Hypercholesterolemia may increase stroke risk by accelerating atherosclerosis, narrowing the luminal diameter in cerebral vessels, and disrupting both vascular endothelial and smooth muscle function. In the present study, we investigated the beneficial effects of combinatorial therapy with probucol and cilostazol on focal cerebral ischemia with hypercholesterolemia. Apolipoprotein E (ApoE) knockout (KO) mice were fed a high-fat diet with or without 0.5% probucol and/or 0.2% cilostazol for 10 weeks. Probucol alone and probucol and cilostazol significantly decreased total, low-density lipoprotein, and high-density lipoprotein cholesterol, whereas cilostazol did not affect the plasma cholesterol levels in ApoE KO mice. Administration of probucol alone and cilostazol alone significantly decreased atherosclerotic lesion area in the aorta, with a significant decrease evident using combinatorial administration. Middle cerebral artery occlusion resulted in significantly larger infarct volumes in ApoE KO mice fed 10 weeks of high-fat diet compared with those in ApoE KO mice fed a regular diet. The infarct volume was reduced significantly using probucol alone or cilostazol alone and even was reduced significantly by their combinatorial administration. Consistent with a larger infarct size, the combinatorial therapy prominently improved neurological function. The combinatorial administration increased cerebral blood flow during ischemia. Expression of endothelial nitric oxide synthase and adiponectin in the cortex were decreased by high-fat diet but were elevated by combinatorial treatment. Adiponectin expression colocalized within the cerebral vascular endothelium. The data suggest that the combination of probucol and cilostazol prevents cerebrovascular damage in focal cerebral ischemic mice with hypercholesterolemia by up-regulation of endothelial nitric oxide synthase and adiponectin.
Hypercholesterolemia is a major underlying cause for ischemic stroke, and therapeutics targeting hypercholesterolemia decrease the risk of stroke in high-risk individuals or in patients with stroke or transient ischemic attack (Amarenco and Labreuche, 2009). Although cerebral ischemia commonly occurs in patients with hypercholesterolemia, studies have been hampered in part because the vessels are smaller and more difficult to access. In contrast, larger arteries such as the aorta and peripheral arteries have been studied extensively (Ross, 1999). In systemic vessels in humans and in animal models, hypercholesterolemia impairs endothelial and smooth muscle function (Casino et al., 1993; d'Uscio et al., 2001). Therefore, the effects of lipid-lowing drugs on cerebrovascular function and pathogenic mechanisms of ischemic stroke with hypercholesterolemia should be elucidated fully in vivo.
Probucol is a potent lipid-soluble antioxidant that possesses antiatherogenic properties (Kuzuya and Kuzuya, 1993) and prevents secondary cardiovascular events in patients with hypercholesterolemia (Yamashita et al., 2008). Cilostazol inhibits platelet aggregation by inhibiting the activity of phosphodiesterase III and has a demonstrated neuroprotective effect against cerebral ischemic injury in experimental studies (Choi et al., 2002; Lee et al., 2007) and clinical studies (Shinohara et al., 2010). In seeking to maximize the effects of these beneficial and useful drugs, it has been suggested that their combinatorial application might reduce atherosclerosis and prevent ischemic damage. The combinatorial strategy is supported by results of a clinical study regarding restenosis (Sekiya et al., 1998) and in vitro and in vivo experimental studies regarding atherosclerosis (Park et al., 2008; Yoshikawa et al., 2008). Moreover, concurrent treatment with probucol and cilostazol produced beneficial synergistic effects against focal cerebral ischemic injury in a rat model by reducing the generation of superoxide (Park et al., 2007). With the aim of developing a more effective therapeutic for focal ischemic brain injury with hypercholesterolemia, the present study was undertaken to examine the efficacy of probucol and cilostazol combinatorial therapy.
The exact mechanisms of cerebrovascular dysfunction to ischemia during hypercholesterolemia are not defined completely but may include reduction in endothelial nitric oxide (NO) production. It has been reported that apolipoprotein E (ApoE) knockout (KO) mice fed a Western high-fat diet (HFD) display endothelial dysfunction via impairment of endothelial nitric-oxide synthase (eNOS)-dependent vasorelaxation (d'Uscio et al., 2001). eNOS contributes to vascular protection via increasing cerebral blood flow (CBF) and plays an important role in the regulation of brain damage after ischemia (Endres et al., 1998), and adiponectin prevents cerebral ischemic injury through an eNOS-dependent mechanism (Nishimura et al., 2008). Thus, agents that regulate adiponectin-mediated eNOS signaling and increase CBF could represent a potential therapeutic target for the prevention of ischemic stroke with hypercholesterolemia.
