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(Stroke. 1996;27:2292-2298.)
© 1996 American Heart Association, Inc.

Articles

Effect of Basic Fibroblast Growth Factor on Experimental Focal Ischemia Studied by Diffusion-Weighted and Perfusion Imaging

Turgut Tatlisumak, MD; Kentaro Takano, MD, PhD; Richard A.D. Carano, MS Marc Fisher, MD

the Department of Neurology, Helsinki University Central Hospital, Finland (T.T.); the Department of Neurology, The Medical Center of Central Massachusetts (T.T., K.T., M.F.), the Department of Biomedical Engineering, Worcester Polytechnic Institute (R.A.D.C.), and the Departments of Radiology and Neurology, University of Massachusetts Medical School (M.F.), Worcester, Mass.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
Background and Purpose Basic fibroblast growth factor (bFGF) has documented neuroprotective properties. This study was performed to evaluate the effects of bFGF on infarct size when administered 30 minutes after induction of focal cerebral ischemia in rats. Diffusion-weighted and perfusion MRI were used during the drug infusion.

Methods We blindly randomized 20 Sprague-Dawley rats to receive either drug (n=10) or vehicle (n=10). The animals underwent middle cerebral artery (MCA) occlusion using the suture model. Diffusion-weighted MRI was initiated 30 minutes after induction of ischemia and repeated frequently for 3.5 hours. Drug (45 µg/kg per hour) or vehicle (saline) infusion began 30 minutes after MCA occlusion and continued for 3 hours. Perfusion images were made at 25, 90, and 150 minutes after MCA occlusion. The animals were killed after 24 hours of permanent MCA occlusion, and brains were stained with 2,3,5-triphenyltetrazolium chloride (TTC).

Results The TTC-derived, corrected infarct volume postmortem in the bFGF-treated group was significantly smaller than that in controls (126.6±51.9 versus 180.2±54.9 mm3, mean±SD, P=.038). Diffusion imaging showed essentially equal lesion volumes 3 hours after MCA occlusion (195.4±61 mm3 in the drug-treated group and 194.4±65 mm3 in controls). At 4 hours, ischemic lesion size was 182.1±56.9 mm3 in treated animals and 222.9±88.7 mm3 in the controls (P=.24, NS). Perfusion imaging did not show a change of cerebral perfusion within ischemic brain regions in the bFGF group during the infusion. No behavioral or physiological side effects were observed.

Conclusions bFGF is a safe and effective treatment for focal cerebral ischemia in rats. We observed a modest delayed difference of ischemic lesion size in vivo with diffusion MRI. The diffusion-weighted MRI findings suggest a potential delayed therapeutic effect of bFGF, and the perfusion imaging findings imply that the effect is not due to increased blood flow to the ischemic region.


Key Words: cerebral ischemia, focal • growth factors • magnetic resonance imaging • rats


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
The FGFs are a family of peptide growth factors originally identified as peptides with mitogenic activity for fibroblasts that exist at high levels in brain extracts.1 Members of the FGF family are potent neural growth factors that have specific effects on the division, differentiation, and survival of specific classes of nerve cells.2 bFGF consists of a 154–amino acid, 18-kD polypeptide with multipotential properties.3 FGFs act via specific receptors, and these receptors occur in many tissues, including muscle cells, endothelial cells, fibroblasts, and primary cultures from several regions of the brain that are enriched in neurons.2 Several studies examined the anti-ischemic effects of bFGF on neurons in vitro and in vivo.3 4 5 6 7 8 9 10 11 12 There are no prior studies using MRI to evaluate the in vivo effects of bFGF on brain ischemia.

