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Angiotensin type 1 receptor blockage improves ischemic injury following transient focal cerebral ischemia
Neuroscience 134 (2005) 225–231
ANGIOTENSIN TYPE 1 RECEPTOR BLOCKAGE IMPROVES ISCHEMIC INJURY FOLLOWING TRANSIENT FOCAL CEREBRAL ISCHEMIA N. HOSOMI,a* A. NISHIYAMA,b C. R. BAN,a T. NAYA,a T. TAKAHASHI,a M. KOHNOa AND J. A. KOZIOLc
I.v. infusion of angiotensin II following transient forebrain ischemia increases cerebral edema and mortality (Hosomi et al., 1999). Furthermore, prior angiotensin type 1 (AT1) receptor blockage in the rat brain improves neurological outcomes and infarct volumes in cerebral ischemia (Nishimura et al., 2000b; Yamakawa et al., 2003). Nevertheless, the tissue-related contributions of angiotensin II to cerebrovascular and neuronal injury during cerebral ischemia have not been detailed. Recently, Ando et al. (2004) showed that AT1 receptor blocker had an anti-inflammatory effect on cerebral vessels in hypertension. Angiotensin II has been reported to be a potent stimulator of the expression of matrix metalloproteinases (MMPs), which degrade the basal lamina constituents (Arenas et al., 2004). The potential of angiotensin II to increase cerebral edema can be mediated by the expression of MMPs. MMP-2 and MMP-9 have been reported to be upregulated in the ischemic area at different time points in acute stroke (Rosenberg et al., 1996). MMP-9 may contribute to increased vascular endothelial permeability through the degradation of extracellular matrix components (Partridge et al., 1993). We have hypothesized that the stimulation of MMP expression with angiotensin II may be associated with this mechanism, since basal lamina cleavage with MMPs is involved in the induction of cerebral microvascular permeability following cerebral ischemia (Gasche et al., 2001; Kim et al., 2003). The major goals of the present study were to ascertain whether brain angiotensin II is upregulated in our experimental model of focal cerebral ischemia, and to quantify the effects of olmesartan, an AT1 receptor blocker, on ischemic injury in the model.
a Second Department of Internal Medicine, Division of Stroke, Kagawa University School of Medicine, 1750-1 Ikenobe, Miki-cho, Kagawa 761-0793, Japan b Department of Pharmacology, Kagawa University School of Medicine, Kagawa 761-0793, Japan c
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA
Abstract—Following cerebral ischemia, i.v. infusion of angiotensin II increases cerebral edema and mortality. Angiotensin type 1 receptor blockage should therefore improve acute cerebral ischemia. Left middle cerebral artery occlusion (120 min) followed by reperfusion was performed with the thread method under halothane anesthesia in Sprague–Dawley rats. Olmesartan (angiotensin type 1 receptor blocker; 0.01 or 0.1 mol/ kg/h) was infused i.p. for 7 days following middle cerebral artery occlusion followed by reperfusion. Stroke index score, infarct volume, specific gravity, and brain angiotensin II and matrix metalloproteinases were quantified in the ischemic and non-ischemic hemispheres. Olmesartan treatment improved stroke index score, infarct volume, and cerebral edema in our cerebral ischemia model. In particular, stroke index score, infarct volume, and cerebral edema were reduced even with a low dose of olmesartan that did not decrease blood pressure. Paralleling these effects on cerebral ischemia, olmesartan treatment also reduced the reactive upregulation in brain angiotensin II, matrix metalloproteinase-2, matrix metalloproteinase-9, and membrane type 1-matrix metalloproteinase in the ischemic area. Angiotensin type 1 receptor stimulation may be one of the important factors that cause cerebral edema following cerebral ischemia, and that its inhibition may be of therapeutic advantage in cerebral ischemia. © 2005 Published by Elsevier Ltd on behalf of IBRO.
EXPERIMENTAL PROCEDURES
Key words: cerebral ischemia, cerebral edema, angiotensin II, matrix metalloproteinases.
