- Email: [email protected]
Therapeutic Efficacy of Basic Fibroblast Growth Factor on Experimental Focal Ischemia Studied by Magnetic Resonance Imaging
Therapeutic Efficacy of Basic Fibroblast Growth Factor on Experimental Focal Ischemia Studied by Magnetic Resonance Imaging Kolammal Nageswari, PhD,*,¶ Shigenori Mizusawa, BS,* Yasushi Kondoh, Kazuhiro Nakamura, PhD,‡,§ and Iwao Kanno, PhD‡
PhD,†
The purpose of this study was to evaluate the effect of intravenous infusion of basic fibroblast growth factor (bFGF) in a permanent ischemia model at the subacute phase (2 weeks) as well as at 24 hours and 1 week using T2-weighted magnetic resonance imaging (MRI). The middle cerebral artery (MCA) in Sprague-Dawley rats was occluded using an intraluminal suture method. The rats were randomly divided into 2 groups to receive either bFGF (45 g/kg/hr) or saline solution. The infusion was started 30 minutes after MCA occlusion (MCAO) and continued for 3 hours. Regional cerebral blood flow (rCBF) was measured using laser Doppler flowmetry throughout the infusion. T2-weighted MRI was carried out before MCAO, 24 hours after MCAO, and days 7 and 14 after MCAO. Although an elevation in rCBF was seen after the infusion, no significant change between the groups was observed. A significant difference between the bFGF and saline groups in T2-derived lesion volume was observed at 24 hours (P ⬍ .05), on day 7 (P ⬍ .05), and on day 14 (P ⬍ .01). The percentage of lesion area calculated from the ipsilateral hemisphere using hematoxylin and eosin staining on day 14 showed a significant difference between the bFGF and saline groups (P ⬍ .05). No significant change in the number of bromodeoxyuridine (BrdU)-labeled cells between the groups was observed. This study demonstrates that bFGF, infused intravenously starting 30 minutes after the induction of permanent MCAO, significantly reduces region volume even at day 14, as well as at days 1 and 7, compared with the corresponding saline group. Key Words: Cerebral ischemia— growth factors— cerebral blood flow—magnetic resonance imaging. © 2005 by National Stroke Association
Neurotrophic factors are essential for the survival and differentiation of normally developing neurons, and also *From the Department of Internal Medicine, †Department of Neurology, ‡Department of Radiology, and §Akita Industry Promotion Foundation, Akita Research Institute of Brain and Blood Vessels, Akita, Japan and ¶School of Biosciences and Bioengineering, Indian Institute of Technology, Bombay, Powai, India. Received December 29, 2004; revised March 30, 2005; accepted May 10, 2005. Address reprint requests to Shigenori Mizusawa, Department of Internal Medicine, Akita Research Institute of Brain and Blood Vessels, 6 –10 Senshu-Kubota machi, Akita 010-0874, Japan. Email: [email protected] 1052-3057/$—see front matter © 2005 by National Stroke Association doi:10.1016/j.jstrokecerebrovasdis.2005.05.004
play important roles in the protection and recovery of mature neurons under pathological conditions.1 Among these factors, the anti-ischemic effects of basic fibroblast growth factor (bFGF) on neurons in vitro and in vivo have been examined.2–9 Endogenous bFGF expression is known to be up-regulated in tissue surrounding focal brain wounds or infarcts, and exogenous application of bFGF reduces the degree of cell death and stimulates neuronal sprouting in models of ischemic brain injury.10,11 bFGF is also a well-known angiogenic agent.12 Several studies have reported on the therapeutic efficacy of bFGF in acute cerebral ischemia. But most experiments have been evaluated for only 24 hours (the observation period), which is insufficient to detect the longterm effects of bFGF, including neoangiogenesis.4 – 6
Journal of Stroke and Cerebrovascular Diseases, Vol. 14, No. 5 (September-October), 2005: pp 187-192
187
K. NAGESWARI ET AL.
188
Some reports have examined the long-term (beyond 24 hours) effects of intravenous infusion of bFGF; however, these studies have evaluated the ischemic lesion volume at only a single time point after bFGF treatment.8,9 The present study is designed to evaluate the effect of bFGF during the subacute phase of 14 days by monitoring T2-weighted magnetic resonance imaging (MRI) findings at 24 hours, 7 days, and 14 days after bFGF administration in the same animals with middle cerebral artery (MCA) occlusion (MCAO) and also comparing it with the histopathological measurement at day 14. The effect of bFGF on proliferating cells in the ipsilateral hemisphere of ischemia was also evaluated by bromodeoxyuridine (BrdU) immunohistochemistry.
Materials and Methods Animal Preparation Twenty-one male Sprague-Dawley rats weighing 320 – 400 g were used. Anesthesia was induced with 3%– 4% halothane in a 7:3 mixture of N2O and O2 delivered through a close-fitting mask. PE-50 polyethylene catheters were inserted into both the tail artery and vein for measuring blood pressure, pH, arterial carbon dioxide tension (PaCO2), and arterial oxygen tension (PaO2) and drug infusion, respectively. The rectal temperature was continuously monitored and was maintained at 37 ⫾ 0.5°C with a thermostatically controlled heating pad. The MCA was occluded using an intraluminal suture method.13 The left common, external, and internal carotid arteries (CCA, ECA, and ICA) were identified through a ventral cervical midline incision. The ECA was ligated with a 6-0 silk suture and cut 2 mm distal to the bifurcation. The ECA stump was punctured with a 27-gauge needle. A 30-mm 3-0 monofilament nylon thread with a rounded tip was introduced into the ECA stump and advanced approximately 20 mm from the bifurcation of the CCA to the ICA. The test group animals (n ⫽ 11) were given bovine bFGF (Sigma, St. Louis, MO) at a concentration of 45 g/kg/hour. The control group animals (n ⫽ 10) were given saline solution (0.9% NaCl). The infusion was started 30 minutes after MCAO and continued for 3 hours. Regional cerebral blood flow (rCBF) was measured bilaterally using laser Doppler flowmetry (LDF) (Omega flow FLO-C1; Omega Wave, Tokyo, Japan) equipped with an LDF probe with a tip diameter of 0.46 mm. The LDF probes were placed bilaterally 5.5 mm lateral to the bregma. The location selected for rCBF monitoring corresponded to the MCA territory area. The relative rCBF to the baseline value measured for 15–20 minutes before MCAO was monitored until the end of the infusion.
