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Basic fibroblast growth factor increases cortical blood flow in vivo
BRAIN RESEARCH ELSEVIER
Brain Research 665 (1994) 155-157
Short communication
Basic fibroblast growth factor increases cortical blood flow in vivo Luca Regli, Robert E. Anderson, Fredric B. Meyer * Thoralf M. Sundt Jr. Neurosurgical Research Laboratory, Department of Neurosurgery, Mayo Clinic and Mayo Graduate School of Medicine, 200 First Street SW,, Rochester, MN 55905, USA Accepted 13 September 1994
Abstract
Basic FGF has recently been shown to produce systemic and cerebral vasodilation. To test the hypothesis that bFGF increases regional cortical blood flow (rCBF), rCBF and brain pH i were measured while bFGF or vehicle was superperfused onto the cortex of rabbits. The results of this study demonstrate that topical application of bFGF causes an increase in rCBF.
Keywords: Regional cortical blood flow; Basic fibroblast growth factor; Brain pH i
Fibroblast growth factors are multifunctional polypeptides [6,12] that have been implicated in the control of cell proliferation and differentiation in a variety of neuronal cell lines [5,13]. The adult brain [7] is the most abundant source of basic fibroblast growth factor (bFGF). b F G F is actively expressed in the adult central nervous system (CNS) [4] despite the fact that there is no cell proliferation or angiogenesis. This suggests that b F G F may have a non-mitogenic function. Recently, Cuevas et al. [3] demonstrated that intravenous infusion of b F G F led to systemic vasodilation in both rats and rabbits. The systemic effect was mediated through both nitric oxide and calcitonin gene-related peptide mechanisms. More recently, Rosenblatt et al. [11] observed that the cortical administration of b F G F produced a dose dependent vasodilatation of pial arterioles. This effect was attenuated by the co-administration of L-NAME, a nitric oxide synthetase inhibitor. Furthermore, there was no vasodilatation when heat inactivated b F G F was used or when their medium was preincubated with a b F G F antibody. These authors suggested that b F G F may have an important role in the regulation of cerebral blood flow. The purpose of this current study was to determine if the topical administration of b F G F in adult white New Zealand rabbits
* Corresponding author. Fax: (1) (507) 284-5206. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 1 0 8 6 - 2
would lead to a corresponding increase in cortical blood flow (CBF). U p o n approval by the Institutional Animal Use and Care Committee, overnight fasted white New Zealand rabbits (n = 30) weighing between 2.5 and 3.5 kg were induced with thiamylal sodium 30 m g / k g i.v. and operated and studied under 1.0% inspired halothane. The experimental paradigm consisted of using cortical suffusion to drip onto the exposed fronto-parietal cortex either vehicle or b F G F at a rate of 0.05 m l / m i n to yield a total volume of 1 ml. The temperature and p H of the perfusate was 37.0 + 0.5°C and 7.00, respectively. Recombinant human b F G F (147 a.a., R & D Systems, Minneapolis, MN) was dissolved in normal saline containing 0.1% bovine serum albumin as a carrier. Twenty animals were divided into 4 groups: a low dose b F G F group (n = 5 ) suffused with 0.25 /~g/min and a high dose b F G F group (n = 5) suffused with 1.25 ~ g / m i n to yield a total dose of 5 /zg or 25 /xg, respectively, and a vehicle group (n = 5) suffused with normal saline containing 0.1% bovine serum albumin. A fourth group of animals (n = 5) received intracarotid injection of b F G F as a bolus of 1.8 ~zg/kg. Measurements of rCBF and brain p H i [1] were done at 1 min, 10 min, and 20 min of suffusion or after intracarotid injection. A PaCO2-rCBF response curve was repeated at 30 min. There were no significant differences in the systemic parameters of PHa, PaCO2, PaO2, hematocrit or core
L. Regli et al. /Brain Research 665 (1994) 155-157
156
body temperature among the four groups. Mean arterial blood pressure declined from 93_+ 3 to 75_+ 5 m m H g ( P < 0.05) during the first minute following intracarotid injection of b F G F and then returned to baseline over 10 min. In all animals blood pressure stayed within the range of rCBF autoregulation. Cortical suffusion of bFGF or vehicle generated no change in mean arterial blood pressure. Intracarotid injection of b F G F demonstrated no effect on rCBF. Control animals suffused with a vehicle showed no change in rCBF. Suffusion of 5 /xg bFGF demonstrated no significant increase in rCBF (from 56.5 _+ 2.9 to 58.3 _+ 1.9 m l / 1 0 0 g / m i n ; P = NS). Suffusion of 25/xg b F G F resulted in a rapid and significant increase in rCBF. After one minute rCBF increased by 23% as compared to vehicle (from 54.4 _+ 2.7 to 66.9 _+ 3.2 m l / 1 0 0 g / m i n ; P < 0.05) and at 10 and 20 min the increase reached 33% (54.4 + 2.7 vs. 72.6 _+ 1.6 m l / 1 0 0 g / m i n ; P < 0.05) and 30% (54.4 + 2.7 vs. 71.0 _+ 1.5 m l / 1 0 0 g / m i n ; P < 0.05), respectively. After administration of bFGF, rCBF remained elevated for up to 60 rain post-suffusion ( P < 0.01) (Fig. 1). p~,COz-rCBF reactivity was normal in all experi-
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Fig. 1. Rapid and persistent (up to 60 min) increase in rCBF by suffusion of 25/xg of bFGF (open triangle, dotted line), compared to 5 #g (closed triangle, dashed line), and vehicle (open circle, solid line), rCBF was measured by the change of the indicator umbelliferone (A) and 133Xenon (B). The difference in increase of rCBF between (A) and (B) may reflect a selective effect of bFGF on the microvasculature. Analysis of variance with Tukey's post-hoc analysis was used (*P <0.05 indicates significance). Error bars indicate standard error of the mean.
mental groups before suffusion. ,adler 25 /x b F G F suffusion the PCO 2 reactivity of the brain was decreased by 34% (0.95 _+ 0.06 to 0.63 + 0.14 m l / 1 0 0 g / m i n / m m H g CO 2) despite the increase in rCBF. Brain p H i measured 7.02 _+ 0.02 and was stable throughout the experiment and administration of b F G F had no effect on pHi. This current study would confirm results recently reported by Rosenblatt et al. [11] which suggest that bFGF may play an important role in cerebral blood flow regulation. In our experiment, the increase in cortical blood flow was rapid starting within a minute of application and after ten min of continuous perfusion reached a maximal effect, b F G F administration did not alter brain pH~ in vivo, as opposed to in vitro results showing that growth factors may activate the N a + / H + antiporter in fibroblasts [8]. Therefore, the increase in rCBF was not dependent on brain cortical p H i changes. An alternative explanation is that some of the components of bFGF could change the buffering capacity of the extracellular space resulting in a change of rCBF. Regulation of rCBF is determined by the p H of the extracellular space [2]. One potential difficulty with this experimental paradigm is the large doses which were used in the cortical suffusion. It should be noted that in this preparation, a closed cranial window technique was not utilized. Therefore, the test solutions suffused onto the cortical surface would not provide a continuous lavage but rather run off the brain's surface over the margins of the craniectomy. Therefore, the dose of bFGF which had the opportunity to rest on the brain's surface and potentially diffuse through the pial barrier was probably far less than the concentration of the irrigating solution. In addition, the doses of b F G F used in this study were chosen based on the low diffusion coefficient of peptides and their susceptibility to metabolic inactivation by brain peptidases. Pardridge [10] predicted that the concentration of a peptide in brain parenchyma at a distance of 1 m m from the ependymal surface approximates a concentration that is less than 0.1% of peptide concentration in the ventricular compartment. We extrapolated this estimate as being the probable maximum fraction of b F G F penetrating into the outer parenchymal layers in the cortical suffusion model. Thus, we predicted that the concentration of b F G F at 1 m m from the cortical surface in this study would be only nanograms. The concentration of endogenous brain hFGF in vivo has been reported to be 33-50 n g / m g of brain [7]. The doses used in this study were also based on prior reports of the expression of b F G F in vivo. [9,14] The authors thank Ms. Patricia Friedrich and Mr. Robert Carlson for their technical assistance and to Ms. Mary Soper for secretarial assistance. One of the
L. Regli et al. /Brain Research 665 (1994) 155-157
authors (LR) was supported by Foundation Sicpa and Decker.
[1] Anderson, R.E., Meyer, F.B. and Tomlinson, F.H., Focal cortical distribution of blood flow and brain pH i determined by in vivo fluorescent imaging, Am. J. Physiol., 263 (1992) H565-H575. [2] Cameron, I.R. and Caronna, J., The effect of local changes on potassium and bicarbonate concentration on hypothalamic blood flow in the rabbit, J. Physiol., 262 (1976) 415-430. [3] Cuevas, P., Carcellar, F., Ortega, S., Zazo, M., Nieto, I. and Gimenez-Gallego, G., Hypotensive activity of fibroblast growth factor, Science, 254 (1991) 1208-1210. [4] Ernfors, P., L6nnerberg, P., Ayer-LeLievre, C. and Persson, H., Developmental and regional expression of basic fibroblast growth factor mRNA in the rat central nervous system, J. Neurosci. Res., 27 (1990) 10-15. [5] Ferrara, N., Ousley, F. and Gospodarowicz, D., Bovine brain astrocytes express basic fibroblast growth factor, a neurotropic and angiogenic mitogen, Brain Res., 462 (1988) 223-232. [6] Folkman, J. and Klagsbrun, M., Angiogenic factors, Science, 235 (1987) 442-447. [7] Gospodarowicz, D., Cheng, J., Lui, G.M., Baird, A. and Bohlent, P., Isolation of brain fibroblast growth factor by heparin-Sep-
157
harose affinity chromatography: identity with pituitary fibroblast growth factor, Proc. Natl. Acad. Sci. USA, 81 (1984) 6963-6967. [8] Moolenaar, W.H., Tsien, R.Y., van der Saag, P.T. and de Laat, S.W., N a + / H + exchange and cytoplasmic pH in the action of growth factors in human fibroblasts, Nature, 304 (1983) 645-648. [9] 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. [10] Pardridge, W.M. (Ed), Transnasal and intraventricular delivery of drugs. In Peptide Drug Delivery to the Brain, Raven Press, New York, 1991, pp. 99-122. [11] Rosenblatt, S., Irikura, K., Caday, C.G., Finklestein, S.P. and Moskowitz, M.A., Basic fibroblast growth factor dilates rat pial arterioles, J. Cereb. Blood Flow Metab., 14 (1994) 70-74. [12] Wagner, J.A., The fibroblast growth factors: an emerging family of neural growth factors, Curt. Top. Microbiol. Immunol., 165 (1991) 95-118. [13] Westermann, R., Grothe, C. and Unsicker, K., Basic fibroblast growth factor (bFGF), a multifunctional growth factor for neuroectodermal cells, J. Cell Sci., Suppl. 13 (1990) 97-117. [14] Yamada, K., Kinoshita, A., Kohmura, E., Sakaguchi, T., Taguchi, J., Kataoka, K. and Hayakawa, T., Basic fibroblast growth factor prevents thalamic degeneration after cortical infarction, J. Cereb. Blood Flow Metab., 11 (1991) 472-478.
Brain Research 665 (1994) 155-157
Short communication
Basic fibroblast growth factor increases cortical blood flow in vivo Luca Regli, Robert E. Anderson, Fredric B. Meyer * Thoralf M. Sundt Jr. Neurosurgical Research Laboratory, Department of Neurosurgery, Mayo Clinic and Mayo Graduate School of Medicine, 200 First Street SW,, Rochester, MN 55905, USA Accepted 13 September 1994
Abstract
Basic FGF has recently been shown to produce systemic and cerebral vasodilation. To test the hypothesis that bFGF increases regional cortical blood flow (rCBF), rCBF and brain pH i were measured while bFGF or vehicle was superperfused onto the cortex of rabbits. The results of this study demonstrate that topical application of bFGF causes an increase in rCBF.
Keywords: Regional cortical blood flow; Basic fibroblast growth factor; Brain pH i
Fibroblast growth factors are multifunctional polypeptides [6,12] that have been implicated in the control of cell proliferation and differentiation in a variety of neuronal cell lines [5,13]. The adult brain [7] is the most abundant source of basic fibroblast growth factor (bFGF). b F G F is actively expressed in the adult central nervous system (CNS) [4] despite the fact that there is no cell proliferation or angiogenesis. This suggests that b F G F may have a non-mitogenic function. Recently, Cuevas et al. [3] demonstrated that intravenous infusion of b F G F led to systemic vasodilation in both rats and rabbits. The systemic effect was mediated through both nitric oxide and calcitonin gene-related peptide mechanisms. More recently, Rosenblatt et al. [11] observed that the cortical administration of b F G F produced a dose dependent vasodilatation of pial arterioles. This effect was attenuated by the co-administration of L-NAME, a nitric oxide synthetase inhibitor. Furthermore, there was no vasodilatation when heat inactivated b F G F was used or when their medium was preincubated with a b F G F antibody. These authors suggested that b F G F may have an important role in the regulation of cerebral blood flow. The purpose of this current study was to determine if the topical administration of b F G F in adult white New Zealand rabbits