DWI and PI are novel imaging technologies that are sensitive for the early detection of focal brain ischemia.13 14 15 16 17 18 DWI is based on the random translational movement of water molecules in tissue.19 Ischemia causes a rapid decrease in water diffusion, and ischemic regions appear hyperintense on DW images. Ischemic changes can be seen on DW images only minutes after the induction of brain ischemia, making it useful for very early detection and evaluation of ischemic stroke, whereas conventional methods do not disclose any changes in the early hours.14 15 16 The decrease in diffusion is probably related to membrane energy failure of brain cells,20 leading to a rapid shift of water from extracellular to intracellular compartments in the ischemic tissue.21 The diffusion rate for intracellular water is lower than for extracellular water, causing a net decrease of tissue diffusion during brain ischemia. The microcirculation of the brain (cerebral perfusion) can be evaluated by PI (dynamic contrast-enhanced MRI).13 Dynamic changes in regional brain microcirculation are demonstrated by giving a rapid intravenous bolus of an MRI contrast agent and then observing the effect on the brain by ultrafast imaging. When the bolus of contrast agent arrives in the brain, the susceptibility difference between the paramagnetic contrast agent and the tissue sets up local magnetic field gradients in the tissue, causing a loss of phase coherence and thus a decrease of signal intensity in the tissue surrounding the vessels that results in darkening of the images. Because of arterial occlusion in ischemic stroke, the contrast agent cannot pass into the ischemic region and the ischemic site does not show signal loss; thus, it appears bright (hyperintense) on the images while the normal brain tissue darkens, causing a well-demarcated lesion on PI.17 PI is a useful tool for evaluating acute stroke patients17 and for studying experimental focal cerebral ischemia.22 With PI, it is possible to quantify the MTT, rCBF, and CBFi.22 This study was designed to evaluate the effect of delayed intravenous infusion of bFGF on focal cerebral ischemia in vivo using DWI and PI studies and at postmortem in rats subjected to permanent MCA occlusion.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
Animal Preparation
This study was approved by the Animal Research Committee (ARC) of the University of Massachusetts Medical School (ARC protocol A-643). Twenty male Sprague-Dawley rats weighing 310 to 350 g were used. Animals were housed under diurnal lighting conditions and allowed free access to food and water before and after the experiment. Anesthesia was induced by the intraperitoneal injection of chloral hydrate (400 mg/kg body wt). PE-50 polyethylene tubing was inserted into the left femoral artery for continuous monitoring of arterial blood pressure (78205D, Hewlett-Packard Inc) throughout the study and for measuring arterial pH, PaO2, and PaCO2 (Corning 170-pH Blood Gas Analyzer, Corning Inc) at baseline and 30 and 180 minutes after the induction of ischemia. Another PE-50 catheter was inserted into the inferior vena cava through the left femoral vein for drug and vehicle infusions and gadolinium (Magnevist, Berlex Laboratories) injections for PI. Rectal (core) temperature was continuously monitored with a rectal probe inserted to a 4-cm depth from the anal ring and maintained at 37.0°C with a thermostatically controlled heating lamp (model 73ATD, Yellow Springs Instruments Inc) during the surgery. After the induction of anesthesia, rats were mounted in the prone position on a homemade stereotaxic surgical board with ear bars and a tooth bar.

Experimental Focal Brain Ischemia
Focal brain ischemia was induced by the intraluminal suture MCA occlusion method.23 Briefly, the right CCA and the right ECA were exposed through a ventral midline neck incision. The proximal CCA and the ECA were ligated proximally and permanently. A 4-0 nylon monofilament (Ethilon, Ethicon Inc), with its tip rounded by heating near a flame and then coated with silicon (Bayer), was inserted through an arteriectomy of the CCA approximately 3 mm below the carotid bifurcation and advanced into the internal carotid artery to a point approximately 17 mm distal to the carotid bifurcation. Mild resistance indicated that the suture entered the anterior cerebral artery, thus occluding the origins of the anterior cerebral artery, the MCA, and the posterior communicating artery. The animals were then placed in a 1H home-built birdcage coil and were quickly placed into the bore of magnet. In the MRI device, anesthesia was maintained with 1.0% of isoflurane delivered in air at 1.0 L/min. During the MRI measurements, body temperature was continuously monitored using a rectal probe with 0.1°C resolution (T-type thermocouple, Omega Engineering Inc) and was maintained at 37.0°C using a thermostatically regulated heated air flow system. Mean arterial blood pressure was continuously monitored and recorded every 30 minutes during the MRI protocol, and arterial blood gas samples were obtained through the left femoral arterial catheter at 30 and 180 minutes after MCA occlusion while the animals were in the scanner.

