the present study, using immunohistochemical techniques, we investigated whether b F G F participates in the healing of damaged tissue following brain ischemia. Male Crj:Wistar rats, weighing 280-300 g and aged 8 weeks, were used. Forebrain ischemia was induced
according to the method reported previously by Pulsinelli and Brierley 16. Briefly, the rats were anesthetized with sodium pentobarbital (40 mg/kg, i.p.) and the bilateral vertebral arteries were electrocauterized at the level of the first vertebra. At the same time, atraumatic clasps (surgical silk strings) were placed around the c o m m o n carotid arteries without interrupting the arterial blood flow. On the following day, forebrain ischemia was induced by tightening the clasps for 20 min. The rats were allowed to recover for 10 days. Then they were anesthetized with sodium pentobarbital (40 mg/kg, i.p.) and their brains were perfused with 50 ml of physiological saline, removed and frozen with dry ice. The brains were then subjected to immunohistochemical staining. The frozen brains were sectioned at a thickness of 10 # m using a cryostat microtome (0-4.0 m m posterior to the bregma) and mounted on gelatin-coated glass slides. The sections were fixed in ice-cold acetone for 10 rain and treated with 0.3% hydrogen peroxide in methanol for 30 min at room temperature to inactivate endogenous peroxidase. They were then immersed in non-immune horse serum for 30 min, washed in 0.02 M phosphatebuffered saline (pH 7.2), and reacted with monoclonal anti-human b F G F (anti-hbFGF) antibody (25/~g/ml) for 2 h at room temperature. Primary antibodies preabsorbed with excess h b F G F or rat b F G F (50/~g/ml) for 3 h at room temperature, normal mouse immunoglobulin
Correspondence: Y. Kiyota, Biology Research Laboratories, Takeda Chemical Ind. Ltd., 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka 532,
Japan.
323
Fig. 1. The immunohistochemical reaction of bFGF in the hippocampus of a normal rat (A) and a rat exposed to 20 min of forebrain ischemia (B). Control staining (C) was carried out on a section consecutive to that used for bFGF staining, using primary antibody preabsorbed with excess immunogen (hbFGF). Counterstaining with hematoxylin was carried out to visualize the cell nuclei. Scale bar = 1 mm.
or phosphate-buffered saline were substituted for the
body was detected using a V E C T A S T A I N A B C Kit
primary antibodies in o r d e r to carry out control staining. A f t e r washing in phosphate-buffered saline, bound anti-
30 min
(Vector Labs., U . S . A . ) . T h e sections w e r e incubated for at r o o m
temperature
in
biotinylated horse
324
Fig. 2. The immunohistochemical reaction of bFGF in the caudate putamen of a normal rat (A) and a rat exposed to 20 min of forebrain ischemia (B). Counterstaining with hematoxylin was carried out. CC, corpus callosum; CP, caudate putamen. Scale bar = 200/zm.
anti-mouse IgG, and then in the ABC reagent (avidinbiotin complex coupled to peroxidase) for 60 min at room temperature 9. To visualize the antigen-antibody complexes, the sections were then incubated for 6 rain at room temperature in 3-amino-9-ethyl-carbazole (Biomeda Corp., U.S.A.). Immunohistochemical staining for glial fibrillary acidic protein (GFAP) was carried out using rabbit polyclonal antibody against GFAP (Biomeda Corp.), and then visualized in the same way as described above. Some sections were counterstained with hematoxylin. Photographs were taken with a Nikon Microphoto FXA lightmicroscope.
