Basic
Fibroblast
Growth Cultured
Growth
Factor
Promote
Cerebellar Kazuho
Abe,
Factor Survival
Neurons Megumi
and
Takayanagi
from
Epidermal of Primary
Neonatal
and Hiroshi
Rats
Saito
Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Universityof Tokyo, Tokyo 113, Japan Received
February
20, 1991
Accepted
April
3, 1991
ABSTkACT-Neurotrophic effects of CS23, a mutein of human basic fibroblast growth factor (bFGF), and recombinant human epidermal growth factor (hEGF) were compared in dissociated cerebellar cultures from embryonic (E20) and early postnatal (P0-5) rats. Both CS23 and hEGF promoted the survival of cerebellar neurons with out distinction of ages of animals used. The maximum effects of CS23 were always larger than those of hEGF. These results suggest that both bFGF and EGF also func tion as neurotrophic factors in postnatal cerebellum.
Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) are well known as potent mitogens for a variety of cell types (1, 2), but both have recently been found to promote the survival of primary cul tured neurons from various brain regions (3 6). Since bFGF and EGF have been demon strated to be present in the brain (7-9), it is possible that they function as neurotrophic factors in the brain. It is of great interest to compare the effects of bFGF and EGF in rela tion to their cellular mechanisms and roles as neurotrophic factors in the brain. Morrison et al. (6) observed that EGF was effective in enhancing the survival of primary cultured cerebellar neurons from neonatal (PO 1) rats, but bFGF was not effective. How ever, contrary results were obtained in our previous study: bFGF strongly enhanced the survival of cultured cerebellar neurons from embryonic (E17-18) rats with larger max imum effects than EGF (10). One of the dis tinct differences between their experiments and ours is different ages of animals used. It is
possible that the responsiveness of the neurons to growth factor(s) varies with developmental stages. Therefore, in the present study, we attempted to compare the effects of bFGF and EGF on the survival of cerebellar neurons obtained from fetal and early postnatal rats under the same culture conditions. In addi tion, they used bovine bFGF, while we used human bFGF. In the present study, the effects of human and bovine bFGF were also com pared. Growth factors used in the present study were CS23 (a gift from Takeda Chemical In dustries, Ltd.), recombinant bovine bFGF (B bFGF, purchased from Amersham) and re combinant human EGF (H-EGF, a gift from Wakunaga Pharmaceutical Co., Ltd.). CS23 is an acid-resistant mutein of recombinant hu man bFGF (11). We have already confirmed that the neurotrophic activities of CS23 in cul tured brain neurons are equivalent to those of wild type of recombinant human bFGF (12). All drugs were first diluted to a concentration of 10 ,ug/ml in phosphate-buffered saline sup
plemented with 1 mg/ml bovine serum albu min to prevent non-specific binding of the pro teins. Small aliquots were stored at -20°C un til use. The stored drugs were further diluted to the desired concentration by adding DF/TIP medium just before application to the cell cultures. The cerebellum was isolated from embry onic (E20) and early postnatal (P0, P2, P5) Wistar rats. The procedures for cell culture and determination of neuronal survival were the same as described in our previous paper (12). Briefly, the cells were dissociated by in cubation with 0.25% trypsin and 0.01% DNase I at 37°C for 20 min, followed by pipetting. The cell suspensions were plated in poly-L-lysine-coated plastic 48-well plates at a density of 100,000 cells/cm2 with modified Eagle's medium supplemented with 10% fetal bovine serum. Twenty-four hours after plat ing, the medium was changed to serum-free DF/TIP medium with or without the desired concentrations of drugs. This point of time was designated as day 0. The cultures were fixed with 4% paraformaldehyde, and the number of surviving neurons in each well was counted under a microscope. The neuronal cells were distinguished from non-neuronal cells by their morphology or immunostaining (an avidin-biotin-peroxidase complex method) with monoclonal antibodies to neurofilaments (12). First the effects of CS23, B-bFGF or H EGF at various concentrations were compared in the cultures from E20, P0, P2 and P5 cer ebellum. All cultures were fixed 3 days after addition of drugs. The results are shown in Fig. 1. Both CS23 (0.01-10 ng/ml) and B bFGF (0.01 10 ng/ml) significantly increased the neuronal survival in all cases. The effects of CS23 and B-bFGF were virtually the same in all groups, except that the minimum effec tive concentration of CS23 was a little lower than that of B-bFGF. The discrepancy be tween the observation of Morrison et al. (6) and ours (10) is unlikely to be due to the different origin of bFGF. Addition of H-EGF (0.001-10 ng/ml) also increased significantly
the neuronal survival in all cultures, but the concentration-dependency was not very clear in the cultures from P2 and P5. It is possible that EGF exhibits neurotrophic effects through at least two or more processes. The results that effects of drugs seemed to be
Fig. 1. Effects of CS23 (open columns: 0.01, 0.1, 1, 10ng/ml), B-bFGF (hatched columns: 0.01, 0.1, 1, 10 ng/ml) or H-EGF (stippled columns: 0.001, 0.01, 0.1, 1, 10ng/ml) on the survival of primary cultured cer ebellar neurons from embryonic (E20) and early post natal (P0, P2, P5) rats. The neurons were cultured for 3 days in serum-free medium with or without drugs. The effects of CS23 and B-bFGF were compared using the same culture plate, while the effects of EGF were observed in separate culture. Ordinated indicate the number of survivingneurons (cells/cm2). The data are represented as the mean ± S.E.M. of 5 to 6 observa tions. Asterisks indicate significant differences from the group without drugs (closed columns): * P < 0.01 (Duncan's multiple range test).