MRI Measurements
The rate of diffusion of water was measured in vivo for each pixel with the use of pulsed field gradient nuclear MR. The ADC24 25 is defined as

(E1)
where k is the wave vector given by the time integral of the diffusion sensitizing gradient, {tau} is the observation time, and M0 is the equilibrium magnetization at k=0. The MRI studies were performed with a General Electric CSI-II 2.0-T/45-cm imaging spectrometer (General Electric Medical System) operating at 85.56 MHz for 1H equipped with ±20 G cm–1 self-shielding gradients (15-cm bore). The ADC maps were obtained from an eight-slice DW echo-planar imaging pulse sequence.26 Half-sine-shaped diffusion gradients were applied along the anterior-posterior (z) axis of the brain, with k={gamma}g{delta}(2/{pi}) and {tau}={Delta}–{delta}/4, where {gamma} is the gyromagnetic ratio and g, {Delta}, and {delta} are the strength, separation, and duration, respectively, of the applied diffusion gradients. All data were acquired with {delta} of 10 milliseconds, {Delta} of 40 milliseconds, repetition time of 4 seconds, and echo time of 92 milliseconds. The image size was 64x64 pixels with a pixel resolution of 400 mm (in-plane) and a slice thickness of 2.0 mm (axial-plane). The echo-planar data acquisition time was 65 milliseconds with two signal averages per image. Eight contiguous slices were acquired using an interleaved slice acquisition pattern to avoid signal contamination from adjacent slices. The ADC maps were generated with the use of 10 b-values (k2{tau}) ranging from 63 to 1898 s/mm2. ADC maps were obtained at 30, 60, 120, 180, 210, and 240 minutes after MCA occlusion.

Data analysis was performed on an Iris Indigo workstation (100 MHz Iris Indigo R4000, Silicon Graphics Inc). The raw image data were filtered by a two-dimensional gaussian low-pass kernel, which had a real-space full width at half maximum of approximately 5 pixels. The ADC value for each pixel was calculated by performing linear regression to obtain the parameters of Equation 1. The threshold value to define abnormal ADC values on ADC maps was evaluated as follows: to define abnormal diffusion values of water in the brain, we compared each pixel in the ischemic hemisphere with its homologous pixel in the normal hemisphere. As previously described, the side-by-side difference of the ADC value from homologous pixels (ie, the ischemic and normal hemispheres that best define the ischemic lesion volume in vivo 2 hours or longer after MCA occlusion) is 29%, highly correlating with postmortem infarct volume.27 Therefore, in this study, an ADC difference value of –29% was used to define abnormal ischemic pixels.

T2*-weighted echo-planar imaging was used to perform dynamic contrast-enhanced PI. A coronal slice thickness of 2 mm, 25.6x25.6-mm field of view, and 64x64-pixel resolution at the optic chiasm were used. The perfusion slice was centered between the fourth and fifth slices of the ADC maps. The echo time of the gradient-recalled echo was 30 milliseconds. A total of 40 images were obtained, one every 0.5 seconds for 20 seconds. Each echo-planar image was completed in 28 milliseconds. The contrast agent gadopentetate dimeglumine (Magnevist, 0.3 mL of 469.01 mg/ms solution, 0.43 to 0.52 mmol/kg) was injected at the seventh imaging time point, beginning 3 seconds after the start of the imaging sequence.

The perfusion data were processed (Iris workstation) to measure the MTT, rCBV, and CBFi as previously described.22 The change in the T2* rate, {Delta}R2*(t), was obtained from the change in signal intensity on the basis of the following relationship:

(E2)
where S(t) is the signal intensity at time t, S0 is the signal intensity before the injection of the contrast agent, and TE is the echo time. An estimate of the tissue concentration as a function of time, c(t), is obtained from {Delta}R2*(t), c(t)={Delta}R2*(t)/k2, where k2 is a proportionality constant. The rCBV was determined for each pixel by the numerical integration of c(t). The proportionality constant k2 and the arterial input function are required to make an absolute measurement of CBV; a relative measure can be obtained in the absence of these parameters. The use of rCBV provides sufficient information to follow temporal changes in CBV. This method was established and shown to be accurate and reliable previously by Hamberg et al.22 The MTT was given for each pixel by the following relationship:

(E3)

The CBFi was determined on a pixel-by-pixel basis from rCBV and MTT as CBFi=rCBV/MTT. CBFi was chosen as a measure of perfusion because it incorporates the information found in both the rCBV and MTT.