For immunohistochemical staining, we used monocional antibodies (MAb 52) against recombinant hbFGF 2°. Previous studies had shown that this antibody recognized the epitope located between amino acid residues 14 and 40 of hbFGF, and did not bind to bovine acidic F G E In the normal rat brain, slight immunoreactivity was observed in the fimbria, hippocampus, corpus caUosum, anterior commissure, optic nerve, internal capsule, medial habenular nucleus, ependymal cells, superficial glial membrane and pia mater. In the hippocampus, slight immunoreactivity was observed in the stratum moleculare (Fig. 1A). Immunoreactivity was rarely found in the
325
Fig. 3. The immunohistochemical reaction of bFGF in the temporal cortex of a normal rat (A) and a rat exposed to 20 min of forebrain ischemia (B). Counterstaining with hematoxylin was carded out. Scale bar = 200 ~tm. caudate putamen and cerebral cortex (Figs. 2A and 3A), and was evident in both astrocyte-like cells and the tissue surrounding capillaries. In rats exposed to 20 min of forebrain ischemia, intense immunoreactivity was observed in the hippocampal CA1 subfield and caudate putamen (Figs. 1B and 2B). Marked neuronal degeneration was also observed in these areas. Furthermore, marked immunoreactivity was observed in the CA4 subfield of the hippocampus, corpus callosum and layers III and IV of the temporal cortex (Figs. 1B, 2B and 3B). A mild increase in immunoreactivity was observed in the internal capsule, anterior commissure, optic nerve and ependymal cells. This immunoreactivity was abolished in control staining. No specific staining was observed when
the specific antibody was preabsorbed with excess immunogen (Fig. 1C), or when normal mouse immunoglobulin or phosphate-buffered saline was used as the primary antibody. GFAP immunohistochemistry showed that bFGF-positive cells in the hippocampal CA1 subfield corresponded well with GFAP-positive cells with regard to shape and size (Fig. 4). In contrast to the control, these GFAP-positive cells were more numerous and their processes were larger. Renko et al. 19 have reported that b F G F has 3 different molecular weight forms: 18, 23 and 24 kDa. We also detected 3 immunoreactive bands in an immunoblot analysis of normal rat brain homogenates. Then, an immunoreactivity of the 18-kDa form was increased after
326
Fig. 4. The immunohistochemical reaction of bFGF (A) and GFAP (B) in the CA1 subfield of the hippocampus of a rat exposed to 20 min of forebrain ischemia. GFAP staining was carried out on a section consecutive to that used for bFGF staining. Or, stratum oriens; Py, stratum pyramidale; Rad, stratum radiatum. Scale bar = 100 am.
forebrain ischemia while the same intensity as normal rat control was observed for the others (data not shown). This finding suggests that the increased immunoreactivity after ischemia m a y be due to an increase of the 18-kDa form of b F G E Some research groups have r e p o r t e d previously that b F G F is localized in neuronal cells 6'15. W e also observed b F G F - l i k e immunoreactivity in neurons. However, this neuronal immunoreactivity was not as m a r k e d as that in astroglial cells. In a recent study, we have recognized that b F G F m R N A appears to be localized primarily within neurons. This finding is in agreement with previous reports 6'15.
O u r immunohistochemical investigation was a i m e d at determining whether b F G F is g e n e r a t e d in rat brain following transient forebrain ischemia. We found that intense b F G F - l i k e immunoreactivity was present in several brain regions, such as the CA1 subfield of the hippocampus and the caudate p u t a m e n , 10 days after forebrain ischemia, whereas only slight immunoreactivity was present in n o r m a l rat brain. It has been well d o c u m e n t e d that m a r k e d n e u r o d e g e n e r a t i o n is o b s e r v e d in the CA1 subfield of the h i p p o c a m p u s , caudate putamen, cerebral cortex and cerebellum following transient forebrain ischemia 1°'17'18. In the present study, m a r k e d neuronal d a m a g e was also o b s e r v e d in these regions, thus