In the present study, we explored the effects of the combinatorial use of probucol and cilostazol on focal cerebral ischemia with hypercholesterolemia using HFD-fed ApoE KO mice as an animal model. The tissue and neurologic outcomes were determined after transient middle cerebral artery (MCA) occlusion in ApoE KO mice fed the HFD for 10 weeks using treatment with probucol and cilostazol. CBF and eNOS expression levels in accordance with adiponectin expression levels were determined as the action mechanisms. The study presents evidence-based cerebrovascular protective effects of combinatorial therapy with probucol and cilostazol on the management of stroke patients with hypercholesterolemia.
Materials and Methods
General Surgical Preparation.
Male ApoE KO mice (Japan SLC Inc., Shizuoka, Japan) having a C57BL/6J genetic background were housed under diurnal lighting conditions and allowed food and tap water ad libitum. All of the animal procedures were in accordance with the institutional guidelines for animal research and were approved by the Pusan National University Animal Care and Use Committee. Four-week-old ApoE KO mice were fed a Western-type HFD (42% of total calories from fat; 0.15% cholesterol; Research Diet, New Brunswick, NJ) containing 0.5% (w/w) probucol, 0.2% (w/w) cilostazol, or 0.5% (w/w) probucol and 0.2% (w/w) cilostazol for 10 weeks. Anesthesia was achieved by isoflurane (2% induction and 1.5% maintenance in 80% N2O and 20% O2) by face mask. The femoral artery was catheterized for the measurement of mean arterial blood pressure using a model MLT844 physiological pressure transducer (AD Instruments, Medford, MA). The data were recorded continuously using a PowerLab data acquisition and analysis system (AD Instruments) and stored in a computer. The depth of anesthesia was checked by the absence of cardiovascular changes in response to tail pinch. Rectal temperature was kept at 36.5 to 37.5°C using a Panlab thermostatically controlled heating mat (Harvard Apparatus, Holliston, MA). Arterial blood gases and pH were measured before ischemia using an i-STAT system (Abbott Laboratories, Abbott Park, IL).
Measurement of Plasma Cholesterol.
Blood was collected from the left ventricle under light anesthesia and stored on ice for 30 min before centrifugation at 13,000 rpm at 4°C for 10 min, and the plasma was separated and kept at −80°C until assayed. Lipoprotein cholesterol distribution of plasma samples was determined enzymatically with reagents from Kyowa Medex (Tokyo, Japan) using a TBA-200FR NEO apparatus (Toshiba Medical Systems, Tokyo, Japan).
Atherosclerotic Lesion Analysis.
After 10 weeks of the HFD, mice were anesthetized and euthanized. Hearts were perfused using 10 ml of phosphate-buffered saline (PBS) followed by 10 ml of 4% paraformaldehyde. After incubation in 4% paraformaldehyde overnight, the adventitia was cleaned thoroughly under a dissecting microscope, and the aorta was cut open longitudinally and pinned onto a silicone plate. To calculate the lesion area, aortas were stained with Oil red O (Sigma-Aldrich, St. Louis, MO). Fifty milliliters of an Oil red O stock solution (0.5% w/v in isopropyl alcohol) was mixed with 30 ml of distilled water and filtered before use. Aortas were rinsed briefly with PBS containing 0.5% Tween 20, incubated in the Oil red O solution for 10 min, and then destained in PBS containing 0.5% Tween 20 for 48 min. Atherosclerotic lesion areas were quantified using iSolution full image analysis software (Image and Microscope Technology, Vancouver, Canada).
Focal Cerebral Ischemia.