The perfusion images were used to determine whether bFGF caused an increase in CBF in a permanently occluded MCA territory. In the ischemic (right) hemisphere, four ROIs, each 2x2 voxels in size, were chosen within the ischemic regions (three ROIs were in the parietal cortex and one in the caudate putamen, Fig 1Down). The MTT, rCBV, and CBFi values were calculated for each ROI at the three PI time points (25, 90, and 150 minutes after MCA occlusion) in each animal. The mean values for both experimental groups were calculated for comparison.



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  		Figure 1. ROIs chosen for PI analysis. Three ROIs (A, B, and C) are located in the cortex and one (D) is located in the caudate putamen. Each ROI is 2x2 voxels in size (1 voxel=0.4 mmx0.4 mmx2 mm=0.32 mm3). Each ROI was standardized by using the same pixel coordinates in the 64x64-pixel image matrix. The placement of each ROI on each image was further confirmed by visual inspection. The left lower pixel for ROI-A was the 33rd pixel from left to right and the 57th pixel from bottom to top. The respective pixel coordinates were for ROI-B, 40-53; for ROI-C, 43-46; and for ROI-D, 36-46.

Drug Infusion
Recombinant bFGF was obtained as concentrated stock (4.3 mg/mL in 20 µmol/L sodium citrate, 0.6 mol/L NaCl, and 4.3 mg/mL bovine albumin, pH 4.5) as a generous gift of Scios-Nova Inc and stored at -80°C before use. Stock solution was melted in room temperature and diluted into 0.9% NaCl, giving a final concentration of 37.5 µg/mL. The infusion was started in the magnet 30 minutes after MCA occlusion in a random and blinded manner and continued for 3 hours at a speed of 0.4 mL/h intravenously. Ten animals received vehicle (saline) and 10 animals received 45 µg/kg per hour bFGF in vehicle. The total infusion volume was approximately 1.2 mL.

Calculation of the Infarct Volume
After the MRI protocol, the animals were removed from the magnet bore, both catheters were removed, operation wounds were sutured, and animals were allowed free recovery from anesthesia in separate cages. Twenty-four hours after MCA occlusion, the animals were scored neurologically using a six-point scale (0 for normal to 5 for death) modified from what was originally proposed by Zea-Longa et al.28 The animals were then anesthetized with chloral hydrate and killed. The brains were quickly removed, inspected to confirm the appropriate placement of the suture occluder, and coronally sectioned into six 2.0-mm-thick slices. The brain slices were incubated for 30 minutes in a 2% solution of TTC at 37°C29 and fixed by immersion in a 10% buffered formalin solution. The unstained areas of the fixed brain sections were defined as infarcted. Brain sections were photographed using a charge couple device camera (EDC-1000HR Computer Camera, Electrim Corp), and images were stored on a microcomputer. Later, by use of an image analysis program (Bio Scan Optimas), the areas of the infarct and the right and left hemisphere were calculated for each brain slice. The corrected infarct area in a slice was calculated to compensate for the effect of brain edema. The corrected infarct area in a slice was calculated by subtracting the area of normal tissue in the ipsilateral hemisphere from the total area of the contralateral hemisphere. The infarct area for each slice was multiplied by slice thickness, and the results for each slice were summed to obtain the total corrected infarct volume for each animal.

Statistical Analyses
Data are expressed as mean±SD. Statistical analyses were performed using unpaired t test or two-factor repeated measures ANOVA for continuous variables and the Mann-Whitney U test for nonparametric variables. A two-tailed value of P<.05 was considered significant.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
Body weight was 327.0±12.3 g for control animals and 330.5±11.2 g for drug-treated animals. The body temperature and the mean arterial blood pressure values remained constant in all animals without any significant difference between the two groups throughout the study. There were no significant differences in arterial pH, PaCO2, or PaO2 between the two groups (data not shown). No behavioral side effects were observed after discontinuation of the anesthesia in any of the rats.