327 supporting previous reports. Our investigations revealed that the regions in which marked neuronal degeneration was observed corresponded well with those showing marked bFGF-like immunoreactivity. Finklestein et al. 6 demonstrated that bFGF-like immunoreactivity was increased at sites of focal brain wounds. These findings suggest that bFGF-like immunoreactivity accompanies neuronal damage. Furthermore, we observed an increase of immunoreactivity in areas where no obvious neuronal degeneration had taken place. This might indicate that histologically undetectable alteration had occurred, and that bFGF plays some role in repairing these mildly damaged areas. Cascade reactions involving cellular changes, such as proliferation of glial and capillary endothelial cells after stroke and brain injury, contribute to recovery from neurological damage. It is known that reactive astroglial cells proliferate around brain wounds one week after injury 4'6. If b F G F is associated with the healing of ischemic brain damage, then bFGF immunoreactivity would be detected during the period of healing. For this purpose, we chose a period of 10 days after ischemia for this preliminary experiment. We carried out GFAP staining on sections consecutive to those used for bFGF immunohistochemical staining and found that the shape and size of bFGF-positive cells resembled those of GFAP-positive cells. Finklestein et al. 6 demonstrated that bFGF-positive cells at the site of focal brain wounds had the morphological characteristics of reactive astrocytes, which are known to proliferate around brain wounds 4. It is unclear whether the increased b F G F immunoreactivity was due to the presence of increased numbers of bFGF-positive cells, or to increased levels of b F G F immunoreactivity within individual cells, or both. However, as shown by the increased numbers of bFGF-positive cells, it is suggested that the increased
immunoreactivity was at least due to the increased numbers of bFGF-positive cells such as reactive astrocytes. It has been shown that glial cells produce and release growth and trophic factors 11'12, which help neurons and other cells to survive and proliferate. This suggests that tissue damage may cause accumulation of astrocytes and the subsequent production of trophic factors. It has been demonstrated that astrocytes protect cultured neurons against anoxia 22, and one possible mechanism of action is considered to be the production and release of trophic factors such as b F G E Taken together, these findings suggest that b F G F is generated in areas that are already damaged and/or are being damaged, contributing to protection against injury such as trauma and ischemia. Recently, some research groups have demonstrated that exogenously administered b F G F prevents neurodegeneration in the medial septum following fimbria fornix transection TM. These findings support the suggestion that endogenous b F G F plays a role in 'self-protection' responses against tissue damage. If bFGF really does have a role in such 'self-protection' responses, then it might be generated before neuronal death. In order to obtain evidence to support this hypothesis, we are now studying the possible timing and location of bFGF generation after forebrain ischemia. In conclusion, we have found that bFGF-like immunoreactivity increases in brain regions where marked neurodegeneration has taken place following transient forebrain ischemia, and we suggest that this increase may be related to the 'self-protection' responses and/or healing of brain damage. We thank Mr. K. Ikeda, Mrs. H. Hamajo and Miss K. Nishida for their valuable technical assistance, and Dr. D.B. Douglas for critical reading of the manuscript.