A fiber-optic probe was affixed to the skull over the MCA for measurement of regional CBF by a PeriFlux Laser Doppler System 5000 (Perimed, Stockholm, Sweden). Baseline values were measured before internal carotid artery ligation (considered to be 100% flow). Focal cerebral ischemia was induced by occluding the MCA by a previously described intraluminal filament technique (Huang et al., 1994). MCA occlusion was induced by a silicon-coated 7-0 monofilament in the internal carotid artery, and the monofilament was advanced to occlude the MCA. In all of the animals, regional CBF was measured to confirm the achievement of consistent and similar levels of ischemic induction. The filament was withdrawn 60 min after occlusion, and reperfusion was confirmed using laser Doppler. The surgical wound was sutured, and mice were allowed to recover from anesthesia. Brains were removed at 24 h after MCA occlusion. Cerebral infarct size was determined on 2,3,5-triphenyltetrazolium chloride-stained, 2-mm-thick brain sections. Infarction areas were quantified with iSolution full image analysis software (Image and Microscope Technology).
Neurological deficit was scored in each mouse at 24 h after the ischemic insult in a blinded fashion according to the following graded scoring system: 0 = no deficit; 1 = forelimb weakness and torso turning to the ipsilateral side when held by tail; 2 = circling to affected side; 3 = unable to bear weight on affected side; 4 = no spontaneous locomotor activity or barrel rolling (Li et al., 2004).
Twenty-four hours after MCA occlusion, mice were anesthetized deeply with thiopental sodium and subsequently perfused transcardially with cold PBS at 4°C followed by 4% paraformaldehyde for fixation. The brain of each mouse then was removed and further fixed for 48 h in 4% paraformaldehyde at 4°C followed by cryoprotection in 20% sucrose for 24 h at 4°C. The isolated brains were frozen and stored in the freezer at −80°C until examined. The frozen brains were cut at a thickness of 10 μm with a Leica CM 3050 cryostat (Leica Microsystems, Nussloch, Germany), immunostained with an antibody against eNOS (BD Biosciences, San Jose, CA), and additionally incubated with fluorescein isothiocyanate-conjugated secondary antibody to detect eNOS. In double-fluorescence staining, sections were stained with anti-CD31 antibody (BD Biosciences), followed by treatment with fluorescein isothiocyanate-conjugated secondary antibody to detect CD31, and subsequently with anti-adiponectin antibody (R&D Systems, Minneapolis, MN), followed by treatment with Texas Red-conjugated secondary antibody to detect adiponectin. Fluorescently stained sections were analyzed by Axio Imager fluorescence microscopy (Carl Zeiss, Jena, Germany).
Probucol [4,4′-(isopropylidenedithio)bis(2,6-di-tert-butylophenol)] and cilostazol [OPC-13013; 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl) butoxy]-3,4-dihydro-2-(1H)-quinolinone] were donated by Otsuka Pharmaceutical (Tokushima, Japan) and were added to the HFD.
The data are expressed as mean ± S.E.M. Control versus vehicle group was compared by unpaired t test. Vehicle versus drug alone or combinatorial groups were compared using Dunnett's test. Pearson's correlation coefficient was calculated between infarct volume and total cholesterol level. The differences were considered statistically significant, when the two-tailed P values were <0.05. Statistical analysis was performed using SAS software (version 9.1; SAS Institute Japan Ltd., Chuo-ku, Tokyo, Japan).
Physiological Parameters and Plasma Lipid Profiles.
The body weights of ApoE KO mice fed a HFD for 10 weeks were slightly higher than those in normal diet-fed mice (control) (25.90 ± 0.97 versus 32.39 ± 0.71 g, respectively, P < 0.01). Probucol and/or cilostazol did not affect the body weights of HFD-fed mice (Table 1). The blood pressure did not differ among mice treated with probucol and/or cilostazol. After 10 weeks of the HFD, large increases in plasma total cholesterol and low-density lipoprotein (LDL) cholesterol were observed in ApoE KO mice (Table 2, P < 0.01 versus control). Probucol alone or in combination with cilostazol significantly decreased both total and LDL cholesterol levels in ApoE KO mice fed the HFD (P < 0.01 versus vehicle). In contrast, cilostazol alone did not affect the plasma total and LDL cholesterol levels at all.
Effect of Combinatorial Therapy on Atherosclerotic Lesions.
Measurements of lesional histomorphometry from the aorta revealed that the atherosclerotic lesion area (control 4.11 ± 0.83 mm2) was increased markedly to 19.41 ± 2.81 mm2 (P < 0.01) in the HFD-treated group, which was reduced significantly by probucol alone (7.30 ± 2.50 mm2, P < 0.01) or cilostazol alone (10.58 ± 2.37 mm2, P < 0.05) (Fig. 1). Moreover, when the two agents were administered in combination, the atherosclerotic lesions were more potently inhibited (4.04 ± 1.58 mm2, P < 0.01).