One animal died prematurely 10 hours after MCA occlusion in the control group, and immediate craniectomy and TTC staining were performed. No animals died prematurely in the treated group. The neurological score at 24 hours was 3.1±0.9 for controls and 2.6±0.5 for treated animals (P=.17, NS). Corrected infarct volume derived from the TTC staining was significantly smaller in the animals that received bFGF (126.6±51.9 mm3) than in the controls (180.2±54.9 mm3, P=.038).

The ischemic lesion volumes for all animals were calculated using the ADC maps derived from the DW MRI data. The ischemic lesion volume at 30 minutes after MCA occlusion and before starting the infusion was 96.9±34.0 mm3 for control animals and 86.2±36.0 mm3 for treated animals (P=.5, NS). The DWI-derived ischemic lesion volumes increased to 194.4±65.0 and 195.4±61.0 mm3 at 180 minutes for controls and treated animals, respectively. The ischemic lesion volumes of controls continued to grow and reached 222.9±88.7 mm3 at 4 hours after MCA occlusion, whereas in drug-treated animals the ischemic lesion volume was 182.1±56.9 mm3 (P=.24, NS) on ADC maps (Fig 2Down).



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  		Figure 2. Serial changes in the evolution of lesion volume over time in the control ({circ}) and the bFGF-treated groups ({bullet}). Values are mean±SD. MCAO indicates MCA occlusion.

MTT, rCBV, and CBFi values were calculated for each ROI at each of the three PI time points and were compared between the two experimental groups (n=9 for each group; 1 animal in each group was excluded from PI data analyses because of technical limitation of MR data acquisition). These PI parameters did not differ between the control and drug-treated groups for the corresponding ROIs at any imaging time point (between group analysis) and over time in the same group (in group analysis). The data are shown in the TableDown.


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  		Table 1. Perfusion Imaging Results*


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
The present study demonstrates that bFGF infused intravenously starting 30 minutes after induction of focal brain ischemia by permanent occlusion of the MCA significantly reduces postmortem infarct size in rats and shows a trend toward delayed ischemic lesion volume shrinkage in vivo. Our results are consistent with several previous reports.3 6 7 9 10 12 In vitro, bFGF increases the survival and growth of neurons in cultures derived from the cortex,30 hippocampus,31 spinal cord,32 and multiple regions including the cortex, hippocampus, thalamus, and septum.33 Furthermore, it protects cultured neurons against nitric oxide–induced toxicity,5 hypoglycemia,34 and excitatory amino acid–induced neurotoxicity.35 36 In vivo, bFGF protects brain neurons against mechanical injury37 and N-methyl-D-aspartate neurotoxicity.7 bFGF reduced ischemic neuronal death in vivo when administered intraventricularly.38 Intraventricular injection of bFGF 2 hours after global forebrain ischemia in gerbils reduced CA1 neuronal loss.39 Nozaki et al7 reported a neuroprotective effect with intraperitoneal pretreatment of bFGF in neonatal rats exposed to hypoxia/ischemia. Koketsu et al9 found that intraventricular administration of bFGF starting 3 days before ischemia reduced infarct volume in a model of focal cerebral ischemia. Fisher et al3 also found a significant reduction in the infarct size (52%) with a permanent focal ischemia model in rats when bFGF was started intravenously 30 minutes after MCA occlusion. Delayed intravenous treatment was also effective in reducing ischemic lesion size in a reperfusion model.10 A study using intracarotid infusion of bFGF in a rat focal ischemia model showed a significant reduction in infarct size.12 A recent study using a different focal ischemia model in rats failed to show any anti-ischemic effect of bFGF when administered intraventricularly.40 This result is likely to be due to the very late commencement of the drug therapy (2 days after MCA occlusion). Kawamata et al41 started intracisternal bFGF (1 µg per injection) treatment in a biweekly regimen 24 hours after permanent proximal MCA occlusion in mature Sprague-Dawley rats. After 4 weeks, the infarct volumes did not differ; however, the rate and the degree of neurological outcome in bFGF-treated rats was significantly better as measured by several behavioral tests.41

