Basic Fibroblast Growth Factor Increases Regional Cerebral Blood Flow and Reduces Infarct Size After Experimental Ischemia in a Rat Model
Background and Purpose The aim of this study was to ascertain whether basic fibroblast growth factors (bFGF) caused reduction in size of cerebral infarcts in Sprague-Dawley rats with experimental ischemia.
Methods In the first experiment we induced permanent occlusion of the left middle cerebral artery (MCA). Within 5 minutes after MCA occlusion, we infused bFGF (100 ng in 0.1 mL of saline) in the bFGF-treated group (n=14) and 0.1 mL of saline alone in the control group (n=7) into the common carotid artery ipsilateral to MCA occlusion. We harvested the brains 24 hours after MCA occlusion and determined infarct size planimetrically as a percentage of hemisphere size. In the second experiment cerebral blood flow (CBF) was continuously measured for 120 minutes after MCA occlusion in the bFGF-treated group (n=9) and in the control group (n=8) with the use of laser-Doppler flowmetry.
Results Infarct size in the bFGF-treated group decreased significantly in comparison with that in the control group (repeated-measures ANOVA, P<.0001). CBF in the transitional areas between the MCA and the anterior cerebral artery significantly increased in the bFGF-treated group in comparison with that in the control group (repeated-measures ANOVA, P<.005). An approximate 58% decrease in infarct size and a 40% increase in regional CBF were seen on bFGF treatment.
Conclusions The present study suggested that intracarotid administration of bFGF (100 ng) can reduce infarct size after MCA occlusion. It was speculated that the increased CBF in the penumbral areas of MCA may contribute to contraction of infarct size.
It has been shown experimentally that bFGF has both vasodilatory1 and angiogenetic effects2 3 and a protective effect on neurons.4 5 6 7 Yanagisawa-Miwa et al8 reported that intra-arterial infusion of FGF into the coronary artery in a canine model of myocardial infarction significantly reduced the extent of the infarct, and Baffour et al9 stated that in a rabbit model of lower limb ischemia, intramuscular injection of bFGF decreased the size of the ischemic area. Also, in an MCA occlusion model in the rat, Yamada et al7 found that intracisternal administration of bFGF was effective against retrograde degeneration in the thalamus secondary to ischemia in the cerebral gray matter, and Nozaki et al6 reported that intraperitoneal administration of bFGF can exert neuroprotective effects in a neonatal rat model of hypoxia-ischemia. In models of focal ischemia, Koketsu et al10 and Morita et al,11 who administered bFGF intraventricularly, and Finklestein et al12 and Jiang et al,13 who used intravenous administration, showed that it reduced infarct size. In view of the existence of such reports, we set out to ascertain whether bFGF would show efficacy in a rat model of cerebral ischemia.
Materials and Methods
The Committee on Animal Research at Kitasato University School of Medicine approved the protocol used in this study. Male Sprague-Dawley rats weighing 330 to 370 g were housed under diurnal lighting conditions and were allowed free access to food and water before experimentation.
Neither of the experiments was conducted in a blinded fashion.
Anesthesia and Monitoring
In the experiments, anesthesia was induced with 3% to 4% halothane in a 1:1 mixture of N2O and O2 delivered through a closely fitting face mask. A maintenance dose of 0.5% to 1.0% halothane was adjusted slightly so that tail pinches caused no reactions such as abrupt changes in heart rate, MAP, and respiratory rate. We introduced a polyethylene catheter into the right femoral artery to allow continuous monitoring of MAP (polygraph 141-6, NEC Sanei) and for blood sampling for blood gas analysis, pH, and hematocrit (Corning Medical 168 pH/blood gas system). Body temperature was monitored by rectal probe (model MGA-III type 219, Nihon Kohden) and maintained at 37°C with a heating lamp. In the second of these experiments (a CBF measurement study), plasma glucose was also determined, and the temperature of the temporal muscle ipsilateral to the occluded MCA, which was considered to be an index of the brain temperature, was monitored by inserting a needle-type thermistor electrode (TN-94015, Unique Medical Co, Ltd) into that muscle. Heart rate was determined from the arterial pulse.
