Vascular endothelial growth factor expression in transient focal cerebral ischemia in the rat

Neuroscience Letters 249 (1998) 79–82

Vascular endothelial growth factor expression in transient focal cerebral ischemia in the rat Charles S. Cobbs a, Jun Chen b, David A. Greenberg b , c, Steven H. Graham b , d ,* a

Department of Neurosurgery, University of Alabama, Birmingham, AL, USA b Department of Neurology, University of Pittsburgh, Pittsburgh, PA, USA c Department of Neurobiology, University of Pittsburgh, Pittsburgh, PA, USA d Neurology Service (127), University Drive, Department of Veterans Affairs Medical Center, Pittsburgh, PA, USA Received 2 April 1998; received in revised form 28 April 1998; accepted 28 April 1998

Abstract Vascular endothelial growth factor (VEGF) has been implicated in hypoxia-induced angiogenesis in tumors and ischemia. We examined VEGF mRNA and protein expression after occlusion of the middle cerebral artery (MCA) in rats. VEGF mRNA expression studied by in situ hybridization was increased in the ischemic border zone 24 h after 30, 60 or 120 min of focal cerebral ischemia. VEGF protein expression measured by Western blots was also increased in this region 24 and 48 h after ischemia, and VEGF immunocytochemistry localized this increased expression to astroglia. Thus, VEGF is induced after focal cerebral ischemia and could have a role in pathophysiology and recovery in the ischemic border zone.  1998 Elsevier Science Ireland Ltd. All rights reserved

Keywords: Vascular endothelial growth factor; Stroke; Middle cerebral artery occlusion; Border zone; Edema; Angiogenesis

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen and enhances vascular permeability during angiogenesis in development, reproduction, and neoplasia. Hypoxic induction of angiogenesis is thought to result largely from increased VEGF expression [14], which is associated with neovascularization and the formation of collateral vessels in ischemic tissues [1,4]. VEGF-mediated enhancement of capillary permeability leads to procoagulant activity, through the release of von Willebrand factor, expression of tissue factor, and monocyte chemotaxis across the endothelium [2]. These effects suggest a role for VEGF in edema formation, hypercoagulability, and angiogenesis associated with physiological and pathological processes. The border zone in ischemic brain contains still-viable tissue with an uncertain prognosis for recovery [6]. Breakdown of the blood–brain barrier, edema, hypercoagul* Corresponding author. Department of Neurology, 526 South Biomedical Science Tower, University of Pittsburgh Medical Center, Pittsburgh, PA 15213, USA. Tel.: +1 412 6483299; fax: +1 412 6481239; e-mail: [email protected]

ability, leukocyte invasion, and angiogenesis [9] have been observed in this region after stroke. Because VEGF is induced by hypoxia and has been implicated in these processes, we investigated its role in experimental stroke produced by middle cerebral artery (MCA) occlusion. Male Sprague–Dawley rats (280–300 g) were induced with 5% isoflurane, intubated, and ventilated with 1.5% isoflurane in 95% air/5% oxygen. Left temporalis muscle temperature was monitored and kept at 37 ± 0.5°C with a heating-pad and lamp. One femoral artery was cannulated for monitoring blood pressure, arterial blood gases and blood glucose. The bifurcation of the common carotid artery was exposed and the external carotid artery (ECA) was isolated and separated from the vagus nerve. The extracranial branch of the internal carotid artery (ICA) was ligated near its origin with 5–0 silk suture. To occlude the origin of the MCA, a 3–0 monofilament nylon suture was introduced into the lumen of the ICA through the stump of the ECA and gently advanced 20–22 mm into the ICA [10]. After occlusion for the indicated time, the suture was gently withdrawn to permit reperfusion, the ECA stump was ligated and the

0304-3940/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00377- 2

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wound was closed. Control rats underwent sham surgery in which the suture was not inserted. For in situ hybridization [13], rats were anesthetized and sacrificed 24 h after the onset of 0 min, 30 min, 60 min, 120 min, or 24 h of ischemia (n = 3 per group). Brains were quickly removed, frozen in 2-methylbutane and stored at −70°C. Sections (20 mm) were cut on a cryostat at −20°C and collected on precleaned slides. The probe used was a synthetic 41-mer oligonucleotide complementary to nt 321– 361 of rat glioma VEGF [3]; this sequence, which recognizes all splice variants of VEGF, is specific for the VEGF gene on Northern blotting [11]. The sense sequence was used as a negative control. Slides were hybridized at 37°C for 18 h with 1 × 106 cpm of 35S-labeled probe, rinsed in 150 mM NaCl/15 mM sodium citrate (pH 7.4) at 52°C for 60 min, dehydrated, and exposed to Kodak SB-5 film for 3 weeks. Immunocytochemistry was performed with a polyclonal rabbit primary antibody against VEGF (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Anesthetized rats were perfused with paraformaldehyde in PBS after 30 or 120 min of MCA occlusion followed by reperfusion for the remainder of 24 h. Brains were removed, postfixed for 1–4 h, sectioned at 20 mm on a vibratome, and processed using the avidinbiotin-horseradish peroxidase technique (Elite Vectastain, Burlingame, CA, USA). Sections were incubated with a biotinylated horse anti-rabbit IgG for 2 h, placed in an avidin-horseradish solution for 3 h, washed twice with PBS, and reacted for horseradish peroxidase using diaminobenzidine (0.015% in PBS) and 0.001% hydrogen peroxide. Sections were then washed for 1 h and mounted on gelatinized slides. Alternating sections were incubated in the absence of primary antibody, or with primary antibody preabsorbed with 1 mg/ml of VEGF peptide (Santa Cruz Biotechnology). Western blotting was performed using the same primary antibody. Rats were sacrificed 24 or 48 h after 15 min of ischemia. The dorsal cortical mantle ipsilateral to the MCA occlusion was dissected, homogenized, and lysed. Protein samples (40 mg) were loaded onto a 12% SDS-polyacrylamide gel. Western blots were performed using a chemiluminescent detection system (Clontech, Palo Alto, CA, USA). As a negative control, the blot was incubated after the VEGF antibody was preabsorbed with VEGF peptide. VEGF mRNA levels were increased 23.5 h after 30 min of MCA occlusion (Fig. 1), with slight induction in the dorsal cortical mantle, where sublethal ischemia occurs. After 60 min of ischemia, there was marked loss of VEGF mRNA in the basal ganglia, which undergoes infarction, while expression was increased in the ischemic border zone. Maximal mRNA induction in the border zone occurred after 120 min of MCA occlusion. After 24 h of MCA occlusion, there was no detectable VEGF mRNA in the infarcted region and only a small band of increased expression adjacent to the infarct boundary. Thus, induction of VEGF mRNA was most prominent after 60–120 min of temporary focal ischemia, but as the duration of ischemia

(and the area of infarction) increased further, VEGF mRNA expression was lost. Immunodetectable VEGF protein was increased in homogenates from the ischemic border zone compared to homogenates from the same brain region of non-ischemic controls. Control lysates exhibited a faint band of immunoreactivity corresponding to VEGF165, consistent with low levels of basal expression (Fig. 2). Its intensity increased with increasing durations of ischemia up to 48 h. Immunoreactivity was abolished by preabsorbing anti-VEGF antibody with VEGF peptide.

Fig. 1. In situ hybridization of rat brain sections (left, through midcaudate; right, through anterior hippocampus) with VEGF probe after unilateral MCA occlusion (on the side to viewer’s left) and reperfusion of various durations. (A) Non-ischemic control. (B) Thirty minutes of occlusion and 23.5 h of reperfusion. (C) One hour of occlusion and 23 h of reperfusion. (D) Two hours of occlusion and 22 h of reperfusion. (E) Permanent occlusion for 24 h without reperfusion.

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Fig. 2. Induction of VEGF protein expression in ischemic rat brain. The Western blot 1–2 days after ischemia shows increased expression corresponding to VEGF165 compared to control brain, while preabsorption with VEGF peptide abolishes immunoreactivity in samples taken 2 days after ischemia.

Brain sections obtained after 2 h of MCA occlusion and 22 h of reperfusion showed necrosis in the MCA territory (Fig. 3). VEGF immunoreactivity was seen primarily in the border zone adjacent to the area of necrosis, and, at higher magnification, was associated with cells exhibiting morphological features of astroglia (stellate shape, numerous branching processes, apolarity, no nucleolus). In contrast, immunoreactivity within the necrotic, infarcted region was restricted to vascular endothelial cells. No immunoreactivity was observed when the primary antibody was omitted (not shown), and levels of immunostaining were low in nonischemic brain. Our findings indicate that VEGF expression is induced by temporary focal cerebral ischemia. Expression of VEGF mRNA and protein was most prominent in the border zone surrounding the area of infarction, and appeared to be associated primarily or exclusively with astroglia. This is consistent with the finding that VEGF is induced by hypoxia in primary astroglial cultures [7]. By comparison, VEGF immunoreactivity within the infarct was localized to vascular endothelial cells. Two previous studies have examined expression of VEGF in cerebral ischemia. Kova´cs et al. reported that VEGF protein expression in rat brain was induced by permanent MCA occlusion [8]. Our findings differ from those of Kova´cs et al. in two respects. First, we found increased VEGF mRNA and protein expression predominantly in the penumbral region, rather than in the infarct. Second, we detected VEGF primarily in cells with glial morphology, as opposed to neurons and endothelial cells. Hayashi et al. studied VEGF mRNA and protein expression after transient MCA occlusion in the rat [5]. They found increased expression of VEGF mRNA and protein after 1–3 h of occlusion followed by reperfusion. VEGF immunoreactivity was associated with neurons and pial cells within the ischemic territory. The reasons for these discrepancies are unclear, but may include differences in duration of ischemia, duration of reperfusion, or sacrifice time in the different studies. The best characterized role of VEGF is its ability to promote angiogenesis – the sprouting of new capillaries from existing vessels. In rats exposed to chronic hypoxia, mean brain microvessel density increased up to 76% in cerebral cortex [12]. In stroke patients at autopsy, the number of

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microvessels in the ischemic border zone was increased compared to vessel density in the opposite hemisphere, and higher blood vessel counts correlated with longer survival [9]. Our finding that VEGF is induced in the ischemic border zone following MCA occlusion suggests that VEGF may mediate angiogenesis in this region in patients with stroke, perhaps resulting in increased collateral blood supply to the ischemic brain, as occurs in ischemic myocardium [1]. Altering expression of VEGF using antisense oligodeoxynucleotides, viral vectors or transgenic animals will be required to determine more definitively whether VEGF is

Fig. 3. Immunocytochemical staining of 40-mm rat brain sections with VEGF antibody after 2 h of MCA occlusion and 22 h of reperfusion. (A) Coronal section of rat brain at 2.5× showing regions from which sections shown in (B–E) were taken. (B) Ischemic border zone (1). (C) Control non-ischemic brain (2), showing increased VEGF immunoreactivity in penumbral glial cells, at 10×. (D) Morphology of immunoreactive glial cells shown in (B), at 20×. (E) Ischemic core (3), showing scattered immunostaining among necrotic cells and staining of vascular endothelial cells, at 20×.

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