1 Abraham, J.A., Mergia, A., Whang, J.L., Tumolo, A., Friedman, J., Hjerrild, K.A., Gospodarowicz, D. and Fiddes, J.C., Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast gl:owth factor, Science, 233 (1986) 545-548. 2 Anderson, K.J., Dam, D., Lee, S. and Cotman, C.W., Basic fibroblast growth factor prevents death of lesioned cholinergic neurons in vivo, Nature, 332 (1988) 360-361. 3 Davidson, J.M., Klagsbrun, M., Hill, K.E., Buckley, A., Sullivan, R., Brewer, P.S. and Woodward, S.C., Accelerated wound repair, cell proliferation, and collagen accumulation are produced by a cartilage-derived growth factor, J. Cell Biol., 100 (1985) 1219-1227. 4 DuBois, M., Bowman, P.D. and Goldstein, G.W., Cell proliferation after ischemic infarction in gerbil brain, Brain Research, 347 (1985) 245-252. 5 Esch, E, Baird, A., Ling, N., Ueno, N., Hill, F., Denoroy, L., Klepper, R., Gospodarowicz, D., Bohlen, P. and Guillemin, R., Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF, Proc. Natl. Acad. Sci. U.S.A., 82
(1985) 6507-6511. 6 Finklestein, S.P., Apostolides, P.J., Caday, C.G., Prosser, J., Philips, M.F. and Klagsbrun, M., Increased basic fibroblast growth factor (bFGF) immunoreactivity at the site of focal brain wounds, Brain Research, 460 (1988) 253-259. 7 Folkman, J. and Klagsbrun, M., Angiogenic factors, Science, 235 (1987) 442-447. 8 Gospodarowicz, D., Massoglia, S., Cheng, J. and Fujii, D.K., Effect of fibroblast growth factor and lipoproteins on the proliferation of endothelial cells derived from bovine adrenal cortex, brain cortex and corpus luteum capillaries, J. Cell Physiol., 127 (1986) 121-136. 9 Hsu, S.M., Raine, L. and Fanger, H., Use of avidin-biotinperoxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures, J. Histoehem. Cytochem., 29 (1981) 577-580. 10 Kiyota, Y., Miyamoto, M. and Nagaoka, A., Relationship between brain damage and memory impairment in rats exposed to transient forebrain ischemia, Brain Research, 538 (1991) 295-302. 11 Manthorpe, M., Varon, S. and Adler, R., Neurite-promoting
328 factor in conditioned medium from RN22 schwannoma cultures. Bioassay, fractionation, and properties, J. Neurochem., 37 (1981) 759-767. 12 McKeefer, EE., Quindlem, E., Banks, M., Williams, U., Kornblith, EL., Laverson, S., Greenwood, M.A. and Smith, B., Biosynthesized products of cultured neuroglia cells. I. Selective release of proteins by cells from human astrocytomas, Neurology, 31 (1981) 1445-1452. 13 Morrison, R.S., Sharma, A., DeVellis, J. and Bradshaw, R.A., Basic fibroblast growth factor supports the survival of cerebral cortical neurons in primary culture, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 7537-7541. 14 Otto, D., Frotscher, M. and Unsicker, K., Basic fibroblast growth factor and nerve growth factor administered in gel foam rescue medial septal neurons after fimbria fornix transection, J. Neurosci. Res., 22 (1989) 83-91. 15 Pettmann, B., Labourdette, G., Weibel, M. and Sensenbrenner, M., The brain fibroblast growth factor (FGF) is localized in neurons, Neurosci. Lett., 68 (1986) 175-180. 16 Pulsinelli, W.A. and Brierley, J.B., A model of bilateral hemispheric ischemia in the unanesthetized rat, Stroke, 10 (1979) 267-272. 17 Pulsinelli, W.A., Brierley, J.B. and Plum, E, Temporal profile of neuronal damage in a model of transient forebrain ischemia, Ann. Neurol. 11 (1982) 491-498.
18 Pulsinelli, W.A., Selective neuronal vulnerability: morphological and molecular characteristics, Progr. Brain. Res., 63 (1985) 29-37. 19 Renko, M., Quarto, N., Morimoto, T. and Rifkin, D.B., Nuclear and cytoplasmic localization of different basic fibroblast growth factor species, J. Cell. Physiol., 144 (1990) 108-114. 20 Seno, M., Iwane, M., Sasada, R., Moriya, N., Kurokawa, T. and Igarashi, K., Monoclonal antibodies against human basic fibroblast growth factor, Hybridoma, 8 (1989) 209-221. 21 Thomas, K.A., Rios Candelore, M., Gim6nez Gallego, G., DiSalvo, J., Bennett, C., Rodkey, J. and Fitzpatrick, S., Pure brain-derived acidic fibroblast growth factor is a potent angiogenic vascular endothelial cell mitogen with sequence homology to interleukin 1, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 6409-6413. 22 Ubulsreth, S., Hefti, E, Ginsberg, M.D., Dietrich, W.D. and Busto, R., Astrocytes protect cultured neurons from degeneration induced by anoxia, Brain Research, 422 (1987) 303-311. 23 Walicke, E, Cowan, W.M., Ueno, N., Baird, A. and GuiUemin, R., Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 3012-3016. 24 Walicke, EA., Basic and acidic fibroblast growth factors have trophic effects on neurons from multiple CNS regions, J. Neurosci., 8 (1988) 2618-2627.