Experiment 1: Effect of bFGF on Brain Infarct Size
The left common carotid artery was exposed through a midline incision, and a silicone tube 0.3 mm in diameter and 5 cm in length and fitted with an indwelling 29-gauge metal needle was inserted into the artery. After this was secured in place, the rat was mounted on a stereotaxic frame. The left proximal MCA was then exposed by a subtemporal approach and occluded from proximal to the olfactory tract to the inferior cerebral vein by electrocauterization.14 15
Within 5 minutes after left MCA occlusion, Sprague-Dawley rats received bFGF (FGF group, n=14, bFGF 100 ng in 0.1 mL of saline, pH 7.0, R&D System) or saline (control group, n=7, saline 0.1 mL) by intra-arterial infusion (ipsilateral common carotid artery) with the use of an inserted metal needle. After surgery the rats were returned to their cages and allowed free access to food and water. They were decapitated under ether anesthesia after 24 hours. The brain was removed carefully and sectioned coronally across the optic chiasma, and subsequent coronal cuts were made at equal intervals, three anterior and three posterior to the first, resulting in six coronal slices. To delineate the infarct areas, we stained all brain slices with the dye 2,3,5-triphenyltetrazolium chloride, which stains only viable tissue brick red.16 Infarct areas were found in all of coronal slices 1 to 5 in each rat, but not in any coronal slice 6. We therefore determined the brain infarct area for each of coronal slices 1 to 5 in each group planimetrically using a Macintosh image analyzer, and we expressed each such area as a percentage of the affected hemisphere.
Experiment 2: Effect of bFGF on CBF
In this experiment, a separate group of 17 rats was used.
We performed laser-Doppler flowmetry using an ALF21N lasermeter (Advance, Inc) equipped with a 3.0-mW semiconductive laser with a wavelength of 780 nm to continuously monitor cortical CBF. The left dorsolateral portion of the calvaria was carefully abraded with a high-speed diamond minidrill under a surgical microscope until only a paper-thin layer of bone remained. The dura and a thin inner layer of bone (approximately 0.1 mm thick) were kept intact. The vessels in the dura and in the pia could be seen clearly through this abraded area. A laser-Doppler flowmetry probe (tip diameter, 3 mm) attached to a micromanipulator was placed on the thin bone layer 4 mm lateral and 2±0.5 mm caudal to the bregma, and their positions were carefully chosen to avoid large dural or pial vessels. The area selected for blood flow monitoring corresponded with the transitional zone between the MCA and the ACA supply areas.17 18 Steps were taken to prevent the entry of extraneous light into the probe. Steady-state baseline values were recorded for 5 to 10 minutes before MCA occlusion. Then the left MCA was occluded by the same method used in experiment 1.14 15 The values of CBF in the control group (n=8) and the FGF group (n=9) after MCA occlusion fell to 56±1% and 60±3%, respectively, of the preocclusion values, but no statistically significant difference was seen in either group. These data indicate that the CBF measurement sites corresponded in the two groups. Three to 5 minutes after MCA occlusion, bFGF (FGF group: bFGF 100 ng in 0.1 mL of saline) or saline (control group: saline 0.1 mL) was administered through the silicone tube with the indwelling needle in the left common carotid artery. The CBF was continuously monitored from before MCA occlusion until 120 minutes after bFGF and saline administration. The CBF values after bFGF or saline administration were expressed in terms of percent changes from the steady-state baseline values after occlusion.
Physiological parameters for both experiments are shown in Tables 1⇓ and 2⇓. There were no within-group or between-group differences in MAP, Paco2, Pao2, pH, hematocrit, rectal temperature, or heart rate in these experiments.