One hundred twenty-two male Sprague–Dawley (SD) rats were obtained from Clea Japan, Inc. (Tokyo, Japan). Our experiments were conducted when the animals were 6 –7 weeks old (weights 180 –250 g). The experimental procedures were approved by the Institutional Animal Care and Use Committee in Kagawa University School of Medicine and were in accordance with the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health. The protocol was planned with the care to minimize the number of animals used and their suffering. Focal cerebral ischemia and reperfusion in the SD rats were induced according to procedures described by Zea-Longa et al. (1989). Briefly, each animal was anesthetized with 4% halothane and spontaneously respired with 1% halothane in a 2:1 N2O/O2 mixture, with the use of a facemask. Under the operating microscope, a 30-mm segment of 4-0 ethylon monofilament, its tip rounded by heating, was gently introduced from the external carotid artery into the internal carotid artery lumen until a slight resistance was felt and the skin incision closed. The rats were reanesthetized and the filament was withdrawn 120 min after induction of ischemia. Animals
Cerebral ischemia is responsible for the loss of microvascular integrity, a consequence of the loss of basal lamina matrix ligands. The major basal lamina constituents decrease roughly in parallel during experimental middle cerebral artery occlusion (MCA:O) followed by reperfusion (MCA:O/R) (Hamann et al., 1995). This disruption of normal cell-matrix adhesion has been associated with cell death, edema, and loss of cell viability (Re et al., 1994). *Corresponding author. Tel: ⫹81-87-891-2150; fax: ⫹81-87-891-2152. E-mail address: [email protected] (N. Hosomi). Abbreviations: AT1, angiotensin type 1; MCA:O, middle cerebral artery occlusion; MCA:O/R, middle cerebral artery occlusion followed by reperfusion; MMP, matrix metalloproteinase; MT, membrane type; SD, Sprague–Dawley. 0306-4522/05$30.00⫹0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2005.03.054
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were then returned to their cages and closely monitored until they recovered from anesthesia. In sham-operated rats, the external carotid artery was surgically prepared for insertion of the filament, but the filament was not inserted. Rectal temperature was monitored continuously and maintained between 36.5 °C and 37.5 °C throughout the anesthesia via a feedback-regulated heating pad placed underneath the rat. Fourteen animals died within 4 h after reperfusion following MCA:O. These were considered operation failures and excluded from all analyses. Animals that survived longer than 4 h after reperfusion had fairly constant significant cerebral infarction with 2,3,5-triphenyltetrazolium chloride staining.
Experimental protocols This study consisted of three parts, each involving the administration of olmesartan or its vehicle as a continuous i.p. injection via an osmotic mini-pump. In this regard, an osmotic mini-pump (Model No. 201, ALZET, Cupertino, CA, USA) was filled with the vehicle (2.5% NaHCO3) or olmesartan (AT1 receptor blocker; Sankyo Co., Tokyo, Japan) and was inserted into the abdomen of experimental animals, and the wound was sutured. The first part of this study was a preliminary dose-finding experiment. Olmesartan at 0.001, 0.01, 0.1, and 1 mol/kg/h and its vehicle as control were administered continuously for 7 days in different groups of the non-ischemic SD rats (n⫽5 per group). During the experimental period, blood pressures were monitored using the tail cuff method. The experiment was terminated 7 days after the osmotic mini-pump implantation under halothane anesthesia by left ventricular transcardiac perfusion of the cranial structures with chilled (4 °C) saline perfusate containing heparin (200 IU/l), nitroprusside (1 mg/l), and bovine serum albumin (50 g/l). Brain perfusion was regarded as complete by the visual absence of blood and the absence of blood elements on microscopic inspection. The removed brains were quickly frozen in 2-methylbutane/dry ice for subsequent quantification of tissue angiotensin II. In the second part of this study, osmotic mini-pumps were implanted just after reperfusion following MCA:O, and olmesartan at 0.01 or 0.1 mol/kg/h or its vehicle was administered continuously from the onset of reperfusion (n⫽12 animals per group). Again, blood pressures were monitored during the experimental period using the tail cuff method. Stroke index scores were evaluated 7 days after MCA:O/R. The experiment was then terminated with decapitation under halothane anesthesia, and infarct volumes (n⫽5 animals per group) and specific gravity measurements (n⫽7 animals per group) were made. In the last part of this study, olmesartan at 0.01 or 0.1 mol/ kg/h or its vehicle was administered as described in the second part of this study. The experiments were terminated 12 h or 7 days after MCA:O/R under halothane anesthesia by left ventricular transcardiac perfusion as described above. The removed brains were quickly frozen in 2-methylbutane/dry ice for subsequent quantification of brain angiotensin II or MMP-2, MMP-9, and membrane type (MT)1-MMP (n⫽7 for each time point, respectively). Plasma was collected at the termination of the experiment.