Hypotension is a well-known side effect of bFGF, likely related to a dose-dependent vasodilating effect.42 At the dose used (45 µg/kg per hour), we did not observe hypotension in any rats. Most studies using bFGF, including the present study, had short-term treatment protocols. Because bFGF is a mitogen for astrocytes,43 44 oligodendrocytes,45 46 and endothelial cells in vitro,47 48 delayed side effects such as induction of neoplasms may be expected. However, up to 1 month of intracerebral application of bFGF did not cause neoplastic changes.49 50 Yamada et al51 induced focal ischemia in Wistar rats and started intracisternal injections of bFGF 24 hours later. They killed the animals 28 days later. In vehicle animals, they observed thalamic degeneration secondary to MCA territory infarction, whereas in bFGF-treated animals the microscopic structure of the thalamus was well preserved. They did not report any microscopic or macroscopic unexpected findings.51 In a recent study, 4 weeks of intracisternal bFGF treatment (1 µg per injection, biweekly injections) did not lead to abnormal cell proliferation in brain.41 These data give further support to the long-term safety of bFGF.

Radiolabeled bFGF does not cross the blood-brain barrier in the intact hemisphere but crosses it in the damaged hemisphere.3 Because bFGF is neurotrophic and neuroprotective in concentrations of 1 to 10 ng/mL in vitro, the appearance of only a small fraction of the administered bFGF to the ischemic injury site should be enough to have a neuroprotective role.33 52

How bFGF reduces infarct size still remains uncertain. The expression of bFGF dramatically increases after traumatic and ischemic brain injury.53 54 Because bFGF is a potent vasodilator, it could dilate the collaterals in the peri-ischemic zone even at doses not promoting systemic hypotension, thus increasing the blood flow to the penumbral regions.12 The observation that topical application of bFGF dilates cerebral pial arteries in rats supports this hypothesis.55 The PI results in our study did not demonstrate that an intravenous infusion of bFGF affected perfusion, implying that the neuroprotective effect with intravenous administration may be independent of an improvement in CBF directly in the ischemic regions. In the rat suture occlusion model, a potential problem could be the effect of a vasodilating agent inducing recirculation around the occluder. Because this would lead to a major improvement in blood flow to the ischemic regions, PI should be able to detect this effect, and it did not. bFGF is an angiogenic agent, but since the animals were killed at 24 hours, the observation period is not sufficient to detect neoangiogenesis. Transient neuroprotective effects of hypothermia are well known, and agents that reduce brain temperature can postpone or diminish ischemic damage.56 Intravenously administered bFGF, initiated 30 minutes after inducing focal brain ischemia in a previous study, did not change brain temperature in rats.3 DWI is sensitive to brain temperature changes,57 and small changes in brain temperature can be demonstrated on ADC maps in rats.58 We did not observe any significant change in ADC values calculated from the intact brain hemispheres of treated animals before and during drug infusion (data not shown). This implies that bFGF does not alter brain temperature. Because bFGF has diverse neuroprotective properties in vitro against anoxia, hypoglycemia, excitatory amino acids, free radicals, and nitric oxide, at least part of its anti-ischemic effect may be related to neuroprotection. The excitatory amino acids have a pivotal role in brain ischemia, and other molecules including free radicals and nitric oxide contribute to ischemic injury. The neuroprotective effect of bFGF may also be related to new neuronal gene transcription, protein synthesis, and inhibition of intracellular influx of calcium during ischemia.35 The suture occlusion model causes large lesions in the MCA territory in rats, and long-term studies with this model are associated with high mortality. We designed our study with death at 24 hours after MCA occlusion. Long-term benefit of bFGF was shown by others using a different focal brain ischemia model that allows good survival.41 51 These studies suggest that the effects of bFGF on focal ischemia are not transient.

The lesion size on ADC maps increased similarly over time in both groups for the initial 3 hours (Fig 2Up). Ischemic lesion size in the treated group decreased slightly at the 3.5-hour imaging time point, whereas the ischemic lesion size continued to increase in the control group. This finding persisted at the 4-hour imaging time point and suggests the possibility of a delayed treatment effect with bFGF beginning around 3 hours after starting the drug infusion and possibly continuing for several hours. This possibility will have to be confirmed by more prolonged MRI protocols.