Effect of Combinatorial Therapy on Infarct Size and Neurological Deficit.
To determine whether combinatorial therapy improved the tissue outcome during cerebral ischemia in hypercholesterolemic mice, the infarct size was measured 23 h after a 1-h transient MCA occlusion. MCA occlusion resulted in 76% larger infarct volumes in ApoE KO mice fed the HFD for 10 weeks compared with those of ApoE KO mice fed the regular diet (101.75 ± 12.06 versus 57.73 ± 15.82 mm3, P < 0.05; Fig. 2, A and B), which were reduced significantly by probucol alone (30.32 ± 11.19 mm3, P < 0.01) or cilostazol alone (52.38 ± 19.06 mm3, P < 0.05) and reduced significantly by probucol and cilostazol (23.65 ± 7.67 mm3, P < 0.01). Consistent with a larger infarct size, the combinatorial therapy showed prominent improvement of neurological function (Fig. 2C). There was a positive correlation between plasma total cholesterol levels and infarct size after probucol treatment, and this positive correlation was noted when probucol was administered with cilostazol (data not shown). Because an increase in CBF protects against stroke, the changes in CBF during ischemia were assessed. When the CBF time course in MCA was measured, MCA occlusion was revealed to cause an abrupt reduction in CBF, and CBF was higher in combination-treated mice than that in age- and diet-matched ApoE KO mice, suggesting that combinatorial treatment with probucol and cilostazol leads to increased CBF during ischemia (Fig. 3).
Effect of Combinatorial Therapy on eNOS and Adiponectin Expression.
To explore the action mechanisms of combinatorial treatment with probucol and cilostazol on focal cerebral ischemia with hypercholesterolemia, we studied eNOS expression level in accordance with adiponectin expression levels. Few eNOS- and adiponectin-positive cells were observed in the vehicle group (Fig. 4, A and B). eNOS- and adiponectin-positive cells were increased in mice that received probucol and cilostazol in combination, whereas probucol alone or cilostazol alone showed only marginal effects on eNOS and adiponectin expression, indicating the beneficial effects of combinatorial therapy. In addition, cilostazol alone or combinatorial treatment increased CD31, an endothelial cell marker (Fig. 4B). Dual-immunofluorescence staining was performed on adiponectin (red) and CD31 (green), and merged images demonstrated that these proteins colocalized (yellow) (Fig. 4C). Taken together, these data support the suggestion that adiponectin accumulates at the endothelium in the cortex.
In this study, we evaluated the neuroprotective potential of combinatorial therapy with probucol and cilostazol to suppress cerebral ischemic injury with hypercholesterolemia. Probucol alone and cilostazol alone significantly reduced the infarct volume in ApoE KO mice fed a HFD, and the combinatorial administration of probucol and cilostazol significantly reduced infarct size with neurological deficits. In addition, cotreatment with probucol and cilostazol increased CBF due to enhancement of eNOS and adiponectin expression during ischemia. These data support the view that the combination of probucol and cilostazol prevents cerebrovascular damage in focal cerebral ischemic mice with hypercholesterolemia at least partly because of an increase of CBF via an increase of eNOS and adiponectin. This effect likely has a role in mediating the beneficial effects of such strategies in cerebrovascular disease, specifically ischemic stroke.