Stroke index score To determine neurological deficits, neurological examinations were performed at 7 days after reperfusion in the second experiment, as mentioned above. The animals were evaluated for a decrease in alertness and movement, ptosis, cocked head, circling behavior, splayed or rotated limbs, seizures, and tremor. Each symptom was assigned a numerical weight, which was used in calculating the stroke index score (McGraw, 1977). The stroke index was weighted as hair roughed up or tremor: 1, obtunded: 1, paucity of movements: 1, head cocked: 3, eye fixed open: 3, ptosis: 2, a splayed out hind limb: 3, extreme rotation of hind: 3, circling behavior: 3, seizures: 2, rolling seizure: 3, extreme weak-
ness: 6, and death: grand sum⫹3. The maximum score was 34, in deceased animals.
Infarct volume Each brain was cut into 2-mm-thick coronal slices with a rodent brain matrix. The extent of ischemic infarction was revealed by reaction with a 2% solution of 2,3,5-triphenyltetrazolium chloride (Sigma Chemical, St. Louis, MO, USA) for 20 –30 min. The border between infarct and non-infarct tissue was outlined with an image analysis system (Image 1.3: National Institute of Mental Health, Bethesda, MD, USA), and the area of infarction was measured by subtracting the area of the non-lesioned ipsilateral hemisphere from that of the contralateral side (Lin et al., 1993). The volume of infarction was calculated by integration of the lesion areas.
Cerebral specific gravity Cerebral specific gravity, an index marker of cerebral edema, was determined at the cortex and caudate putamen by microgravimetry as described previously (Hosomi et al., 1999).
Brain and plasma angiotensin II Brain and plasma angiotensin II levels were measured by radioimmunoassay, as previously described (Nishiyama et al., 2002).
Brain MMP-2, MMP-9, MT1-MMP Brain was minced and homogenized in lysis buffer (50 mmol/l Tris–HCl [pH 7.6], 1.5 mmol/l NaCl, 0.5 mmol/l CaCl2, 1 mol/l ZnCl2, 1.0% Triton X-100, 0.01% Brij 35, and 0.25% Triton X-100) on ice and centrifuged at 4 °C for 20 min at 9000 r.p.m. Supernatants were divided into aliquots and stored at ⫺80 °C. Preliminary experiments determined the optimal conditions for homogenization and activity extraction. MMP-2, MMP-9, and MT1-MMP in brain extract were determined by commercially available activity assay system kits (MMP-2, MMP-9, and MT1-MMP; Amersham Biosciences UK Limited; Buckinghamshire, UK). Assays were performed according to the manufacturer’s instructions. Concentrations were calculated in nanograms per milligram protein. The linearity ranges for the assays were 0.19 –3 ng/ml in MMP-2, 0.125– 4 ng/ml in MMP-9, and 0.125– 4 ng/ml in MT1-MMP. Both latent and active forms of the MMPs are detected in the assays. The assays are applicable to different species, but not to other MMPs. The protein contents of tissue and plasma samples were determined according to the Bradford method with bovine serum albumin as the standard (Hosomi et al., 2001). The reproducibility of the angiotensin II, MMP-2, MMP-9, and MT1-MMP assays was reasonably good: coefficient of variation of angiotensin II, MMP-2, MMP-9, and MT1-MMP measurements was 8.8%, 9.2%, 9.6%, and 8.2% respectively.