Our findings support prior observations that bFGF is a safe and effective drug when given intravenously after the onset of focal cerebral ischemia; it is without detectable effects on cerebral perfusion and may have a delayed treatment effect. bFGF represents a novel potential approach to acute ischemic stroke therapy.


*    Selected Abbreviations and Acronyms
 
ADC = apparent diffusion coefficient
(b)FGF = (basic) fibroblast growth factor
CBFi = cerebral blood flow index
CCA = common carotid artery
DWI = diffusion-weighted MRI
ECA = external carotid artery
MCA = middle cerebral artery
MTT = mean transit time
PI = perfusion imaging
rCBV = relative cerebral blood volume
ROI = region of interest
TTC = 2,3,5-triphenyltetrazolium chloride


*    Acknowledgments
 
This study was partly supported by Scios-Nova Inc, Mountain View, Calif.


*    Footnotes
 
Reprint requests to Dr Turgut Tatlisumak, Department of Neurology, Helsinki University Central Hospital, Haartmaninkatu 4, 00290 Helsinki, Finland.

Received June 5, 1996; revision received August 1, 1996; accepted September 6, 1996.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
down arrowIntroduction 
 down arrowReferences 
 

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Editorial Comment

Chang Y. Hsu, MD, PhD, Guest Editor

Cerebrovascular Disease SectionDepartment of NeurologyWashington University School of MedicineSt Louis, Mo


*    Introduction 
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 up arrowReferences
 *Introduction 
down arrowReferences 
 
Potential neuroprotective effects of trophic factors have been extensively tested in animal stroke models. Among the recently identified trophic factors, bFGF has been consistently noted to reduce ischemic brain injury in a number of laboratories.1R 2R 3R 4R In the preceding article, Tatlisumak and associates confirmed the bFGF effect in reducing infarct volumes in a rat stroke model.5R An important aspect of this study was the application of advanced MR technology to assess the brain region at risk of developing infarction. The lesion volumes based on DWI were essentially equal between control and treatment groups before infusion of bFGF. Four hours after bFGF administration, the lesion volumes defined by DWI were significantly reduced in the treatment group. This treatment effect was confirmed 24 hours later by a conventional method using TTC stain.

The applicability of animal stroke models to the development of new therapies has been addressed in a series of editorials in Stroke.6R 7R 8R 9R 10R A recurrent critique is a "serious mismatch" between animal experiments and therapeutic interventions in stroke patients. The former frequently are based on morphological assessment of infarct volumes, while the latter usually are based on functional outcomes.6R 7R 8R 9R 10R The application of new MR technology as shown in the study by Tatlisumak et al promises to narrow the gap in outcome measures between animals and stroke patients. DWI offers the advantage of early detection of ischemic brain lesion.11R DWI already has been successfully applied to confirm therapeutic efficacies of a group of glutamate antagonists in preclinical drug testing.12R 13R 14R DWI is currently under study to confirm its usefulness in the evaluation of patients with ischemic stroke.15R It is likely that future clinical trials in stroke patients may add MR therapeutic end points similar to those used in animal stroke models.


*    Selected Abbreviations and Acronyms
 
ADC = apparent diffusion coefficient
(b)FGF = (basic) fibroblast growth factor
CBFi = cerebral blood flow index
CCA = common carotid artery
DWI = diffusion-weighted MRI
ECA = external carotid artery
MCA = middle cerebral artery
MTT = mean transit time
PI = perfusion imaging
rCBV = relative cerebral blood volume
ROI = region of interest
TTC = 2,3,5-triphenyltetrazolium chloride

*PI data include 9 animals per group (1 animal from each group was discarded because of defective data).


*    References 
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 up arrowReferences
 up arrowIntroduction 
 *References 
 

  1. Koketsu N, Berlove DJ, Moskowitz MA, Kowall NW, Caday CG, Finklestein SP. Pretreatment with intraventricular basic fibroblast growth factor decreases infarct size following focal cerebral ischemia in rats. Ann Neurol.. 1994;35:451-457.
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  4. Jiang N, Finklestein SP, Do TY, 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 in the rat. J Neurol Sci. 1996;139:173-179.[Medline] [Order article via Infotrieve]
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