200, Varian, Palo Alto, CA). Anesthesia was maintained with halothane (0.5–1%) mixed with a 3:7 ratio of O2 and N2O. Rectal temperature, heart rate, and blood oxygen saturation were continuously monitored. Images were acquired with a custom-made surface coil (30-mm diameter). T2-weighted images were taken with a spin-echo sequence (repetition time, 1.5 sec; echo time, 10, 40, and 80 msec; field of view, 45 ⫻ 45 mm; matrix size, 128 ⫻ 128). Six contiguous slices (1.6-mm thick; 0.4-mm gap) were set, with the position of the second slice position placed at the level of the bregma. MRI examination was carried out before and 24 hours after MCAO and repeated on days 7 and 14. T2 values were estimated voxel by voxel. Each T2 value was calculated by a linear least squares fit of the logarithm of the T2-weighted intensity versus echo time (TE). The abnormal lesioned voxels were defined from a threshold value of 30% higher (corresponding to a 3 ⫻ standard deviation difference) than the normal value. The T2-derived lesion volume was defined as a summation of the abnormal lesioned voxels across all slices and expressed as the percent volume relative to the ipsilateral hemisphere. These calculations were performed using custom-made software.
Histopathological Examination BrdU (20 mg/kg) was administered intraperitoneally at 24 hours after MCAO, on day 7, and on day 14. After 1 hour of BrdU injection on day 14, the rats were anesthetized with sodium pentobarbital and their brains transcardially perfused with a 4% paraformaldehyde in phosphate-buffered saline. Paraffin-embedded tissue blocks were sectioned at 4 m. Hematoxylin and eosin (HE) staining was done to measure the ischemic area in the test and control groups. The HEderived lesion area was defined as a summation across 6 slices (corresponding to the slices of MRI measurement) and multiplied by slice thickness (2 mm) to provide lesion volume, which was then expressed as the percent volume relative to the ipsilateral hemisphere. Immunostaining for BrdU, glial fibrillary acidic protein (GFAP), microtubule-associated protein-2 (MAP2), and ED-1 (a marker of microglial/phagocytic cells) was carried out using the avidin-biotin peroxidase complex method.14 –16 To determine the number of BrdU-labeled cells, the region of interest (ROI) (size, 94 ⫻ 118 m) was selected from the border area of the lesioned cortex of the ipsilateral hemisphere.
Statistical Analyses MRI Measurements The animals were placed on the homemade cradle and then set in a 4.7-T imaging spectrometer (UNITY, INOVA
The data are expressed as mean ⫾ standard deviation (SD). Statistical analyses were performed using the unpaired t-test. T2 data were analyzed by analysis of
EFFICACY OF bFGF IN MCAO
189
Table 1. Physiological parameters before MCAO and during infusion in the saline and bFGF groups
PaCO2 (mm Hg) Before MCAO After 1 hour of After 2 hour of After 3 hour of PaO2 (mm Hg) Before MCAO After 1 hour of After 2 hour of After 3 hour of pH Before MCAO After 1 hour of After 2 hour of After 3 hour of MABP (mm Hg) Before MCAO After 1 hour of After 2 hour of After 3 hour of
Saline group (n ⫽ 6)
bFGF group (n ⫽ 7)
MCAO MCAO MCAO
51.8 ⫾ 12.5 44.0 ⫾ 9.5 43.7 ⫾ 13.6 43.9 ⫾ 12.5
52.4 ⫾ 9.2 47.1 ⫾ 6.1 43.3 ⫾ 4.6 48.0 ⫾ 5.0
MCAO MCAO MCAO
113.6 ⫾ 34.6 114.1 ⫾ 20.5 118.0 ⫾ 23.0 126.7 ⫾ 18.6
109.5 ⫾ 15.6 104.7 ⫾ 9.0 103.5 ⫾ 12.2 105.4 ⫾ 16.7
MCAO MCAO MCAO
7.37 ⫾ 0.05 7.38 ⫾ 0.04 7.41 ⫾ 0.05 7.40 ⫾ 0.03
7.37 ⫾ 0.05 7.40 ⫾ 0.02 7.41 ⫾ 0.01 7.40 ⫾ 0.02
MCAO MCAO MCAO
87.8 ⫾ 3.9 105.6 ⫾ 6.6 103.3 ⫾ 22.4 95.7 ⫾ 15.9
85.2 ⫾ 11.2 84.6 ⫾ 12.1 85.9 ⫾ 14.8 84.2 ⫾ 12.2
Values are mean ⫾ SD.
variance with Bonferroni’s post hoc analysis. A 2-tailed test value of P ⬍ .05 was considered significant.
Results Physiological parameters for both experiments are given in Table 1. There were no within-group or between-group differences in mean arterial blood pressure (MABP), PaCO2, PaO2 or pH in these experiments. The mortality rates for the bFGF and saline groups were 27% and 40%, respectively. Figure 1 illustrates the body weight of the rats on different days. The rats started losing weight after MCAO, and weight loss continued until day 7, after which they started gaining weight. The bFGF group regained body weight to the
Figure 1. SD.
Change in body weight on different days. Values are mean ⫾
pre-MCAO level on day 14. However, the saline group demonstrated a decrease in body weight on day 14 compared with the pre-MCAO weight. Figure 2 shows the representive time courses of T2 maps, and HE staining and MAP-2 staining done 14 days after MCAO. The lesions in the T2 maps agreed with those in the histological staining. In the bFGF group, the lesion area was reduced mainly in the cerebral cortex compared with the saline group. Figure 3 demonstrates the changes in the T2-derived lesion volume measured after 24 hours of MCAO, on day 7, and on day 14. Lesion volume was significantly different between the bFGF and saline groups at 24 hours (P ⬍ .05), on day 7 (P ⬍ .05), and on day 14 (P ⬍ .01). Changes in rCBF after bFGF or saline infusion after MCAO are shown in Figure 4. Although there was an elevation in CBF after infusion, no significant change was observed between the bFGF and saline groups. The percentage of lesion volume calculated from the ipsilateral hemisphere using HE staining showed a significant difference (P ⬍ .05) between the saline and bFGF groups (Fig. 5). Figure 6 shows the number of BrdU-labeled cells in the border area of the lesioned cortex of the ipsilateral hemisphere. No significant change in the number of BrdU-labeled cells was observed between the bFGF and saline groups. The ROI that we selected showed negative MAP-2, positive ED-1, and positive GFAP immunohistochemical staining.
190
K. NAGESWARI ET AL.
Figure 4. Changes in rCBF ipsilateral hemisphere as measured by LDF. Values are mean ⫾ SD. Shaded bar indicates the period of bFGF infusion.
Figure 2. Time courses of T2 maps and HE staining and MAP-2 staining done 14 days after MCAO.
Discussion The present study demonstrates that bFGF infused intravenously, starting 30 minutes after induction of focal brain ischemia by permanent MCAO (pMCAO), signifi-
Figure 3. Changes in the T2-derived lesion volume measured 24 hours 7 days, and Day 14 days after MCAO. Values are mean ⫾ SD. *P ⬍ .05 versus the bFGF group on days 1 and 7. **P ⬍ .01 versus the bFGF group on day 14.
cantly reduced the lesion volume at 24 hours, 7 days, and 14 days after MCAO compared with the corresponding saline group as determined by T2-derived MRI measurements of lesion volume. Histopathological assessment using HE staining also showed that bFGF significantly reduced the lesion volume at day 14. Several studies have reported the therapeutic efficacy of bFGF in acute cerebral ischemia,4 – 6 but few reports have examined the long-term effects of bFGF. These reports include the behavioral recovery in rat after 4 weeks of pMCAO, 7 the neovascularization in normal rat cerebral cortex 2 weeks after topical application of bFGF,12 and the reduction of infarct volume at a only single time point (7 days or 3 months) after intravenous bFGF treatment in rat with MCAO.8,9 Our present results indicate the long-term continuous efficacy of bFGF at the
Figure 5. HE-derived lesion volume of the saline and bFGF groups. Values are mean ⫾ SD.
EFFICACY OF bFGF IN MCAO
Figure 6. Number of BrdU-labeled cells in the ipsilateral hemisphere in the saline and bFGF groups. Values are mean ⫾ SD.
subacute phase (day 14) throughout 24 hours and 7 days by monitoring T2-weighted MRI images. T2-derived ischemic volumes in this study were significantly lower on day 7 than those for the corresponding 24 hours after MCAO for both groups. Edema formation may cause an overestimation of lesion volume at 24 hours after MCAO. There was a tendency toward a slight elevation in T2-derived ischemic volumes observed in the saline group on day 14 compared with that on day 7, although this elevation was not statistically significant. The tendency observed in our results corresponds to that noted in an earlier report of Virley17 demonstrating that the size of the lesion initially reduces from 1 to 7 days but remains constant from 7 to 28 days after MCAO using a strict threshold criterion. One of the possible mechanisms of action of growth factors includes the effects on rCBF. It has been reported that intracarotid administration of bFGF reduced infarct size after MCAO by increasing rCBF in the peri-ischemic zone, measured using LDF.4 However, the present study did not show any significant difference in rCBF after either intravenous bFGF or saline infusion. Also, the dose of bFGF infusion used in our study did not alter the physiological parameters significantly. Our results are consistent with the report that an intravenous infusion of bFGF does not affect CBF at all.18 Hence the effect of bFGF in this study may be independent of an improvement in CBF directly in the ischemic regions. bFGF protects neurons in vitro against a number of insults that play crucial roles in the pathophysiology of ischemia, such as anoxia, hypoglycemia, excitatory amino acids, Ca2⫹ ionophores, free radicals, and nitric oxide.19 –22 The excitatory amino acids play a pivotal role in brain ischemia, and other molecules, including free radicals and nitric oxide, also contribute to the 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.19 Thus the reduction in ischemic vol-
191
ume after bFGF infusion may be due to the neuroprotective property of bFGF. BrdU is an analogue of thymidine, which is incorporated into the DNA of cells during the S-phase and has been used to investigate cell proliferation.23,24 For analyzing the BrdU-labeled cells in the bFGF and saline groups, we selected the ROI from the border area of the lesioned cortex. This area seemed to show the progress of the repair process, indicated by ED-1- and GFAP-positive staining. Because bFGF has angiogenic properties, we expected a larger number of BrdU-labeled cells in the bFGF-treated group. However, contrary to our expectation, we found no significant change in the number of BrdU-labeled cells between the bFGF and saline groups (Fig. 6). One possible explanation for this result is that a single infusion of bFGF early after MCAO may not affect cell proliferation. Another possible reason is the failure or loss of endothelial cell labeling. Zhang et al25 reported that the number of BrdU-labeled cells remained at 90% of the initial value for 14 days and that the largest numbers of BrdU-positive cells were observed 7 days after pMCAO.25 Moreover, the intraventricular injection of bFGF increased the number of BrdU-positive cells significantly, even on the next day of single administration of BrdU.26 We injected BrdU 24 hours, 7 days, and 14 days after MCAO in this study, so the BrdU-positive cells could be detectable 14 days after MCAO. Thus it is likely that a single intravenous infusion of bFGF early after MCAO may not affect cell proliferation. However, a single topical application of bFGF was reportedly effective for neovascularization in rat.12 In addition, the implantation of nylon mesh including bFGF and collagen gel mixture to the surface of the cerebral cortex induced angiogenesis and promoted neocapillary formation in mice.27 The discrepancy in findings may be attributed to the differences in the route and method of bFGF administration.