322
BRES 24598
Increase in basic fibroblast growth factor-like immunoreactivity in rat brain after forebrain ischemia Yoshihiro Kiyota 1, Kenji Takami 1, Makoto Iwane 2, Akio Shino 1, Masaomi Miyamoto 1, Ryoichi Tsukuda I and A k i n o b u Nagaoka 1 Biology Research Laboratories and 2Biotechnology Research Laboratories, Takeda Chemical Ind. Ltd., Osaka 532 (Japan)
(Accepted 8 January 1991) Key words: Basic fibroblast growth factor; Immunohistochemistry; Forebrain ischemia; Hippocampus; Caudate putamen; Rat
Using immunohistochemical techniques, a study was conducted to determine whether basic fibroblast growth factor (bFGF) is generated as one of the 'self-repair' responses in rat brain following transient forebrain ischemia. In normal brain, slight bFGF-like immunoreaetivity was observed. However, in rats exposed to 20 min of forebrain ischemia, intense bFGF-like immunoreactivity was observed in the CA1 subfield of the hippocampus and the eaudate putamen, and marked activity was evident in the temporal cortex, corpus callosum and the CA4 subfield of the hippocampus. Marked neuronal degeneration was also observed in these brain regions following forebrain ischemia. These results suggest that induction of bFGF-like immunoreactivity may be related to the healing which follows brain ischemia. Several 'trophic factors' are known to regulate the survival and growth of neurons in the brain and peripheral tissues. Fibroblast growth factor (FGF), which can exist in both acidic and basic forms, has been purified and sequenced as one these trophic factors 1'5'21. It has been demonstrated that basic F G F (bFGF) promotes the fibroblast and capillary proliferation that accompanies the healing of wounds in peripheral tissues 3. b F G F also has potent effects on the survival and outgrowth of neurons from various brain regions 13'23"24 as well as on the survival and proliferation of endothelial cells 7'8. Furthermore, b F G F has been demonstrated to protect neurons from retrograde degeneration induced by axonal transection 2"14. These findings suggest that b F G F has neurotrophic effects on brain neurons and may function in their normal maintenance. Thus, b F G F may play an important role in the 'self-repair' responses that follow injury such as trauma and brain ischemia. Recently, it has been demonstrated that bFGF-like immunoreactivity increases at the site of focal brain wounds 6, suggesting that b F G F may contribute to the repair of damaged tissue. In the present study, using immunohistochemical techniques, we investigated whether b F G F participates in the healing of damaged tissue following brain ischemia. Male Crj:Wistar rats, weighing 280-300 g and aged 8 weeks, were used. Forebrain ischemia was induced
according to the method reported previously by Pulsinelli and Brierley 16. Briefly, the rats were anesthetized with sodium pentobarbital (40 mg/kg, i.p.) and the bilateral vertebral arteries were electrocauterized at the level of the first vertebra. At the same time, atraumatic clasps (surgical silk strings) were placed around the c o m m o n carotid arteries without interrupting the arterial blood flow. On the following day, forebrain ischemia was induced by tightening the clasps for 20 min. The rats were allowed to recover for 10 days. Then they were anesthetized with sodium pentobarbital (40 mg/kg, i.p.) and their brains were perfused with 50 ml of physiological saline, removed and frozen with dry ice. The brains were then subjected to immunohistochemical staining. The frozen brains were sectioned at a thickness of 10 # m using a cryostat microtome (0-4.0 m m posterior to the bregma) and mounted on gelatin-coated glass slides. The sections were fixed in ice-cold acetone for 10 rain and treated with 0.3% hydrogen peroxide in methanol for 30 min at room temperature to inactivate endogenous peroxidase. They were then immersed in non-immune horse serum for 30 min, washed in 0.02 M phosphatebuffered saline (pH 7.2), and reacted with monoclonal anti-human b F G F (anti-hbFGF) antibody (25/~g/ml) for 2 h at room temperature. Primary antibodies preabsorbed with excess h b F G F or rat b F G F (50/~g/ml) for 3 h at room temperature, normal mouse immunoglobulin
Correspondence: Y. Kiyota, Biology Research Laboratories, Takeda Chemical Ind. Ltd., 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka 532,
Japan.