The areas occupied by brain infarcts in coronal slices 1 to 5, expressed as percentages of the affected hemispheres (mean±SEM), are shown in Fig 1⇓. On analysis with the linear contrasts method (repeated-measures ANOVA, type III), infarct size in the FGF group decreased significantly more than that in the control group (P<.0001), and the infarct size in coronal slices 1, 2, and 3 was significantly smaller (P<.01) in the FGF group than in the controls. The total calculated infarct volumes were 13.4±2.9% and 31.6±2.5% of the affected hemisphere volumes, respectively, showing a significant difference by unpaired Student’s t test (P<.005).
Changes in CBF after bFGF administration are shown in Fig 2⇓.
The repeated-measures ANOVA showed a significant intergroup difference (P<.005; Fig 2⇑). Also, in the FGF group the CBF from 20 minutes after bFGF administration was significantly greater than that in the controls, as indicated by unpaired Student’s t tests, and a significant difference (P<.05, then P<.01; Fig 2⇑) persisted until the end of the experiment.
Experiments 1 and 2 showed that the infarct volume decreased by 58% and that CBF in the transitional areas between the MCA and the ACA increased by approximately 40% in the FGF group in comparison with the corresponding changes in the control group.
There are a number of methods of determining the extent of an experimental infarction. We used 2,3,5- triphenyltetrazolium chloride staining to measure the size of the infarcts directly.16 Twenty-four hours after MCA occlusion, the infarcts in the bFGF-administered group had decreased significantly in volume in comparison with those in the control group. The present study is the first to describe size reduction of cerebral infarcts as a result of infusion of bFGF into the common carotid artery. However, since we made our estimates of infarct size 24 hours after MCA occlusion, we cannot exclude the possibility that bFGF simply delays the eventual development of infarcts instead of causing a permanent decrease in their size. To resolve this problem, we will perform an additional experiment to demonstrate whether infarct volume is still reduced after a delay of a few days.
Mechanism of Cerebral Infarct Size Reduction
We have not yet been able to elucidate the mechanism whereby cerebral infarcts are reduced in size by bFGF, but the following are possible explanations.
Angiogenesis by bFGF
It is clear that bFGF possesses angiogenic properties.2 3 Intermittent administration of bFGF into the cerebral ventricles of rats whose carotid arteries had been ligated bilaterally was followed by a dose-dependent increase in the number of capillary vessels,19 and this effect was reported to bring about neovascularization 2 weeks after the single topical administration of bFGF in the cerebral cortex of normal rats.20
Angiogenesis consists of a series of processes whereby the budding of endothelial cells from existing blood vessels takes place, followed by the formation of a network of new capillaries. In the present experiment infarcts were examined and measured 24 hours after bFGF administration, and therefore it would be difficult to accept the proposition that their reduction in size could be due to neoangiogenesis alone.
Dilatation of Collateral Vessels by bFGF
We observed that bFGF, on administration into the common carotid artery, caused a marked increase in the CBF (Fig 2⇑). The site of our measurement of CBF in the present study was the transitional zone between the MCA and the ACA supply areas17 18 ; the degree of blood flow reduction after MCA occlusion (56±1% reduction in the control group, 60±3% reduction in the FGF group), which was approximately half that found by Takagi et al21 and by Morikawa et al,22 suggested that this site was one in which the ACA blood supply had a major effect.
Cuevas et al23 observed a dose-dependent decrease in the systemic blood pressure on intravenous administration of FGF in the rat, indicating that FGF dilates the systemic vessels. However, several reports have focused on the reaction of the cerebral vessels to bFGF. For instance, it has recently been reported that topically applied bFGF caused dilation of the pial arterioles of the normal rat as a result of a nitric oxide–dependent mechanism1 and also that topically applied bFGF dose-dependently improved the regional CBF of normal rabbits. However, it was found that intracarotid bFGF injection caused no increase in normal rabbits.24
Species differences and differences in the bFGF administration route must be taken into account, but the results of the above studies suggest the possibility that although intraluminal administration of bFGF induces dilation of the systemic vessels, this effect may not extend to the cerebral vessels. However, we would further suggest, on the basis of the findings in the above paragraph, that local abluminal administration of bFGF does bring about dilation of the cerebral vessels.
On the basis of these reports, we speculated as follows on the mechanism whereby the infarcts were reduced in size in the present study. First, the bFGF administered by carotid injection may have leaked out of the patent ACA. The escaping bFGF may next have caused dilation of the collateral channels originating in the ACA, then dilating the arterioles in the penumbral tissue around the occluded MCA, which would have caused an increase in the CBF in the penumbral tissue. It was thought that such an increase in penumbral tissue CBF might possibly be a contributory factor in the decrease in infarct size.
Role of bFGF as a Trophic Factor
It has been shown that bFGF prolongs the survival of neurons25 and stimulates the synthesis of DNA in astrocytes.26 In addition to this neurotrophic action, bFGF inhibits the neurotoxic effect of glutamate, reduces N-methyl-d-aspartate–induced excitotoxic damage, and also suppresses intracellular calcium overload.27 28 It is believed that bFGF acts directly as a trophic factor on neurons and astrocytes, and the possibility of contributions by these mechanisms to cerebral infarct size reduction cannot be ruled out in the present study.25 26
Selection of bFGF Dose
The decision to use a 100-ng dose in this study was made for the following reasons: Cuevas et al23 showed that the intravenous injection of bFGF in mature rats lowered MAP dose dependently with doses of 190 to 929 ng; in addition, Yanagisawa-Miwa et al8 in the canine model of myocardial infarction mentioned above used approximately 1 μg/kg of bFGF without affecting blood pressure. To avoid inappropriate alteration of the blood pressure, we decided to use a dose comparable to that of the latter report.
Penetration of BBB by bFGF
According to Regli et al,24 the effect of bFGF was not generated on the luminal side of the vessels but rather from the abluminal compartment. Therefore, the bFGF that was injected into the intracarotid artery must pass through the BBB.
Since bFGF consists of charged polypeptides of high molecular weight (molecular weight, 16 to 18 kD), it is probable that it cannot pass through the BBB under normal circumstances. It is reported that in normal rabbits receiving intracarotid administration of bFGF, no change in CBF was seen.24 However, in our study bFGF was administered intra-arterially after MCA occlusion, and therefore the results of the above study, in which intra-arterial infusion was performed when the BBB conditions were normal, do not apply directly.
Gotoh et al29 judged the period of BBB permeability in a model of MCA occlusion from the passage of albumin (molecular weight, 69 kD) and reported that this passage took place 72 hours after MCA occlusion. On the basis of these results, we considered that during the period of bFGF administration in our experiment, the damage to the BBB was probably slight. Therefore, although the failure of albumin to pass through the BBB in any significant quantity was not perhaps unexpected, it is quite possible that bFGF, having a far smaller molecular weight than albumin, did penetrate the BBB during the first 24 hours.
Selected Abbreviations and Acronyms
|ACA||=||anterior cerebral artery|
|bFGF||=||basic fibroblast growth factor|
|CBF||=||cerebral blood flow|
|FGF||=||fibroblast growth factor|
|MAP||=||mean arterial blood pressure|
|MCA||=||middle cerebral artery<\/.>|
The authors wish to thank Sigeyoshi Maruyama and Hiroshi Ishikawa for technical assistance and C.W.P. Reynolds for linguistic assistance.
- Received January 20, 1995.
- Revision received June 30, 1995.
- Accepted July 12, 1995.
- Copyright © 1995 by American Heart Association
Thompson JA, Anderson KD, DiPietro JM, Zwiebel JA, Zametta M, Anderson WF, Maciag T. Site-directed neovessel formation in vivo. Science. 1988;241:1349-1352.
MacMillan V, Judge D, Wiseman A, Settles D, Swain J, Davis J. Mice expressing a bovine basic fibroblast growth factor transgene in the brain show increased resistance to hypoxemic-ischemic cerebral damage. Stroke. 1993;24:1735-1739.
Yanagisawa-Miwa A, Uchida Y, Nakamura F, Tomaru T, Kido H, Kamijo T, Sugimoto T, Kaji K, Utsuyama M, Kurashima C, Ito H. Salvage of infarcted myocardium by angiogenic action of basic fibroblast growth factor. Science. 1992;257:1401-1403.
Baffour R, Berman J, Garb JL, Rhee SSW, Kaufman J, Friedmann P. Enhanced angiogenesis and growth of collaterals by in vivo administration of recombinant basic fibroblast growth factor in a rabbit model of acute lower limb ischemia: dose-response effect of basic fibroblast growth factor. J Vasc Surg. 1992;16:181-191.
Morita Y, Murayama N, Inoue T, Ogino R, Ohno T. Protective effects of bFGF against neuronal damages in vitro and in vivo. Soc Neurosci Abstr. 1993;19:1644. Abstract.
Finklestein SP, Meadows ME, Fisher M, Do T, Charette M. Intravenous basic fibroblast growth factor (bFGF) reduces infarct size following focal cerebral ischemia in rats. Soc Neurosci Abstr. 1994;20:181. Abstract.
Jiang N, Finklestein SP, Do T, Caday CG, Charette M, Chopp M. Delayed intravenous administration of basic fibroblast growth factor (bFGF) reduces infarct volume in a model of focal cerebral ischemia/reperfusion. Stroke. 1995;26:165. Abstract.
Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL, Bartkowski HM. Rat middle cerebral artery occlusion: evaluation of a neurologic examination. Stroke. 1986;17:472-476.
Bederson JB, Pitts LH, Germano SM, Nishimura MC, Davis RL, Bartkowski HM. Evaluation of 2.3.5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats. Stroke. 1986;17:1304-1308.
Nakayama H, Ginsberg MD, Dietrich WD. (S)-Emopamil, a novel calcium channel blocker and serotonin S2 antagonist, markedly reduces infarct size following middle cerebral artery occlusion in the rat. Neurology. 1988;38:1667-1673.
Takagi K, Ginsberg MD, Globus MY-T, Dietrich WD, Martinez E, Kraydieh S, Busto R. Changes in amino acid neurotransmitters and cerebral blood flow in the ischemic penumbral region following middle cerebral artery occlusion in the rat: correlation with histopathology. J Cereb Blood Flow Metab. 1993;13:575-585.
Morikawa E, Moskowitz MA, Huang Z, Yoshida T, Irikura K, Dalkara T. l-Arginine infusion promotes nitric oxide–dependent vasodilation, increases regional cerebral blood flow, and reduces infarction volume in the rat. Stroke. 1994;25:429-435.
Cuevas P, Carceller F, Ortega S, Zazo M, Nieto I, Gimenez-Gallego G. Hypotensive activity of fibroblast growth factor. Science. 1991;254:1208-1210.
Regli L, Anderson RE, Meyer FB. Basic fibroblast growth factor increases cerebral blood flow in vivo. J Cereb Blood Flow Metab. 1993;13(suppl 1):S66. Abstract.
Mattson MP, Murrain M, Guthrie PB, Kater SB. Fibroblast growth factor and glutamate: opposing roles in the generation and degeneration of hippocampal neuroarchitecture. J Neurosci. 1989;9:3728-3740.
Gotoh O, Asano T, Koide T, Takakura K. Ischemic brain edema following occlusion of the middle cerebral artery in the rat, I: the time courses of the brain water, sodium and potassium contents and blood-brain barrier permeability to 125I-albumin. Stroke. 1985;16:101-109.