Statistical analysis Summary statistics are expressed as the mean⫾standard deviation. Differences in changes in blood pressure among groups were examined using repeated measures analysis of variance (ANOVA). Differences in blood pressure at each time point, infarct volume, and specific gravity among groups were examined using one way-ANOVA. Differences in brain angiotensin II, MMP-2, MMP-9, and MT1-MMP among groups were examined using two way-ANOVA. When the overall ANOVA P-value was ⬍0.05, Bonferroni’s correction for multiple comparisons was then used to assess individual group differences. Overall differences in stroke index scores among groups were examined using the KruskalWallis test, followed by Mann-Whitney U tests for individual comparisons. Differences between the ischemic and non-ischemic hemispheres were examined using paired t-tests.
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Fig. 1. Time courses of mean blood pressure in vehicle rats olmesartan rats at two dose levels (0.01 and 0.1 and mol/kg/h); n⫽12 rats per cohort. O-1, 1 h after MCA:O; O-2, 2 h after MCA:O; Pre, preliminary; R-1h, 1 h after MCA:O/R; R-4h, 4 h after MCA:O/R; R-1d, 1 day after MCA:O/R; R-4d, 4 days after MCA:O/R; R-7d, 7 days after MCA:O/R. ‡ P⬍0.001 to the vehicle and olmesartan (0.01 mol/kg/h) rats with Bonferroni’s correction following repeated measure ANOVA.
RESULTS In the dose-finding experiment, mean blood pressures decreased significantly in the 0.1 and 1 mol/kg/h cohorts (F4,20⫽7.760, P⬍0.001 with repeated measure-ANOVA, P⬍0.005 with Bonferroni’s correction, n⫽5; respectively), but did not decrease in the 0.01 and 0.001 mol/kg/h cohorts. Brain angiotensin II was decreased in a dosedependent manner relative to olmesartan infusion dose (F4,20⫽10.294, P⬍0.001 with one way-ANOVA, n⫽5 per cohort). In subsequent experiments examining the effects of olmesartan on cerebral ischemic injury, we therefore chose the doses of olmesartan in 0.1 mol/kg/h, the minimum dose to decrease blood pressure, and 0.01 mol/ kg/h, the maximal dose not to decrease blood pressure. In the first MCA:O/R experiments, mean blood pressure did not decrease in the olmesartan (0.01 mol/kg/h) rats through the experimental period. Mean blood pressure started to decrease in the olmesartan (0.1 mol/kg/h) rats from 1 h after MCA:O/R, reached to the significantly lower level at 4 h after MCA:O/R compared with the vehicle and olmesartan (0.01 mol/kg/h) rats, and was stable afterward through the experimental period (F2,33⫽10.512, P⬍0.001 with repeated measure-ANOVA, P⬍0.001 with Bonferroni’s correction, n⫽12; respectively; Fig. 1). Stroke index scores at 7 days after MCA:O/R were significantly decreased in both olmesartan (0.01 and 0.1 mol/kg/h) rats (P⬍0.001 with Mann-Whitney U test, n⫽12; respectively; Fig. 2). There was no significant difference in stroke index score between the olmesartan (0.01 and 0.1 mol/kg/h) rats. Infarct volumes at 7 days after MCA:O/R were significantly reduced in both olmesartan (0.01 and 0.1 mol/kg/h) rats (F2,12⫽8.110, P⬍0.01 with one way-ANOVA, P⫽0.012 and 0.002 with Bonferroni’s correction, n⫽5; respectively; Fig. 3). There was no significant difference in infarct volume between the olmesartan (0.01 and 0.1 mol/kg/h) rats. In all three cohorts, specific
gravity, a marker of cerebral edema, significantly decreased in both the cortex and the caudate putamen in the infarct areas compared with the non-ischemic hemispheres (P⬍0.01 or 0.001 with paired t-test, n⫽7; Fig. 4). When comparing cohorts, specific gravity was significantly higher at both olmesartan doses (0.01 and 0.1 mol/kg/h) in the infarct areas of both the cortex (F2,18⫽6.307, P⬍0.01 with one way-ANOVA, P⫽0.010 and 0.004 with Bonferroni’s correction, n⫽7; respectively) and the caudate putamen (F2,18⫽9.795, P⬍0.005 with one way-
Fig. 2. Stroke index score of vehicle rats and olmesartan rats (0.01 and 0.1 mol/kg/h) 7 days after MCA:O/R (n⫽12 per cohort). The bar showed the median value. * P⬍0.001 to the vehicle rats with MannWhitney U test following Kruskal-Wallis test.