323
Fig. 1. The immunohistochemical reaction of bFGF in the hippocampus of a normal rat (A) and a rat exposed to 20 min of forebrain ischemia (B). Control staining (C) was carried out on a section consecutive to that used for bFGF staining, using primary antibody preabsorbed with excess immunogen (hbFGF). Counterstaining with hematoxylin was carried out to visualize the cell nuclei. Scale bar = 1 mm.
or phosphate-buffered saline were substituted for the
body was detected using a V E C T A S T A I N A B C Kit
primary antibodies in o r d e r to carry out control staining. A f t e r washing in phosphate-buffered saline, bound anti-
30 min
(Vector Labs., U . S . A . ) . T h e sections w e r e incubated for at r o o m
temperature
in
biotinylated horse
324
Fig. 2. The immunohistochemical reaction of bFGF in the caudate putamen of a normal rat (A) and a rat exposed to 20 min of forebrain ischemia (B). Counterstaining with hematoxylin was carried out. CC, corpus callosum; CP, caudate putamen. Scale bar = 200/zm.
anti-mouse IgG, and then in the ABC reagent (avidinbiotin complex coupled to peroxidase) for 60 min at room temperature 9. To visualize the antigen-antibody complexes, the sections were then incubated for 6 rain at room temperature in 3-amino-9-ethyl-carbazole (Biomeda Corp., U.S.A.). Immunohistochemical staining for glial fibrillary acidic protein (GFAP) was carried out using rabbit polyclonal antibody against GFAP (Biomeda Corp.), and then visualized in the same way as described above. Some sections were counterstained with hematoxylin. Photographs were taken with a Nikon Microphoto FXA lightmicroscope.
For immunohistochemical staining, we used monocional antibodies (MAb 52) against recombinant hbFGF 2°. Previous studies had shown that this antibody recognized the epitope located between amino acid residues 14 and 40 of hbFGF, and did not bind to bovine acidic F G E In the normal rat brain, slight immunoreactivity was observed in the fimbria, hippocampus, corpus caUosum, anterior commissure, optic nerve, internal capsule, medial habenular nucleus, ependymal cells, superficial glial membrane and pia mater. In the hippocampus, slight immunoreactivity was observed in the stratum moleculare (Fig. 1A). Immunoreactivity was rarely found in the
325
Fig. 3. The immunohistochemical reaction of bFGF in the temporal cortex of a normal rat (A) and a rat exposed to 20 min of forebrain ischemia (B). Counterstaining with hematoxylin was carded out. Scale bar = 200 ~tm. caudate putamen and cerebral cortex (Figs. 2A and 3A), and was evident in both astrocyte-like cells and the tissue surrounding capillaries. In rats exposed to 20 min of forebrain ischemia, intense immunoreactivity was observed in the hippocampal CA1 subfield and caudate putamen (Figs. 1B and 2B). Marked neuronal degeneration was also observed in these areas. Furthermore, marked immunoreactivity was observed in the CA4 subfield of the hippocampus, corpus callosum and layers III and IV of the temporal cortex (Figs. 1B, 2B and 3B). A mild increase in immunoreactivity was observed in the internal capsule, anterior commissure, optic nerve and ependymal cells. This immunoreactivity was abolished in control staining. No specific staining was observed when
the specific antibody was preabsorbed with excess immunogen (Fig. 1C), or when normal mouse immunoglobulin or phosphate-buffered saline was used as the primary antibody. GFAP immunohistochemistry showed that bFGF-positive cells in the hippocampal CA1 subfield corresponded well with GFAP-positive cells with regard to shape and size (Fig. 4). In contrast to the control, these GFAP-positive cells were more numerous and their processes were larger. Renko et al. 19 have reported that b F G F has 3 different molecular weight forms: 18, 23 and 24 kDa. We also detected 3 immunoreactive bands in an immunoblot analysis of normal rat brain homogenates. Then, an immunoreactivity of the 18-kDa form was increased after
326
Fig. 4. The immunohistochemical reaction of bFGF (A) and GFAP (B) in the CA1 subfield of the hippocampus of a rat exposed to 20 min of forebrain ischemia. GFAP staining was carried out on a section consecutive to that used for bFGF staining. Or, stratum oriens; Py, stratum pyramidale; Rad, stratum radiatum. Scale bar = 100 am.
forebrain ischemia while the same intensity as normal rat control was observed for the others (data not shown). This finding suggests that the increased immunoreactivity after ischemia m a y be due to an increase of the 18-kDa form of b F G E Some research groups have r e p o r t e d previously that b F G F is localized in neuronal cells 6'15. W e also observed b F G F - l i k e immunoreactivity in neurons. However, this neuronal immunoreactivity was not as m a r k e d as that in astroglial cells. In a recent study, we have recognized that b F G F m R N A appears to be localized primarily within neurons. This finding is in agreement with previous reports 6'15.