Hypercholesterolemia may increase the risk of stroke by accelerating atherosclerosis and segmental vessel narrowing or occlusion involving several vascular beds as well as by disrupting vascular endothelial and smooth muscle function. The stroke–cholesterol relationship is complex and contains several paradoxes (Amarenco, 2001; Goldstein et al., 2006). Some studies showed a direct relationship between dyslipidemia (high total and LDL cholesterol levels and low high-density lipoprotein cholesterol level) and stroke (Jürgens et al., 1995; Tanne et al., 2001), whereas other studies have not identified such an association (Tanizaki et al., 2000; Sacco et al., 2001). Few studies have evaluated ischemic stroke subtypes and patient subgroups (Tirschwell et al., 2004). Cholesterol-lowering trials have shown a decrease in the risk of cerebral infarction among patients assigned to statin treatment (White et al., 2000). In the present study, 1-h MCA occlusion and 2-h reperfusion resulted in significantly larger infarct volumes by 76% in ApoE KO mice fed a HFD for 10 weeks compared with those in ApoE KO mice on regular diet, which is consistent with a previous report (Mogi et al., 2006). This larger infarct volume was reduced significantly in accordance with significantly decreased cholesterol levels and atherosclerotic lesion areas in the aorta by probucol alone or by a combinatorial treatment with probucol and cilostazol. Taking into consideration the association between the infarct volume after MCA occlusion and the serum lipid profile (total cholesterol), there was a positive linear relationship (r = 0.77, P < 0.01) in the control, vehicle, probucol alone, and combinatorial groups. When we observed Oil red O staining in cerebral arteries of ApoE KO mice fed a HFD for 10 weeks, no atherosclerotic lesions were observed in cerebral arteries (data not shown). Therefore, we believed that the increase in ischemic brain damage in ApoE KO mice fed a HFD may not be directly due to the structure of cerebral arteries by atherosclerotic plaque formation.
Lipid-lowering therapy significantly reduces the risk of stroke (Collins et al., 2003). In part, this improved outcome has been attributed to slowed progression of intracranial (carotid) atherosclerosis consequent to a decrease in hypercholesterolemia (Amarenco and Labreuche, 2009). Some investigators have suggested that the beneficial effects of statins in stroke may be only in part due to lipid-lowering properties, with the primary benefit derived from improved endothelial function as well as the anti-inflammatory and antithrombotic actions of the drugs (Vaughan et al., 2001; Ishikawa et al., 2004). Probucol (0.5%) and cilostazol (0.2%) treatment significantly decreased total and LDL cholesterol levels and ameliorated the progression of atheroma formation in the entire aorta in ApoE KO mice fed the HFD. A clinical study (Sekiya et al., 1998) and experimental studies (Yoshikawa et al., 2008) have suggested the potential beneficial effects of probucol and cilostazol on restenosis and atherosclerosis. In LDL receptor-deficient mice, the combination of probucol and cilostazol more significantly decreased the atherosclerotic lesion area than either probucol or cilostazol alone (Yoshikawa et al., 2008). The clinical study showed that treatment with a combination of probucol and cilostazol was safe and effective in preventing acute poststenting complications and suppressing chronic restenosis (Sekiya et al., 1998). Presently, the combinatorial treatment with probucol and cilostazol modulated plasma cholesterol levels. It is possible that these effects may have inhibited atherosclerotic lesions and stroke development in ApoE KO mice fed the HFD.
Both probucol and cilostazol have been approved for use. Both are safe and efficient in their respective therapeutic categories with some different and similar action mechanisms. Probucol, which is a potent lipid-soluble antioxidant, possesses antiatherogenic properties (Kuzuya and Kuzuya, 1993). Cilostazol increases intracellular cAMP levels by inhibiting phosphodiesterase III and has a demonstrated in vivo neuroprotective effect against cerebral ischemic injury via antiapoptotic and anti-inflammatory effects (Choi et al., 2002; Lee et al., 2007). Moreover, cilostazol has been shown to increase plasma HDL cholesterol levels and inhibit atherosclerosis formation in ApoE KO mice fed with a Western diet and LDL receptor-deficient mice fed with a HFD (Lee et al., 2005; da Rosa et al., 2006). It is likely that two drugs that possess different mechanisms of action may result in an effective therapy. In the previous in vitro study, we assessed the synergistic efficacy of combinatorial therapy with probucol and cilostazol on antioxidant and anti-inflammatory actions in cultured human coronary artery endothelial cells (Park et al., 2008). Concurrent treatment with probucol and cilostazol had beneficial synergistic effects against focal cerebral ischemic injury in rats via reduced superoxide generation (Park et al., 2007). Consistent with these reports, we presently observed that a combinatorial therapy with probucol and cilostazol reduced infarct size with neurological deficits, providing a potential strategy for preventing ischemic stroke with hypercholesterolemia. In addition, cilostazol alone or combinatorial treatment accelerated angiogenesis, which is demonstrated by increased microvessels (CD31 staining; Fig. 4B). Our results are consistent with the reported findings that long-term treatment with cilostazol increased angiogenesis, which can increase CBF and improve brain tissue recovery (Ye et al., 2007). Therefore, an additional use of cilostazol could improve brain tissue recovery and functional recovery in addition to the antioxidant action of probucol.