smaller in the cultures from postnatal rats is very probably due to the fact that neuronal survival was relatively higher even without drugs. Further investigations are necessary to clarify whether the differences of survival in the medium without drugs are related to in vivo cerebellum development. In different cul tures from Fig. 1, the maximum effects of CS23 and H-EGF were compared. The max imum number of surviving neurons in the pres ence of CS23 (10 ng/ml) was larger than those of hEGF (10 ng/ml) in all cases. Taking the maximum effects of CS23 (differences between number of surviving neurons with and without CS23) as 100%, the maximum effects of H EGF (differences between number of surviv ing neurons with and without H-EGF) were 57%, 49%, 58% and 45% in the cultures from E20, P0, P2 and P5 cerebellum, respec tively. The effect of concomitant application of H EGF with CS23 was investigated in cultures from P2 cerebellum. As shown in Fig. 2, H EGF (1 ng/ml) increased the effects of a low er concentration of CS23 in an additive man ner, but did not significantly potentiate the maximum effects of CS23. These results are virtually the same as previously observed in the culture from E17 18 cerebellum (10). The neuronal survival 6 days after addition of CS23 (1 ng/ml) or H-EGF (1 ng/ml) was also determined in the cultures from P2 cer ebellum. The serum-free DF/TIP medium and drugs were renewed on day 3. Without CS23 or H-EGF, the number of surviving neurons greatly decreased, but both CS23 and H-EGF also promoted the survival after 6 days. The number of surviving neurons (cells/cm2) after 6 days in the absence of drugs was 3,429 ± 562 (n = 10), while those in the presence of 1 ng/ml CS23 and 1 ng/ml H-EGF were 24,964 ± 1,540 (n = 9) and 15,582 ± 2,845 (n = 9), respectively. The present results suggest that difference in the ages of animals used or origin of bFGF cannot account for the discrepancy between the data of Morrison et al. (6) and our pre vious study (10). The difference might be re
lated to the state of the neurons after dissec tion or the culture conditions in which the neurons were grown. Kinoshita et al. (13) have recently shown that survival promoting effects of EGF were obvious under anoxic condition rather than under normal culture conditions. It is possible that the effects of bFGF are also influenced by culture condition. In addition, Morrison et al. (6) used the cul ture medium supplemented with 5 ,uM cyto sine arabinoside (araC) as an antimitotic drug to prepare relatively pure populations of neuronal cells. They seem to believe that the treatment with araC killed proliferating cells selectively, but araC is clearly toxic to the neurons also, judging from the data of our preliminary experiments: the number of sur viving neurons (cells/cm2) after 3 days in cul ture without and with araC (2 ,uM) was 17,750 ± 1,082 (n = 5) and 5,313 ± 968 (n = 5), re spectively. Furthermore, it has very recently been found that araC inhibited specifically the intraneuronal signal transduction mechanisms that are necessary for the neurons to respond to exogenous neurotrophic factor (14). The use of araC is not appropriate for investigating the action of neurotrophic factors. In our
Fig. 2. Effects of CS23alone (0) and in combination with H-EGF (1 ng/ml, •) on the survival of cultured cerebellar neurons from P2 rats. Abscissas indicate the concentration of CS23 added. Ordinates are as in Fig. 1. All data are expressed as the mean ± S.E.M. of 5 observations. Asterisks indicate significant differences from the group without H-EGF (0): *P < 0.05 (Stu dent's t-test).
present culture, possibly contaminating non neuronal cells were limited to less than 10% without using araC, as previously described (10, 12). However, influence of proliferation of fibroblasts or glial cells cannot be complete ly ruled out, especially when the culture was exposed to growth factors for a long period of time. It is necessary to determine the number of glial cells by immunostaining with antibody to glial fibrillary acidic protein (GFAP) or to do experiments with antimitotics other than araC or any treatment that removes non neuronal cells specifically and completely. Further investigation about the factor(s) affecting the responsiveness of brain neurons to bFGF and EGF should give important in formation about the mechanisms underlying the selective development and maintenance of the brain nervous system. Acknowledgments The authors are gratefulto Takeda ChemicalIndus tries, Ltd. and WakunagaPharmaceutical Co., Ltd. for their generousgiftsof drugs. REFERENCES
1 Gospodarowicz, D., Neufeld, G. and Schweiger, L.: Fibroblast growth factor. Mol. Cell. Endocri nol. 46, 187-204 (1986) 2 Carpenter, G. and Cohen, S.: Epidermal growth factor. Annu. Rev. Biochem. 48, 193-216 (1979) 3 Walicke, P., Cowan, W.M., Ueno, N., Baird, A. and Guillemin, R.: Fibroblast growth factor pro motes survival of dissociated hippocampal neurons and enhances neurite extension. Proc. Natl. Acad. Sci. U.S.A. 83, 7537-7541 (1986) 4 Morrison, R.S., Sharma, A., de Vellis, 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, 7537-7541 (1986) 5 Morrison, R.S., Kornblum, H.I., Leslie, F.M. and Bradshaw, R.A.: Trophic stimulation of cultured neurons from neonatal rat brain by epidermal growth factor. Science 238, 72-75 (1987)
6
Morrison, R.S., Keating, R.F. and Moskal, J.R.: Basic fibroblast growth factor and epidermal
growth factor exert differential trophic effects on CNS neurons. J. Neurosci. 21, 71-79 (1988) 7 Gospodarowicz, D., Cheng, J., Lui, G.-M., Baird, A. and Bohlen, P.: Isolation of brain fibroblast growth factor by heparin-Sepharose affinity chro matography: identity with pituitary fibroblast growth factor. Proc. Natl. Acad. Sci. U.S.A. 81, 6963 6967 (1984) 8 Fallon, J.H., Seroogy, K.B., Loughlin, S.E., Mor rison, R.S., Bradshaw, R.A., Knauer, D.J. and Cunningham, D.D.: Epidermal growth factor im munoreactive material in the central nervous system: location and development. Science 224, 1107 1109 (1984) 9 Lakshmanan, J., Weichsel, M.E. and Fisher, D.A.: Epidermal growth factor in synaptosomal fractions of mouse cerebral cortex. J. Neurochem. 46, 1081-1085 (1986) 10 Abe, K., Takayanagi, M. and Saito, H.: A com parison of neurotrophic effects of epidermal growth factor and basic fibroblast growth factor in primary cultured neurons from various regions of fetal rat brain. Japan. J. Pharmacol. 54, 45-54 (1990) 11 Seno, M., Sasada, R., Iwane, M., Sudo, K., Kurokawa, T., Ito, K. and Igarashi, K.: Stabilizing basic fibroblast growth factor using protein en gineering. Biochem. Biophys. Res. Commun. 151, 701 708 (1988) 12 Abe, K., Takayanagi, M. and Saito, H.: Effects of recombinant human basic fibroblast growth factor and its modified protein, CS23, on survival of primary cultured neurons from various regions of fetal rat brain. Japan. J. Pharmacol. 53, 221-227 (1990) 13 Kinoshita, A., Yamada, K., Hayakawa, T., Kataoka, K., Mushiroi. T., Kohmura, E. and Mogami, H.: Modification of anoxic neuronal in jury by human recombinant epidermal growth fac tor and its possible mechanism. J. Neurosci. Res. 25, 324-330 (1990) 14 Martin, D.P., Wallace T.L. and Johnson, E.M., Jr.: Cytosine arabinoside kills postmitotic neurons in a fashion resembling trophic factor deprivation: evidence that a deoxycytidine-dependent process may be required for nerve growth factor signal transduction. J. Neurosci. 10, 184 193 (1990)
Fibroblast
Growth Cultured
Growth
Factor
Promote
Cerebellar Kazuho
Abe,
Factor Survival
Neurons Megumi
and
Takayanagi
from
Epidermal of Primary
Neonatal
and Hiroshi
Rats
Saito
Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Universityof Tokyo, Tokyo 113, Japan Received
February
20, 1991
Accepted
April
3, 1991
ABSTkACT-Neurotrophic effects of CS23, a mutein of human basic fibroblast growth factor (bFGF), and recombinant human epidermal growth factor (hEGF) were compared in dissociated cerebellar cultures from embryonic (E20) and early postnatal (P0-5) rats. Both CS23 and hEGF promoted the survival of cerebellar neurons with out distinction of ages of animals used. The maximum effects of CS23 were always larger than those of hEGF. These results suggest that both bFGF and EGF also func tion as neurotrophic factors in postnatal cerebellum.
Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) are well known as potent mitogens for a variety of cell types (1, 2), but both have recently been found to promote the survival of primary cul tured neurons from various brain regions (3 6). Since bFGF and EGF have been demon strated to be present in the brain (7-9), it is possible that they function as neurotrophic factors in the brain. It is of great interest to compare the effects of bFGF and EGF in rela tion to their cellular mechanisms and roles as neurotrophic factors in the brain. Morrison et al. (6) observed that EGF was effective in enhancing the survival of primary cultured cerebellar neurons from neonatal (PO 1) rats, but bFGF was not effective. How ever, contrary results were obtained in our previous study: bFGF strongly enhanced the survival of cultured cerebellar neurons from embryonic (E17-18) rats with larger max imum effects than EGF (10). One of the dis tinct differences between their experiments and ours is different ages of animals used. It is
possible that the responsiveness of the neurons to growth factor(s) varies with developmental stages. Therefore, in the present study, we attempted to compare the effects of bFGF and EGF on the survival of cerebellar neurons obtained from fetal and early postnatal rats under the same culture conditions. In addi tion, they used bovine bFGF, while we used human bFGF. In the present study, the effects of human and bovine bFGF were also com pared. Growth factors used in the present study were CS23 (a gift from Takeda Chemical In dustries, Ltd.), recombinant bovine bFGF (B bFGF, purchased from Amersham) and re combinant human EGF (H-EGF, a gift from Wakunaga Pharmaceutical Co., Ltd.). CS23 is an acid-resistant mutein of recombinant hu man bFGF (11). We have already confirmed that the neurotrophic activities of CS23 in cul tured brain neurons are equivalent to those of wild type of recombinant human bFGF (12). All drugs were first diluted to a concentration of 10 ,ug/ml in phosphate-buffered saline sup
plemented with 1 mg/ml bovine serum albu min to prevent non-specific binding of the pro teins. Small aliquots were stored at -20°C un til use. The stored drugs were further diluted to the desired concentration by adding DF/TIP medium just before application to the cell cultures. The cerebellum was isolated from embry onic (E20) and early postnatal (P0, P2, P5) Wistar rats. The procedures for cell culture and determination of neuronal survival were the same as described in our previous paper (12). Briefly, the cells were dissociated by in cubation with 0.25% trypsin and 0.01% DNase I at 37°C for 20 min, followed by pipetting. The cell suspensions were plated in poly-L-lysine-coated plastic 48-well plates at a density of 100,000 cells/cm2 with modified Eagle's medium supplemented with 10% fetal bovine serum. Twenty-four hours after plat ing, the medium was changed to serum-free DF/TIP medium with or without the desired concentrations of drugs. This point of time was designated as day 0. The cultures were fixed with 4% paraformaldehyde, and the number of surviving neurons in each well was counted under a microscope. The neuronal cells were distinguished from non-neuronal cells by their morphology or immunostaining (an avidin-biotin-peroxidase complex method) with monoclonal antibodies to neurofilaments (12). First the effects of CS23, B-bFGF or H EGF at various concentrations were compared in the cultures from E20, P0, P2 and P5 cer ebellum. All cultures were fixed 3 days after addition of drugs. The results are shown in Fig. 1. Both CS23 (0.01-10 ng/ml) and B bFGF (0.01 10 ng/ml) significantly increased the neuronal survival in all cases. The effects of CS23 and B-bFGF were virtually the same in all groups, except that the minimum effec tive concentration of CS23 was a little lower than that of B-bFGF. The discrepancy be tween the observation of Morrison et al. (6) and ours (10) is unlikely to be due to the different origin of bFGF. Addition of H-EGF (0.001-10 ng/ml) also increased significantly
the neuronal survival in all cultures, but the concentration-dependency was not very clear in the cultures from P2 and P5. It is possible that EGF exhibits neurotrophic effects through at least two or more processes. The results that effects of drugs seemed to be
Fig. 1. Effects of CS23 (open columns: 0.01, 0.1, 1, 10ng/ml), B-bFGF (hatched columns: 0.01, 0.1, 1, 10 ng/ml) or H-EGF (stippled columns: 0.001, 0.01, 0.1, 1, 10ng/ml) on the survival of primary cultured cer ebellar neurons from embryonic (E20) and early post natal (P0, P2, P5) rats. The neurons were cultured for 3 days in serum-free medium with or without drugs. The effects of CS23 and B-bFGF were compared using the same culture plate, while the effects of EGF were observed in separate culture. Ordinated indicate the number of survivingneurons (cells/cm2). The data are represented as the mean ± S.E.M. of 5 to 6 observa tions. Asterisks indicate significant differences from the group without drugs (closed columns): * P < 0.01 (Duncan's multiple range test).
smaller in the cultures from postnatal rats is very probably due to the fact that neuronal survival was relatively higher even without drugs. Further investigations are necessary to clarify whether the differences of survival in the medium without drugs are related to in vivo cerebellum development. In different cul tures from Fig. 1, the maximum effects of CS23 and H-EGF were compared. The max imum number of surviving neurons in the pres ence of CS23 (10 ng/ml) was larger than those of hEGF (10 ng/ml) in all cases. Taking the maximum effects of CS23 (differences between number of surviving neurons with and without CS23) as 100%, the maximum effects of H EGF (differences between number of surviv ing neurons with and without H-EGF) were 57%, 49%, 58% and 45% in the cultures from E20, P0, P2 and P5 cerebellum, respec tively. The effect of concomitant application of H EGF with CS23 was investigated in cultures from P2 cerebellum. As shown in Fig. 2, H EGF (1 ng/ml) increased the effects of a low er concentration of CS23 in an additive man ner, but did not significantly potentiate the maximum effects of CS23. These results are virtually the same as previously observed in the culture from E17 18 cerebellum (10). The neuronal survival 6 days after addition of CS23 (1 ng/ml) or H-EGF (1 ng/ml) was also determined in the cultures from P2 cer ebellum. The serum-free DF/TIP medium and drugs were renewed on day 3. Without CS23 or H-EGF, the number of surviving neurons greatly decreased, but both CS23 and H-EGF also promoted the survival after 6 days. The number of surviving neurons (cells/cm2) after 6 days in the absence of drugs was 3,429 ± 562 (n = 10), while those in the presence of 1 ng/ml CS23 and 1 ng/ml H-EGF were 24,964 ± 1,540 (n = 9) and 15,582 ± 2,845 (n = 9), respectively. The present results suggest that difference in the ages of animals used or origin of bFGF cannot account for the discrepancy between the data of Morrison et al. (6) and our pre vious study (10). The difference might be re
lated to the state of the neurons after dissec tion or the culture conditions in which the neurons were grown. Kinoshita et al. (13) have recently shown that survival promoting effects of EGF were obvious under anoxic condition rather than under normal culture conditions. It is possible that the effects of bFGF are also influenced by culture condition. In addition, Morrison et al. (6) used the cul ture medium supplemented with 5 ,uM cyto sine arabinoside (araC) as an antimitotic drug to prepare relatively pure populations of neuronal cells. They seem to believe that the treatment with araC killed proliferating cells selectively, but araC is clearly toxic to the neurons also, judging from the data of our preliminary experiments: the number of sur viving neurons (cells/cm2) after 3 days in cul ture without and with araC (2 ,uM) was 17,750 ± 1,082 (n = 5) and 5,313 ± 968 (n = 5), re spectively. Furthermore, it has very recently been found that araC inhibited specifically the intraneuronal signal transduction mechanisms that are necessary for the neurons to respond to exogenous neurotrophic factor (14). The use of araC is not appropriate for investigating the action of neurotrophic factors. In our
Fig. 2. Effects of CS23alone (0) and in combination with H-EGF (1 ng/ml, •) on the survival of cultured cerebellar neurons from P2 rats. Abscissas indicate the concentration of CS23 added. Ordinates are as in Fig. 1. All data are expressed as the mean ± S.E.M. of 5 observations. Asterisks indicate significant differences from the group without H-EGF (0): *P < 0.05 (Stu dent's t-test).
present culture, possibly contaminating non neuronal cells were limited to less than 10% without using araC, as previously described (10, 12). However, influence of proliferation of fibroblasts or glial cells cannot be complete ly ruled out, especially when the culture was exposed to growth factors for a long period of time. It is necessary to determine the number of glial cells by immunostaining with antibody to glial fibrillary acidic protein (GFAP) or to do experiments with antimitotics other than araC or any treatment that removes non neuronal cells specifically and completely. Further investigation about the factor(s) affecting the responsiveness of brain neurons to bFGF and EGF should give important in formation about the mechanisms underlying the selective development and maintenance of the brain nervous system. Acknowledgments The authors are gratefulto Takeda ChemicalIndus tries, Ltd. and WakunagaPharmaceutical Co., Ltd. for their generousgiftsof drugs. REFERENCES
1 Gospodarowicz, D., Neufeld, G. and Schweiger, L.: Fibroblast growth factor. Mol. Cell. Endocri nol. 46, 187-204 (1986) 2 Carpenter, G. and Cohen, S.: Epidermal growth factor. Annu. Rev. Biochem. 48, 193-216 (1979) 3 Walicke, P., Cowan, W.M., Ueno, N., Baird, A. and Guillemin, R.: Fibroblast growth factor pro motes survival of dissociated hippocampal neurons and enhances neurite extension. Proc. Natl. Acad. Sci. U.S.A. 83, 7537-7541 (1986) 4 Morrison, R.S., Sharma, A., de Vellis, 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, 7537-7541 (1986) 5 Morrison, R.S., Kornblum, H.I., Leslie, F.M. and Bradshaw, R.A.: Trophic stimulation of cultured neurons from neonatal rat brain by epidermal growth factor. Science 238, 72-75 (1987)
6
Morrison, R.S., Keating, R.F. and Moskal, J.R.: Basic fibroblast growth factor and epidermal
growth factor exert differential trophic effects on CNS neurons. J. Neurosci. 21, 71-79 (1988) 7 Gospodarowicz, D., Cheng, J., Lui, G.-M., Baird, A. and Bohlen, P.: Isolation of brain fibroblast growth factor by heparin-Sepharose affinity chro matography: identity with pituitary fibroblast growth factor. Proc. Natl. Acad. Sci. U.S.A. 81, 6963 6967 (1984) 8 Fallon, J.H., Seroogy, K.B., Loughlin, S.E., Mor rison, R.S., Bradshaw, R.A., Knauer, D.J. and Cunningham, D.D.: Epidermal growth factor im munoreactive material in the central nervous system: location and development. Science 224, 1107 1109 (1984) 9 Lakshmanan, J., Weichsel, M.E. and Fisher, D.A.: Epidermal growth factor in synaptosomal fractions of mouse cerebral cortex. J. Neurochem. 46, 1081-1085 (1986) 10 Abe, K., Takayanagi, M. and Saito, H.: A com parison of neurotrophic effects of epidermal growth factor and basic fibroblast growth factor in primary cultured neurons from various regions of fetal rat brain. Japan. J. Pharmacol. 54, 45-54 (1990) 11 Seno, M., Sasada, R., Iwane, M., Sudo, K., Kurokawa, T., Ito, K. and Igarashi, K.: Stabilizing basic fibroblast growth factor using protein en gineering. Biochem. Biophys. Res. Commun. 151, 701 708 (1988) 12 Abe, K., Takayanagi, M. and Saito, H.: Effects of recombinant human basic fibroblast growth factor and its modified protein, CS23, on survival of primary cultured neurons from various regions of fetal rat brain. Japan. J. Pharmacol. 53, 221-227 (1990) 13 Kinoshita, A., Yamada, K., Hayakawa, T., Kataoka, K., Mushiroi. T., Kohmura, E. and Mogami, H.: Modification of anoxic neuronal in jury by human recombinant epidermal growth fac tor and its possible mechanism. J. Neurosci. Res. 25, 324-330 (1990) 14 Martin, D.P., Wallace T.L. and Johnson, E.M., Jr.: Cytosine arabinoside kills postmitotic neurons in a fashion resembling trophic factor deprivation: evidence that a deoxycytidine-dependent process may be required for nerve growth factor signal transduction. J. Neurosci. 10, 184 193 (1990)