O u r immunohistochemical investigation was a i m e d at determining whether b F G F is g e n e r a t e d in rat brain following transient forebrain ischemia. We found that intense b F G F - l i k e immunoreactivity was present in several brain regions, such as the CA1 subfield of the hippocampus and the caudate p u t a m e n , 10 days after forebrain ischemia, whereas only slight immunoreactivity was present in n o r m a l rat brain. It has been well d o c u m e n t e d that m a r k e d n e u r o d e g e n e r a t i o n is o b s e r v e d in the CA1 subfield of the h i p p o c a m p u s , caudate putamen, cerebral cortex and cerebellum following transient forebrain ischemia 1°'17'18. In the present study, m a r k e d neuronal d a m a g e was also o b s e r v e d in these regions, thus
327 supporting previous reports. Our investigations revealed that the regions in which marked neuronal degeneration was observed corresponded well with those showing marked bFGF-like immunoreactivity. Finklestein et al. 6 demonstrated that bFGF-like immunoreactivity was increased at sites of focal brain wounds. These findings suggest that bFGF-like immunoreactivity accompanies neuronal damage. Furthermore, we observed an increase of immunoreactivity in areas where no obvious neuronal degeneration had taken place. This might indicate that histologically undetectable alteration had occurred, and that bFGF plays some role in repairing these mildly damaged areas. Cascade reactions involving cellular changes, such as proliferation of glial and capillary endothelial cells after stroke and brain injury, contribute to recovery from neurological damage. It is known that reactive astroglial cells proliferate around brain wounds one week after injury 4'6. If b F G F is associated with the healing of ischemic brain damage, then bFGF immunoreactivity would be detected during the period of healing. For this purpose, we chose a period of 10 days after ischemia for this preliminary experiment. We carried out GFAP staining on sections consecutive to those used for bFGF immunohistochemical staining and found that the shape and size of bFGF-positive cells resembled those of GFAP-positive cells. Finklestein et al. 6 demonstrated that bFGF-positive cells at the site of focal brain wounds had the morphological characteristics of reactive astrocytes, which are known to proliferate around brain wounds 4. It is unclear whether the increased b F G F immunoreactivity was due to the presence of increased numbers of bFGF-positive cells, or to increased levels of b F G F immunoreactivity within individual cells, or both. However, as shown by the increased numbers of bFGF-positive cells, it is suggested that the increased
immunoreactivity was at least due to the increased numbers of bFGF-positive cells such as reactive astrocytes. It has been shown that glial cells produce and release growth and trophic factors 11'12, which help neurons and other cells to survive and proliferate. This suggests that tissue damage may cause accumulation of astrocytes and the subsequent production of trophic factors. It has been demonstrated that astrocytes protect cultured neurons against anoxia 22, and one possible mechanism of action is considered to be the production and release of trophic factors such as b F G E Taken together, these findings suggest that b F G F is generated in areas that are already damaged and/or are being damaged, contributing to protection against injury such as trauma and ischemia. Recently, some research groups have demonstrated that exogenously administered b F G F prevents neurodegeneration in the medial septum following fimbria fornix transection TM. These findings support the suggestion that endogenous b F G F plays a role in 'self-protection' responses against tissue damage. If bFGF really does have a role in such 'self-protection' responses, then it might be generated before neuronal death. In order to obtain evidence to support this hypothesis, we are now studying the possible timing and location of bFGF generation after forebrain ischemia. In conclusion, we have found that bFGF-like immunoreactivity increases in brain regions where marked neurodegeneration has taken place following transient forebrain ischemia, and we suggest that this increase may be related to the 'self-protection' responses and/or healing of brain damage. We thank Mr. K. Ikeda, Mrs. H. Hamajo and Miss K. Nishida for their valuable technical assistance, and Dr. D.B. Douglas for critical reading of the manuscript.
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