Hypercholesterolemia is associated with decreased NO bioavailability and endothelial dysfunction, which may be very important in the altered CBF evident in stroke. Endothelial dysfunction via impairment of eNOS-dependent vasorelaxation in the aorta, carotid artery, and cerebral arterioles of ApoE KO mice fed a HFD has been reported (d'Uscio et al., 2001) and also in the forearm and coronary arteries of hypercholesterolemic patients (Casino et al., 1993). eNOS and vascular NO maintain CBF and protect against brain injury after ischemia (Endres et al., 1998). Probucol improves the endothelium-dependent relaxation in aortic balloon injury in rabbits and a swine model of left ventricular hypertrophy by increasing NO-mediated vasodilation (Lau et al., 2003). In addition, cilostazol causes vasodilation through an endothelial NO-dependent pathway in rat aorta (Nakamura et al., 2001) and increases the phosphorylation of eNOS at Ser1177 in human aortic endothelial cells (Hashimoto et al., 2006). In this study, we observed an increase in CBF by combinatorial treatment with probucol and cilostazol during cerebral ischemia in ApoE KO mice fed a HFD. Moreover, hypercholesterolemia down-regulated eNOS and adiponectin in the cortex, and these effects were increased by the combinatorial long-term application of probucol and cilostazol. However, we could not observe changes in phosphorylated eNOS by combinatorial treatment with probucol and cilostazol (data not shown), suggesting that probucol and cilostazol treatment do not activate eNOS acutely by posttranslational modification including phosphorylation, which is an approach for acute stroke treatment. We suggested that long-term probucol and cilostazol treatment leads to up-regulation of eNOS, which has a combined effect of stroke prevention and prophylactic treatment for ischemic stroke with hypercholesterolemia.
Adiponectin exerts beneficial actions on cerebrovascular disease. Adiponectin is cerebroprotective by an eNOS-dependent mechanism (Nishimura et al., 2008). Adiponectin KO mice develop impaired ischemia-induced angiogenesis in a mouse model of vascular insufficiency and an excessive vascular remodeling response to injury (Matsuda et al., 2002). Recent results of a clinical study suggested an association between hypoadiponectinemia and increased mortality after ischemic stroke and a negative correlation between adiponectin levels and initial infarct volume (Chen et al., 2005). As well, hyperadiponectinemia is neuroprotective against ischemic stroke (Ouchi et al., 2003). Furthermore, adiponectin accumulates in the vascular endothelium during cerebral ischemia (Nishimura et al., 2008). Thus, agents that increase circulating adiponectin levels could represent a potential therapeutic target for the prevention of ischemic stroke. Probucol can significantly elevate serum adiponectin concentrations in diabetic rats (Zhang et al., 2009), and cilostazol increases adiponectin levels in type 2 diabetic patients and in a diabetic animal model (Hsieh and Wang, 2009). In the present study, the accumulation of adiponectin with eNOS in the vasculature in the combinatorial therapy group may have served to protect the vasculature. Taken together, these observations suggest that combinatorial treatment with probucol and cilostazol regulates the adiponectin-eNOS signaling axis functions to modulate vascular function, protecting against cerebral injury after stroke.
In summary, combinatorial therapy with probucol and cilostazol prevents ischemic stroke with hypercholesterolemia through an increase of CBF via an increase of eNOS and adiponectin. This finding may provide convincing evidence to support cerebrovascular protective effects of combinatorial therapy on focal cerebral ischemic injury with hypercholesterolemia.
Participated in research design: J.H. Kim and Shin.
Conducted experiments: J.H. Kim and S.H. Park.
Contributed new reagents or analytic tools: Hong, Y.D. Kim, and K.P. Park.
Performed data analysis: J.H. Kim, Choi, and Shin.
Wrote or contributed to the writing of the manuscript: J.H. Kim, Bae, and Shin.
This work was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology [Grants 2009-0066654, 2010-0007470]; and Otsuka Pharmaceutical (Tokushima, Japan).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- nitric oxide
- apolipoprotein E
- cerebral blood flow
- endothelial nitric-oxide synthase
- high-fat diet
- low-density lipoprotein
- middle cerebral artery
- phosphate-buffered saline.
- Received March 2, 2011.
- Accepted May 4, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics