Attenuation of Focal Brain Ischemia by Telmisartan, an Angiotensin II Type 1 Receptor Blocker, in Atherosclerotic Apolipoprotein E−Deficient Mice

161 Hypertens Res Vol.31 (2008) No.1 p.161-168 Original Article Attenuation of Focal Brain Ischemia by Telmisartan, an Angiotensin II Type 1 Receptor Blocker, in Atherosclerotic Apolipoprotein E–Deficient Mice Masaru IWAI1), Shinji INABA1), Yumiko TOMONO1), Harumi KANNO1), Jun IWANAMI1), Masaki MOGI1), and Masatsugu HORIUCHI1) The effects of an angiotensin II (Ang II) type 1 (AT1) receptor blocker (ARB) on focal brain ischemia and atherosclerotic lesions were explored in atherosclerotic apolipoprotein E–deficient (ApoEKO) mice treated with a high-cholesterol diet (HCD). The ischemic brain area and neurological deficit 24 h after middle cerebral artery (MCA) occlusion were significantly greater in ApoEKO mice treated with HCD for 10 weeks than in those with a normal standard diet. The reduction of cerebral surface blood flow in the penumbral region and the increase in superoxide production in the ischemic area were exaggerated in HCD-treated ApoEKO mice. Histological analysis showed atherosclerotic changes in the proximal aorta and deposition of lipid droplets in the arterial wall in the brain. Administration of an ARB, telmisartan (0.3 mg/kg/day), for the last 2 weeks after 8 weeks of HCD feeding attenuated the ischemic brain area, the neurological deficit, the superoxide production in the ischemic area, and the reduction of cerebral blood flow in the penumbra, without significantly changing blood pressure or serum cholesterol level. Telmisartan also decreased atherosclerotic lesion formation in the proximal aorta of HCD-treated ApoEKO mice, although it did not remarkably change lipid deposition in the cerebral arteries. These results suggest that the blockade of the AT1 receptor attenuates ischemic brain damage induced in an atherosclerosis model. This inhibitory action is mediated through the attenuation of the reduction in cerebral blood flow and of oxidative stress in the brain; it also mediated through telmisartan’s anti-atherosclerotic effect. (Hypertens Res 2008; 31: 161–168) Key Words: cerebral ischemia, atherosclerosis, angiotensin, receptors, oxidative stress Introduction Atherosclerosis is associated with various cardiovascular events, such as coronary heart disease and stroke. Recent clinical studies have indicated a close relationship between atherosclerosis of the carotid and coronary arteries and the risk of stroke (1–3). In these studies, regression of atherosclerosis seemed to lower the risk of stroke. However, the pathogenesis and effective prevention of ischemic brain damage in athero- sclerosis patients have not been well studied. Previous studies also suggested that the renin-angiotensin system plays an important role in stroke. Angiotensin II was found to be significantly involved in ischemic brain damage in a study using a mouse model of brain infarction induced by middle cerebral artery (MCA) occlusion (4–6). Focal brain ischemia was reduced in angiotensin II type 1 (AT1) receptor–deficient mice (4), whereas it was exaggerated in angiotensin II type 2 (AT2) receptor–deficient mice (5). Peripheral administration of an AT1 receptor blocker (ARB) significantly reduced the From the 1)Department of Molecular Cardiovascular Biology and Pharmacology, Ehime University Graduate School of Medicine, Tohon, Japan. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Cardiovascular Research Foundation, the Takeda Science Foundation, and the Mitsubishi Pharma Research Foundation. Address for Reprints: Masatsugu Horiuchi, M.D., Ph.D., Department of Molecular Cardiovascular Biology and Pharmacology, Ehime University Graduate School of Medicine, Shitsukawa, Tohon 791–0295, Japan. E-mail: Received June 1, 2007; Accepted in revised form August 5, 2007. 162 Hypertens Res Vol. 31, No. 1 (2008) Fig. 1. Focal brain ischemic area 24 h after MCA occlusion in apolipoprotein E–deficient (ApoEKO) mice treated with a highcholesterol diet (HCD). ApoEKO mice were fed for 10 weeks with either a normal standard diet (ND) or a HCD containing 1.25% cholesterol. Coronal sections were stained with 2,3,5-triphenyltetrazolium chloride (TTC). Brain sections were numbered from frontal (section 1) to caudal (section 6). Some mice were treated with telmisartan (Telm; 0.3 mg/kg/day) for 14 days using an osmotic minipump after 8 weeks of HCD feeding. A: TTC staining of brain sections from ApoEKO mice 24 h after MCA occlusion. B: Morphometry of ischemic area determined with TTC staining and expressed as a percentage of total area. n= 7–8 for each group. *p< 0.05 vs. ND. †p< 0.05 vs. HCD without telmisartan. Values are means ± SEM. ischemic brain area and neurological deficit after MCA occlusion (5, 7). On the other hand, apolipoprotein E–deficient (ApoEKO) mice treated with a high-cholesterol diet (HCD) developed atherosclerotic changes. Blockade of the AT1 receptor markedly attenuated atherosclerotic changes in ApoEKO mice treated with HCD (8). In addition, atherosclerotic lesion formation was lower in ApoE/AT1a receptor– deficient mice than in ApoEKO mice (9), while it was higher in ApoE/AT2 receptor–deficient mice (10). Moreover, previous reports suggested that suppression of oxidative stress is important in ARBs’ inhibitory effects on ischemic brain damage as well as atherosclerosis (6, 8, 10). The present study was designed to explore 1) the influence of atherosclerosis on ischemic brain damage using ApoEKO mice; 2) whether or not ARB inhibits ischemic brain damage in ApoEKO mice after treatment with HCD, in which atherosclerotic formation has been observed; and 3) whether or not the inhibition of ischemic brain damage is related to the inhibition of atherosclerosis. Methods Animals Adult male ApoEKO mice (B6.129P2-Apoetm1Unc, The Jackson Laboratory, Bar Harbor, USA; 10 × backcrossed) were Iwai et al: Ischemic Brain Damage in Atherosclerotic Mice Fig. 2. Neurological score after MCA occlusion in ApoEKO mice treated with HCD. MCA occlusion was performed, and the neurological score was used to evaluate neurological deficit 24 h after the operation. ND, normal standard diet; HCD, high-cholesterol diet; Telm, telmisartan. n= 7–8 for each group. *p < 0.05 vs. ND. †p< 0.05 vs. HCD without telmisartan. Values are means ± SEM. housed in a room where lighting was controlled with a 12-h light/dark cycle and room temperature was kept at 25°C (10). They were given a standard diet (MF, Oriental Yeast, Tokyo, Japan) or HCD (1.25% cholesterol, 10% coconut oil in MF) for 10 weeks from 6 weeks of age and water ad libitum. Telmisartan (0.3 mg/kg/day; provided by Nippon Boehringer Ingelheim Co., Ltd., Kawanishi, Japan), an AT1 receptor– selective ARB, was administered for the last 2 weeks after 8 weeks of HCD feeding, using an osmotic minipump (Alzet model 1002, Durect, Cupertino, USA) implanted intraperitoneally. Serum cholesterol level was measured by the cholesterol oxidase method (Cholesterol E-test, Wako Pure Chemic (...truncated)