* Corresponding author. Fax: (1) (507) 284-5206. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 1 0 8 6 - 2
would lead to a corresponding increase in cortical blood flow (CBF). U p o n approval by the Institutional Animal Use and Care Committee, overnight fasted white New Zealand rabbits (n = 30) weighing between 2.5 and 3.5 kg were induced with thiamylal sodium 30 m g / k g i.v. and operated and studied under 1.0% inspired halothane. The experimental paradigm consisted of using cortical suffusion to drip onto the exposed fronto-parietal cortex either vehicle or b F G F at a rate of 0.05 m l / m i n to yield a total volume of 1 ml. The temperature and p H of the perfusate was 37.0 + 0.5°C and 7.00, respectively. Recombinant human b F G F (147 a.a., R & D Systems, Minneapolis, MN) was dissolved in normal saline containing 0.1% bovine serum albumin as a carrier. Twenty animals were divided into 4 groups: a low dose b F G F group (n = 5 ) suffused with 0.25 /~g/min and a high dose b F G F group (n = 5) suffused with 1.25 ~ g / m i n to yield a total dose of 5 /zg or 25 /xg, respectively, and a vehicle group (n = 5) suffused with normal saline containing 0.1% bovine serum albumin. A fourth group of animals (n = 5) received intracarotid injection of b F G F as a bolus of 1.8 ~zg/kg. Measurements of rCBF and brain p H i [1] were done at 1 min, 10 min, and 20 min of suffusion or after intracarotid injection. A PaCO2-rCBF response curve was repeated at 30 min. There were no significant differences in the systemic parameters of PHa, PaCO2, PaO2, hematocrit or core
L. Regli et al. /Brain Research 665 (1994) 155-157
156
body temperature among the four groups. Mean arterial blood pressure declined from 93_+ 3 to 75_+ 5 m m H g ( P < 0.05) during the first minute following intracarotid injection of b F G F and then returned to baseline over 10 min. In all animals blood pressure stayed within the range of rCBF autoregulation. Cortical suffusion of bFGF or vehicle generated no change in mean arterial blood pressure. Intracarotid injection of b F G F demonstrated no effect on rCBF. Control animals suffused with a vehicle showed no change in rCBF. Suffusion of 5 /xg bFGF demonstrated no significant increase in rCBF (from 56.5 _+ 2.9 to 58.3 _+ 1.9 m l / 1 0 0 g / m i n ; P = NS). Suffusion of 25/xg b F G F resulted in a rapid and significant increase in rCBF. After one minute rCBF increased by 23% as compared to vehicle (from 54.4 _+ 2.7 to 66.9 _+ 3.2 m l / 1 0 0 g / m i n ; P < 0.05) and at 10 and 20 min the increase reached 33% (54.4 + 2.7 vs. 72.6 _+ 1.6 m l / 1 0 0 g / m i n ; P < 0.05) and 30% (54.4 + 2.7 vs. 71.0 _+ 1.5 m l / 1 0 0 g / m i n ; P < 0.05), respectively. After administration of bFGF, rCBF remained elevated for up to 60 rain post-suffusion ( P < 0.01) (Fig. 1). p~,COz-rCBF reactivity was normal in all experi-
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Fig. 1. Rapid and persistent (up to 60 min) increase in rCBF by suffusion of 25/xg of bFGF (open triangle, dotted line), compared to 5 #g (closed triangle, dashed line), and vehicle (open circle, solid line), rCBF was measured by the change of the indicator umbelliferone (A) and 133Xenon (B). The difference in increase of rCBF between (A) and (B) may reflect a selective effect of bFGF on the microvasculature. Analysis of variance with Tukey's post-hoc analysis was used (*P <0.05 indicates significance). Error bars indicate standard error of the mean.
mental groups before suffusion. ,adler 25 /x b F G F suffusion the PCO 2 reactivity of the brain was decreased by 34% (0.95 _+ 0.06 to 0.63 + 0.14 m l / 1 0 0 g / m i n / m m H g CO 2) despite the increase in rCBF. Brain p H i measured 7.02 _+ 0.02 and was stable throughout the experiment and administration of b F G F had no effect on pHi. This current study would confirm results recently reported by Rosenblatt et al. [11] which suggest that bFGF may play an important role in cerebral blood flow regulation. In our experiment, the increase in cortical blood flow was rapid starting within a minute of application and after ten min of continuous perfusion reached a maximal effect, b F G F administration did not alter brain pH~ in vivo, as opposed to in vitro results showing that growth factors may activate the N a + / H + antiporter in fibroblasts [8]. Therefore, the increase in rCBF was not dependent on brain cortical p H i changes. An alternative explanation is that some of the components of bFGF could change the buffering capacity of the extracellular space resulting in a change of rCBF. Regulation of rCBF is determined by the p H of the extracellular space [2]. One potential difficulty with this experimental paradigm is the large doses which were used in the cortical suffusion. It should be noted that in this preparation, a closed cranial window technique was not utilized. Therefore, the test solutions suffused onto the cortical surface would not provide a continuous lavage but rather run off the brain's surface over the margins of the craniectomy. Therefore, the dose of bFGF which had the opportunity to rest on the brain's surface and potentially diffuse through the pial barrier was probably far less than the concentration of the irrigating solution. In addition, the doses of b F G F used in this study were chosen based on the low diffusion coefficient of peptides and their susceptibility to metabolic inactivation by brain peptidases. Pardridge [10] predicted that the concentration of a peptide in brain parenchyma at a distance of 1 m m from the ependymal surface approximates a concentration that is less than 0.1% of peptide concentration in the ventricular compartment. We extrapolated this estimate as being the probable maximum fraction of b F G F penetrating into the outer parenchymal layers in the cortical suffusion model. Thus, we predicted that the concentration of b F G F at 1 m m from the cortical surface in this study would be only nanograms. The concentration of endogenous brain hFGF in vivo has been reported to be 33-50 n g / m g of brain [7]. The doses used in this study were also based on prior reports of the expression of b F G F in vivo. [9,14] The authors thank Ms. Patricia Friedrich and Mr. Robert Carlson for their technical assistance and to Ms. Mary Soper for secretarial assistance. One of the
L. Regli et al. /Brain Research 665 (1994) 155-157
authors (LR) was supported by Foundation Sicpa and Decker.
[1] Anderson, R.E., Meyer, F.B. and Tomlinson, F.H., Focal cortical distribution of blood flow and brain pH i determined by in vivo fluorescent imaging, Am. J. Physiol., 263 (1992) H565-H575. [2] Cameron, I.R. and Caronna, J., The effect of local changes on potassium and bicarbonate concentration on hypothalamic blood flow in the rabbit, J. Physiol., 262 (1976) 415-430. [3] Cuevas, P., Carcellar, F., Ortega, S., Zazo, M., Nieto, I. and Gimenez-Gallego, G., Hypotensive activity of fibroblast growth factor, Science, 254 (1991) 1208-1210. [4] Ernfors, P., L6nnerberg, P., Ayer-LeLievre, C. and Persson, H., Developmental and regional expression of basic fibroblast growth factor mRNA in the rat central nervous system, J. Neurosci. Res., 27 (1990) 10-15. [5] Ferrara, N., Ousley, F. and Gospodarowicz, D., Bovine brain astrocytes express basic fibroblast growth factor, a neurotropic and angiogenic mitogen, Brain Res., 462 (1988) 223-232. [6] Folkman, J. and Klagsbrun, M., Angiogenic factors, Science, 235 (1987) 442-447. [7] Gospodarowicz, D., Cheng, J., Lui, G.M., Baird, A. and Bohlent, P., Isolation of brain fibroblast growth factor by heparin-Sep-
157
harose affinity chromatography: identity with pituitary fibroblast growth factor, Proc. Natl. Acad. Sci. USA, 81 (1984) 6963-6967. [8] Moolenaar, W.H., Tsien, R.Y., van der Saag, P.T. and de Laat, S.W., N a + / H + exchange and cytoplasmic pH in the action of growth factors in human fibroblasts, Nature, 304 (1983) 645-648. [9] 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. [10] Pardridge, W.M. (Ed), Transnasal and intraventricular delivery of drugs. In Peptide Drug Delivery to the Brain, Raven Press, New York, 1991, pp. 99-122. [11] Rosenblatt, S., Irikura, K., Caday, C.G., Finklestein, S.P. and Moskowitz, M.A., Basic fibroblast growth factor dilates rat pial arterioles, J. Cereb. Blood Flow Metab., 14 (1994) 70-74. [12] Wagner, J.A., The fibroblast growth factors: an emerging family of neural growth factors, Curt. Top. Microbiol. Immunol., 165 (1991) 95-118. [13] Westermann, R., Grothe, C. and Unsicker, K., Basic fibroblast growth factor (bFGF), a multifunctional growth factor for neuroectodermal cells, J. Cell Sci., Suppl. 13 (1990) 97-117. [14] Yamada, K., Kinoshita, A., Kohmura, E., Sakaguchi, T., Taguchi, J., Kataoka, K. and Hayakawa, T., Basic fibroblast growth factor prevents thalamic degeneration after cortical infarction, J. Cereb. Blood Flow Metab., 11 (1991) 472-478.