Conclusion The present study demonstrates the efficacy of intravenous infusion of bFGF in a permanent ischemia model during the subacute phase using T2-weighted MRI images. The lesion volume in rats with bFGF infusion was significantly lowered at all time points of MRI measurement—24 hours, 7 days, and 14 days after MCAO. Our MRI findings were supported by the histological assessment of ischemic area using HE staining. Acknowledgment: We acknowledge the technical assistance of Nobuko Suzuki, Yozo Ito, David Wright, and Jeff Kershaw and also the administrative efforts of Dr. Yukihiko Ono. One of the authors (K.N.) was supported by the JSPS Invitation Fellowship for Research in Japan (long-term). This study was also supported by the Akita Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence, Japanese Science and Technology Agency.
K. NAGESWARI ET AL.
192
References 1. Lin LH, Doherty DH, Lile JD, et al. GDNF: a glial cell line- derived neurotrophic factor for midbrain dopaminergic neurons. Science 1993;260:1130-1132. 2. Schäbitz WR, Li F, Irie K, et al. Synergistic effects of a combination of low-dose basic fibroblast growth factor and citicoline after temporary experimental focal ischemia. Stroke 1999;30:427-432. 3. Nozaki K, Finklestein SP, Beal MF. Basic fibroblast growth factor protects against hypoxia-ischemia and NMDA neurotoxicity in neonatal rats. J Cereb Blood Flow Metab 1993;13:221-228. 4. Tanaka R, Miyasaka Y, Yada K, et al. Basic fibroblast growth factor increases regional cerebral blood flow and reduces infarct size after experimental ischemia in a rat model. Stroke 1995;26:2154-2159. 5. Koketsu N, Berlove DJ, Moskowitz MA, et al. Pretreatment with intraventricular basic fibroblast growth factor decreases infarct size following focal cerebral ischemia in rats. Ann Neurol 1994;35:451-457. 6. Fisher M, Meadows ME, Do T, et al. Delayed treatment with intravenous basic fibroblast growth factor reduces infarct size following permanent focal cerebral ischemia in rats. J Cereb Blood Flow Metab 1995;15:953-959. 7. Kawamata T, Alexis NE, Dietrich WD, et al. Intracisternal basic fibroblast growth factor (bFGF) enhances behavioral recovery following focal cerebral infarction in the rat. J Cereb Blood Flow Metab 1996;16:542-547. 8. Jiang N, Finklestein SP, Do T, et al. 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. 9. Sugimori H, Speller H, Finklestein SP. Intravenous basic fibroblast growth factor produces a persistent reduction in infarct volume following permanent focal ischemia in rats. J Neurosci Lett 2001;300;13-16. 10. Berlove DJ, Finklestein SP. Growth factors and brain injury. In: Moody TW, eds. Growth factors, peptides and receptors. New York, Plenum Press, 1993:137-143. 11. Finklestein SP. Growth Factors in stroke. In: Caplan LR, ed. Brain ischemia: basic concepts and clinical relevance. London: Springer-Verlag, 1995:37-41. 12. Cuevas P, Gimenez-Gallego G, Carceller F, et al. Single topical application of human recombinant basic fibroblast growth factor (rbFGF) promotes neovascularization in rat cerebral cortex. Surg Neurol 1993;39:380-384. 13. Kawamura S, Yasui N, Shirasawa M, et al. Rat middle cerebral artery occlusion using an intraluminal thread technique. Acta Neurochir (Wien) 1991;109:126-132. 14. Imam A, Tokes ZA. Immunoperoxidase localization of a glycoprotein on plasma membrane of secretory epithe-
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
lium from human breast. J Histochem Cytochem 1981;29:581-584. Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem Cytochem 1981;29:577580. Sugihara H, Hattori T, Fukuda M. Immunohistochemical detection of bromodeoxyuridine in formalin-fixed tissues. Histochemistry 1986;85:193-195. Virley D, Beech JS, Smart SC, et al. A temporal MRI assessment of neuropathology after transient middle cerebral artery occlusion in the rat: correlations with behavior. J Cereb Blood Flow Metab 2000;20:563-582. Tatlisumak T, Takano K, Carano RA, et al. Effect of basic fibroblast growth factor on experimental focal ischemia studied by diffusion-weighted and perfusion imaging. Stroke 1996;27:2292-2298. Mattson MP, Murrain M, Guthrie PB, et al. Fibroblast growth factor and glutamate: opposing roles in the generation and degeneration of hippocampal neuroarchitecture. J Neurosci 1989;9:3728-40. Finklestein SP, Kemmou A, Caday CG, et al. Basic fibroblast growth factor protects cerebrocortical neurons against excitatory amino acid toxicity in vitro. Stroke 1993;24(suppl):I-141-I-143. Freese A, Finklestein SP, DiFiglia M. Basic fibroblast growth factor protects striatal neurons in vitro from NMDA-receptor mediated excitotoxicity. Brain Res 1992; 575:351-355. Maiese K, Boniece I, DeMeo D, et al. Peptide growth factors protect against ischemia in culture by preventing nitric oxide toxicity. J Neurosci 1993;13:3034-3040. Dolbeare F. Bromodeoxyuridine: a diagnostic tool in biology and medicine, Part III. Proliferation in normal, injured and diseased tissue, growth factors, differentiation, DNA replication sites and in situ hybridisation. Histochem J 1996;28:531-575. Kuhn HG, Dickinson-Anson H, Gage FH. Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J Neurosci 1996;16: 2027-2033. Zhang RL, Zhang ZG, Zhang L, et al. Proliferation and differentiation of progenitor cells in the cortex and the subventricular zone in the adult rat after focal cerebral ischemia. Neuroscience 2001;105:33-41. Matsuoka N, Nozaki K, Takagi Y, et al. Adenovirusmediated gene transfer of fibroblast growth factor-2 increases BrdU-positive cells after forebrain ischemia in gerbils. Stroke 2003;34:1519-1525. Nageswari K, Yamaguchi S, Yamakawa T, et al. Quantitative assessment of cerebral neocapillary network and its remodeling in mice using intravital fluorescence videomicroscopy. Angiogenesis 2002;5:99-105.
PhD,†
The purpose of this study was to evaluate the effect of intravenous infusion of basic fibroblast growth factor (bFGF) in a permanent ischemia model at the subacute phase (2 weeks) as well as at 24 hours and 1 week using T2-weighted magnetic resonance imaging (MRI). The middle cerebral artery (MCA) in Sprague-Dawley rats was occluded using an intraluminal suture method. The rats were randomly divided into 2 groups to receive either bFGF (45 g/kg/hr) or saline solution. The infusion was started 30 minutes after MCA occlusion (MCAO) and continued for 3 hours. Regional cerebral blood flow (rCBF) was measured using laser Doppler flowmetry throughout the infusion. T2-weighted MRI was carried out before MCAO, 24 hours after MCAO, and days 7 and 14 after MCAO. Although an elevation in rCBF was seen after the infusion, no significant change between the groups was observed. A significant difference between the bFGF and saline groups in T2-derived lesion volume was observed at 24 hours (P ⬍ .05), on day 7 (P ⬍ .05), and on day 14 (P ⬍ .01). The percentage of lesion area calculated from the ipsilateral hemisphere using hematoxylin and eosin staining on day 14 showed a significant difference between the bFGF and saline groups (P ⬍ .05). No significant change in the number of bromodeoxyuridine (BrdU)-labeled cells between the groups was observed. This study demonstrates that bFGF, infused intravenously starting 30 minutes after the induction of permanent MCAO, significantly reduces region volume even at day 14, as well as at days 1 and 7, compared with the corresponding saline group. Key Words: Cerebral ischemia— growth factors— cerebral blood flow—magnetic resonance imaging. © 2005 by National Stroke Association
Neurotrophic factors are essential for the survival and differentiation of normally developing neurons, and also *From the Department of Internal Medicine, †Department of Neurology, ‡Department of Radiology, and §Akita Industry Promotion Foundation, Akita Research Institute of Brain and Blood Vessels, Akita, Japan and ¶School of Biosciences and Bioengineering, Indian Institute of Technology, Bombay, Powai, India. Received December 29, 2004; revised March 30, 2005; accepted May 10, 2005. Address reprint requests to Shigenori Mizusawa, Department of Internal Medicine, Akita Research Institute of Brain and Blood Vessels, 6 –10 Senshu-Kubota machi, Akita 010-0874, Japan. Email: [email protected] 1052-3057/$—see front matter © 2005 by National Stroke Association doi:10.1016/j.jstrokecerebrovasdis.2005.05.004
play important roles in the protection and recovery of mature neurons under pathological conditions.1 Among these factors, the anti-ischemic effects of basic fibroblast growth factor (bFGF) on neurons in vitro and in vivo have been examined.2–9 Endogenous bFGF expression is known to be up-regulated in tissue surrounding focal brain wounds or infarcts, and exogenous application of bFGF reduces the degree of cell death and stimulates neuronal sprouting in models of ischemic brain injury.10,11 bFGF is also a well-known angiogenic agent.12 Several studies have reported on the therapeutic efficacy of bFGF in acute cerebral ischemia. But most experiments have been evaluated for only 24 hours (the observation period), which is insufficient to detect the longterm effects of bFGF, including neoangiogenesis.4 – 6
Journal of Stroke and Cerebrovascular Diseases, Vol. 14, No. 5 (September-October), 2005: pp 187-192
187
K. NAGESWARI ET AL.
188
Some reports have examined the long-term (beyond 24 hours) effects of intravenous infusion of bFGF; however, these studies have evaluated the ischemic lesion volume at only a single time point after bFGF treatment.8,9 The present study is designed to evaluate the effect of bFGF during the subacute phase of 14 days by monitoring T2-weighted magnetic resonance imaging (MRI) findings at 24 hours, 7 days, and 14 days after bFGF administration in the same animals with middle cerebral artery (MCA) occlusion (MCAO) and also comparing it with the histopathological measurement at day 14. The effect of bFGF on proliferating cells in the ipsilateral hemisphere of ischemia was also evaluated by bromodeoxyuridine (BrdU) immunohistochemistry.
Materials and Methods Animal Preparation Twenty-one male Sprague-Dawley rats weighing 320 – 400 g were used. Anesthesia was induced with 3%– 4% halothane in a 7:3 mixture of N2O and O2 delivered through a close-fitting mask. PE-50 polyethylene catheters were inserted into both the tail artery and vein for measuring blood pressure, pH, arterial carbon dioxide tension (PaCO2), and arterial oxygen tension (PaO2) and drug infusion, respectively. The rectal temperature was continuously monitored and was maintained at 37 ⫾ 0.5°C with a thermostatically controlled heating pad. The MCA was occluded using an intraluminal suture method.13 The left common, external, and internal carotid arteries (CCA, ECA, and ICA) were identified through a ventral cervical midline incision. The ECA was ligated with a 6-0 silk suture and cut 2 mm distal to the bifurcation. The ECA stump was punctured with a 27-gauge needle. A 30-mm 3-0 monofilament nylon thread with a rounded tip was introduced into the ECA stump and advanced approximately 20 mm from the bifurcation of the CCA to the ICA. The test group animals (n ⫽ 11) were given bovine bFGF (Sigma, St. Louis, MO) at a concentration of 45 g/kg/hour. The control group animals (n ⫽ 10) were given saline solution (0.9% NaCl). The infusion was started 30 minutes after MCAO and continued for 3 hours. Regional cerebral blood flow (rCBF) was measured bilaterally using laser Doppler flowmetry (LDF) (Omega flow FLO-C1; Omega Wave, Tokyo, Japan) equipped with an LDF probe with a tip diameter of 0.46 mm. The LDF probes were placed bilaterally 5.5 mm lateral to the bregma. The location selected for rCBF monitoring corresponded to the MCA territory area. The relative rCBF to the baseline value measured for 15–20 minutes before MCAO was monitored until the end of the infusion.
200, Varian, Palo Alto, CA). Anesthesia was maintained with halothane (0.5–1%) mixed with a 3:7 ratio of O2 and N2O. Rectal temperature, heart rate, and blood oxygen saturation were continuously monitored. Images were acquired with a custom-made surface coil (30-mm diameter). T2-weighted images were taken with a spin-echo sequence (repetition time, 1.5 sec; echo time, 10, 40, and 80 msec; field of view, 45 ⫻ 45 mm; matrix size, 128 ⫻ 128). Six contiguous slices (1.6-mm thick; 0.4-mm gap) were set, with the position of the second slice position placed at the level of the bregma. MRI examination was carried out before and 24 hours after MCAO and repeated on days 7 and 14. T2 values were estimated voxel by voxel. Each T2 value was calculated by a linear least squares fit of the logarithm of the T2-weighted intensity versus echo time (TE). The abnormal lesioned voxels were defined from a threshold value of 30% higher (corresponding to a 3 ⫻ standard deviation difference) than the normal value. The T2-derived lesion volume was defined as a summation of the abnormal lesioned voxels across all slices and expressed as the percent volume relative to the ipsilateral hemisphere. These calculations were performed using custom-made software.
Histopathological Examination BrdU (20 mg/kg) was administered intraperitoneally at 24 hours after MCAO, on day 7, and on day 14. After 1 hour of BrdU injection on day 14, the rats were anesthetized with sodium pentobarbital and their brains transcardially perfused with a 4% paraformaldehyde in phosphate-buffered saline. Paraffin-embedded tissue blocks were sectioned at 4 m. Hematoxylin and eosin (HE) staining was done to measure the ischemic area in the test and control groups. The HEderived lesion area was defined as a summation across 6 slices (corresponding to the slices of MRI measurement) and multiplied by slice thickness (2 mm) to provide lesion volume, which was then expressed as the percent volume relative to the ipsilateral hemisphere. Immunostaining for BrdU, glial fibrillary acidic protein (GFAP), microtubule-associated protein-2 (MAP2), and ED-1 (a marker of microglial/phagocytic cells) was carried out using the avidin-biotin peroxidase complex method.14 –16 To determine the number of BrdU-labeled cells, the region of interest (ROI) (size, 94 ⫻ 118 m) was selected from the border area of the lesioned cortex of the ipsilateral hemisphere.
Statistical Analyses MRI Measurements The animals were placed on the homemade cradle and then set in a 4.7-T imaging spectrometer (UNITY, INOVA
The data are expressed as mean ⫾ standard deviation (SD). Statistical analyses were performed using the unpaired t-test. T2 data were analyzed by analysis of
EFFICACY OF bFGF IN MCAO
189
Table 1. Physiological parameters before MCAO and during infusion in the saline and bFGF groups
PaCO2 (mm Hg) Before MCAO After 1 hour of After 2 hour of After 3 hour of PaO2 (mm Hg) Before MCAO After 1 hour of After 2 hour of After 3 hour of pH Before MCAO After 1 hour of After 2 hour of After 3 hour of MABP (mm Hg) Before MCAO After 1 hour of After 2 hour of After 3 hour of
Saline group (n ⫽ 6)
bFGF group (n ⫽ 7)
MCAO MCAO MCAO
51.8 ⫾ 12.5 44.0 ⫾ 9.5 43.7 ⫾ 13.6 43.9 ⫾ 12.5
52.4 ⫾ 9.2 47.1 ⫾ 6.1 43.3 ⫾ 4.6 48.0 ⫾ 5.0
MCAO MCAO MCAO
113.6 ⫾ 34.6 114.1 ⫾ 20.5 118.0 ⫾ 23.0 126.7 ⫾ 18.6
109.5 ⫾ 15.6 104.7 ⫾ 9.0 103.5 ⫾ 12.2 105.4 ⫾ 16.7
MCAO MCAO MCAO
7.37 ⫾ 0.05 7.38 ⫾ 0.04 7.41 ⫾ 0.05 7.40 ⫾ 0.03
7.37 ⫾ 0.05 7.40 ⫾ 0.02 7.41 ⫾ 0.01 7.40 ⫾ 0.02
MCAO MCAO MCAO
87.8 ⫾ 3.9 105.6 ⫾ 6.6 103.3 ⫾ 22.4 95.7 ⫾ 15.9
85.2 ⫾ 11.2 84.6 ⫾ 12.1 85.9 ⫾ 14.8 84.2 ⫾ 12.2
Values are mean ⫾ SD.
variance with Bonferroni’s post hoc analysis. A 2-tailed test value of P ⬍ .05 was considered significant.
Results Physiological parameters for both experiments are given in Table 1. There were no within-group or between-group differences in mean arterial blood pressure (MABP), PaCO2, PaO2 or pH in these experiments. The mortality rates for the bFGF and saline groups were 27% and 40%, respectively. Figure 1 illustrates the body weight of the rats on different days. The rats started losing weight after MCAO, and weight loss continued until day 7, after which they started gaining weight. The bFGF group regained body weight to the
Figure 1. SD.
Change in body weight on different days. Values are mean ⫾
pre-MCAO level on day 14. However, the saline group demonstrated a decrease in body weight on day 14 compared with the pre-MCAO weight. Figure 2 shows the representive time courses of T2 maps, and HE staining and MAP-2 staining done 14 days after MCAO. The lesions in the T2 maps agreed with those in the histological staining. In the bFGF group, the lesion area was reduced mainly in the cerebral cortex compared with the saline group. Figure 3 demonstrates the changes in the T2-derived lesion volume measured after 24 hours of MCAO, on day 7, and on day 14. Lesion volume was significantly different between the bFGF and saline groups at 24 hours (P ⬍ .05), on day 7 (P ⬍ .05), and on day 14 (P ⬍ .01). Changes in rCBF after bFGF or saline infusion after MCAO are shown in Figure 4. Although there was an elevation in CBF after infusion, no significant change was observed between the bFGF and saline groups. The percentage of lesion volume calculated from the ipsilateral hemisphere using HE staining showed a significant difference (P ⬍ .05) between the saline and bFGF groups (Fig. 5). Figure 6 shows the number of BrdU-labeled cells in the border area of the lesioned cortex of the ipsilateral hemisphere. No significant change in the number of BrdU-labeled cells was observed between the bFGF and saline groups. The ROI that we selected showed negative MAP-2, positive ED-1, and positive GFAP immunohistochemical staining.
190
K. NAGESWARI ET AL.
Figure 4. Changes in rCBF ipsilateral hemisphere as measured by LDF. Values are mean ⫾ SD. Shaded bar indicates the period of bFGF infusion.
Figure 2. Time courses of T2 maps and HE staining and MAP-2 staining done 14 days after MCAO.
Discussion The present study demonstrates that bFGF infused intravenously, starting 30 minutes after induction of focal brain ischemia by permanent MCAO (pMCAO), signifi-
Figure 3. Changes in the T2-derived lesion volume measured 24 hours 7 days, and Day 14 days after MCAO. Values are mean ⫾ SD. *P ⬍ .05 versus the bFGF group on days 1 and 7. **P ⬍ .01 versus the bFGF group on day 14.
cantly reduced the lesion volume at 24 hours, 7 days, and 14 days after MCAO compared with the corresponding saline group as determined by T2-derived MRI measurements of lesion volume. Histopathological assessment using HE staining also showed that bFGF significantly reduced the lesion volume at day 14. Several studies have reported the therapeutic efficacy of bFGF in acute cerebral ischemia,4 – 6 but few reports have examined the long-term effects of bFGF. These reports include the behavioral recovery in rat after 4 weeks of pMCAO, 7 the neovascularization in normal rat cerebral cortex 2 weeks after topical application of bFGF,12 and the reduction of infarct volume at a only single time point (7 days or 3 months) after intravenous bFGF treatment in rat with MCAO.8,9 Our present results indicate the long-term continuous efficacy of bFGF at the
Figure 5. HE-derived lesion volume of the saline and bFGF groups. Values are mean ⫾ SD.
EFFICACY OF bFGF IN MCAO
Figure 6. Number of BrdU-labeled cells in the ipsilateral hemisphere in the saline and bFGF groups. Values are mean ⫾ SD.
subacute phase (day 14) throughout 24 hours and 7 days by monitoring T2-weighted MRI images. T2-derived ischemic volumes in this study were significantly lower on day 7 than those for the corresponding 24 hours after MCAO for both groups. Edema formation may cause an overestimation of lesion volume at 24 hours after MCAO. There was a tendency toward a slight elevation in T2-derived ischemic volumes observed in the saline group on day 14 compared with that on day 7, although this elevation was not statistically significant. The tendency observed in our results corresponds to that noted in an earlier report of Virley17 demonstrating that the size of the lesion initially reduces from 1 to 7 days but remains constant from 7 to 28 days after MCAO using a strict threshold criterion. One of the possible mechanisms of action of growth factors includes the effects on rCBF. It has been reported that intracarotid administration of bFGF reduced infarct size after MCAO by increasing rCBF in the peri-ischemic zone, measured using LDF.4 However, the present study did not show any significant difference in rCBF after either intravenous bFGF or saline infusion. Also, the dose of bFGF infusion used in our study did not alter the physiological parameters significantly. Our results are consistent with the report that an intravenous infusion of bFGF does not affect CBF at all.18 Hence the effect of bFGF in this study may be independent of an improvement in CBF directly in the ischemic regions. bFGF protects neurons in vitro against a number of insults that play crucial roles in the pathophysiology of ischemia, such as anoxia, hypoglycemia, excitatory amino acids, Ca2⫹ ionophores, free radicals, and nitric oxide.19 –22 The excitatory amino acids play a pivotal role in brain ischemia, and other molecules, including free radicals and nitric oxide, also contribute to the 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.19 Thus the reduction in ischemic vol-
191
ume after bFGF infusion may be due to the neuroprotective property of bFGF. BrdU is an analogue of thymidine, which is incorporated into the DNA of cells during the S-phase and has been used to investigate cell proliferation.23,24 For analyzing the BrdU-labeled cells in the bFGF and saline groups, we selected the ROI from the border area of the lesioned cortex. This area seemed to show the progress of the repair process, indicated by ED-1- and GFAP-positive staining. Because bFGF has angiogenic properties, we expected a larger number of BrdU-labeled cells in the bFGF-treated group. However, contrary to our expectation, we found no significant change in the number of BrdU-labeled cells between the bFGF and saline groups (Fig. 6). One possible explanation for this result is that a single infusion of bFGF early after MCAO may not affect cell proliferation. Another possible reason is the failure or loss of endothelial cell labeling. Zhang et al25 reported that the number of BrdU-labeled cells remained at 90% of the initial value for 14 days and that the largest numbers of BrdU-positive cells were observed 7 days after pMCAO.25 Moreover, the intraventricular injection of bFGF increased the number of BrdU-positive cells significantly, even on the next day of single administration of BrdU.26 We injected BrdU 24 hours, 7 days, and 14 days after MCAO in this study, so the BrdU-positive cells could be detectable 14 days after MCAO. Thus it is likely that a single intravenous infusion of bFGF early after MCAO may not affect cell proliferation. However, a single topical application of bFGF was reportedly effective for neovascularization in rat.12 In addition, the implantation of nylon mesh including bFGF and collagen gel mixture to the surface of the cerebral cortex induced angiogenesis and promoted neocapillary formation in mice.27 The discrepancy in findings may be attributed to the differences in the route and method of bFGF administration.
Conclusion The present study demonstrates the efficacy of intravenous infusion of bFGF in a permanent ischemia model during the subacute phase using T2-weighted MRI images. The lesion volume in rats with bFGF infusion was significantly lowered at all time points of MRI measurement—24 hours, 7 days, and 14 days after MCAO. Our MRI findings were supported by the histological assessment of ischemic area using HE staining. Acknowledgment: We acknowledge the technical assistance of Nobuko Suzuki, Yozo Ito, David Wright, and Jeff Kershaw and also the administrative efforts of Dr. Yukihiko Ono. One of the authors (K.N.) was supported by the JSPS Invitation Fellowship for Research in Japan (long-term). This study was also supported by the Akita Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence, Japanese Science and Technology Agency.
K. NAGESWARI ET AL.
192
References 1. Lin LH, Doherty DH, Lile JD, et al. GDNF: a glial cell line- derived neurotrophic factor for midbrain dopaminergic neurons. Science 1993;260:1130-1132. 2. Schäbitz WR, Li F, Irie K, et al. Synergistic effects of a combination of low-dose basic fibroblast growth factor and citicoline after temporary experimental focal ischemia. Stroke 1999;30:427-432. 3. Nozaki K, Finklestein SP, Beal MF. Basic fibroblast growth factor protects against hypoxia-ischemia and NMDA neurotoxicity in neonatal rats. J Cereb Blood Flow Metab 1993;13:221-228. 4. Tanaka R, Miyasaka Y, Yada K, et al. Basic fibroblast growth factor increases regional cerebral blood flow and reduces infarct size after experimental ischemia in a rat model. Stroke 1995;26:2154-2159. 5. Koketsu N, Berlove DJ, Moskowitz MA, et al. Pretreatment with intraventricular basic fibroblast growth factor decreases infarct size following focal cerebral ischemia in rats. Ann Neurol 1994;35:451-457. 6. Fisher M, Meadows ME, Do T, et al. Delayed treatment with intravenous basic fibroblast growth factor reduces infarct size following permanent focal cerebral ischemia in rats. J Cereb Blood Flow Metab 1995;15:953-959. 7. Kawamata T, Alexis NE, Dietrich WD, et al. Intracisternal basic fibroblast growth factor (bFGF) enhances behavioral recovery following focal cerebral infarction in the rat. J Cereb Blood Flow Metab 1996;16:542-547. 8. Jiang N, Finklestein SP, Do T, et al. 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. 9. Sugimori H, Speller H, Finklestein SP. Intravenous basic fibroblast growth factor produces a persistent reduction in infarct volume following permanent focal ischemia in rats. J Neurosci Lett 2001;300;13-16. 10. Berlove DJ, Finklestein SP. Growth factors and brain injury. In: Moody TW, eds. Growth factors, peptides and receptors. New York, Plenum Press, 1993:137-143. 11. Finklestein SP. Growth Factors in stroke. In: Caplan LR, ed. Brain ischemia: basic concepts and clinical relevance. London: Springer-Verlag, 1995:37-41. 12. Cuevas P, Gimenez-Gallego G, Carceller F, et al. Single topical application of human recombinant basic fibroblast growth factor (rbFGF) promotes neovascularization in rat cerebral cortex. Surg Neurol 1993;39:380-384. 13. Kawamura S, Yasui N, Shirasawa M, et al. Rat middle cerebral artery occlusion using an intraluminal thread technique. Acta Neurochir (Wien) 1991;109:126-132. 14. Imam A, Tokes ZA. Immunoperoxidase localization of a glycoprotein on plasma membrane of secretory epithe-
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
lium from human breast. J Histochem Cytochem 1981;29:581-584. Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem Cytochem 1981;29:577580. Sugihara H, Hattori T, Fukuda M. Immunohistochemical detection of bromodeoxyuridine in formalin-fixed tissues. Histochemistry 1986;85:193-195. Virley D, Beech JS, Smart SC, et al. A temporal MRI assessment of neuropathology after transient middle cerebral artery occlusion in the rat: correlations with behavior. J Cereb Blood Flow Metab 2000;20:563-582. Tatlisumak T, Takano K, Carano RA, et al. Effect of basic fibroblast growth factor on experimental focal ischemia studied by diffusion-weighted and perfusion imaging. Stroke 1996;27:2292-2298. Mattson MP, Murrain M, Guthrie PB, et al. Fibroblast growth factor and glutamate: opposing roles in the generation and degeneration of hippocampal neuroarchitecture. J Neurosci 1989;9:3728-40. Finklestein SP, Kemmou A, Caday CG, et al. Basic fibroblast growth factor protects cerebrocortical neurons against excitatory amino acid toxicity in vitro. Stroke 1993;24(suppl):I-141-I-143. Freese A, Finklestein SP, DiFiglia M. Basic fibroblast growth factor protects striatal neurons in vitro from NMDA-receptor mediated excitotoxicity. Brain Res 1992; 575:351-355. Maiese K, Boniece I, DeMeo D, et al. Peptide growth factors protect against ischemia in culture by preventing nitric oxide toxicity. J Neurosci 1993;13:3034-3040. Dolbeare F. Bromodeoxyuridine: a diagnostic tool in biology and medicine, Part III. Proliferation in normal, injured and diseased tissue, growth factors, differentiation, DNA replication sites and in situ hybridisation. Histochem J 1996;28:531-575. Kuhn HG, Dickinson-Anson H, Gage FH. Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J Neurosci 1996;16: 2027-2033. Zhang RL, Zhang ZG, Zhang L, et al. Proliferation and differentiation of progenitor cells in the cortex and the subventricular zone in the adult rat after focal cerebral ischemia. Neuroscience 2001;105:33-41. Matsuoka N, Nozaki K, Takagi Y, et al. Adenovirusmediated gene transfer of fibroblast growth factor-2 increases BrdU-positive cells after forebrain ischemia in gerbils. Stroke 2003;34:1519-1525. Nageswari K, Yamaguchi S, Yamakawa T, et al. Quantitative assessment of cerebral neocapillary network and its remodeling in mice using intravital fluorescence videomicroscopy. Angiogenesis 2002;5:99-105.