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Fig. 3. Infarct volumes of vehicle rats and olmesartan rats (0.01 and 0.1 mol/kg/h) 7 days after MCA:O/R (n⫽5 per cohort). : mean⫾standard deviation, * P⬍0.05, ** P⬍0.01 to the vehicle rats with Bonferroni’s correction following one-way ANOVA.
ANOVA, P⫽0.007 and P⬍0.001 with Bonferroni’s correction, n⫽7; respectively). In the vehicle rats, brain angiotensin II increased 12 h after MCA:O/R in the ischemic hemisphere compared with the non-ischemic hemisphere (P⬍0.001 with paired t-test, n⫽7; Fig. 5), and decreased to the similar level in the non-ischemic hemisphere 7 days after MCA:O/R. In the
olmesartan (0.01 mol/kg/h) rats, angiotensin II was elevated 12 h after MCA:O/R in the ischemic hemispheres relative to the non-ischemic hemispheres (P⫽0.008 with paired t-test, n⫽7), and decreased to the similar level in the non-ischemic hemisphere 7 days after MCA:O/R. There were significant dose-dependent declines in angiotensin II relative to the vehicle in both hemispheres at 12 h after MCA:O/R, maintained out to 7 days after MCA:O/R (F2,36⫽446.566, P⬍0.001 with two-way ANOVA, n⫽7 per cohort at each time point). Plasma angiotensin II was 12.6⫾2.4 fmol/g protein and did not change over the time course of the experiment in the vehicle rats. In the olmesartan (0.01 and 0.1 mol/kg/h) rats, plasma angiotensin II increased to 18.4⫾3.1 and 31.4⫾2.4 fmol/g protein at 12 h after reperfusion (F2,18⫽102.9, P⬍0.001 with one-way ANOVA, n⫽7 per cohort). In the vehicle rats, brain MMP-2 significantly increased in the ischemic hemispheres at 7 days after MCA:O/R (P⫽0.012 with paired t-test, n⫽7; Fig. 6) but not at 12 h after MCA:O/R compared with the non-ischemic hemispheres. There were significant increases in MMP-9 and MT1-MMP at 12 h after MCA:O/R (P⫽0.017 and 0.022 with paired t-test, n⫽7; respectively) but not at 7 days after MCA:O/R. In olmesartan rats at dose 0.01 and 0.1 mol/ kg/h, there was no significant elevation in MMP-2, MMP-9 or MT1-MMP in the ischemic hemispheres relative to the non-ischemic hemispheres, at either 12 h or 7 days after MCA:O/R, except MMP-2 at 7 days after MCA:O/R in the olmesartan (0.01 mol/kg/h) rats (P⬍0.05 with paired t-test, n⫽7). Lastly, there were significant dose-dependent reductions in MMP-2 levels in the olmesartan rats com-
Fig. 4. Specific gravity in the cortex and caudate putamen of vehicle rats and olmesartan rats (0.01 and 0.1 mol/kg/h) 7 days after MCA:O/R (n⫽7 per cohort). White circle: non-ischemic hemisphere (Non-I); Black circle: ischemic hemisphere (I); : mean⫾standard deviation, ⫹ P⬍0.01, ⫹⫹ P⬍0.001 to the non-ischemic hemisphere with paired t-test. * P⬍0.05, ** P⬍0.01, *** P⬍0.001 to the vehicle rats with Bonferroni’s correction following one-way ANOVA.
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Fig. 5. Brain angiotensin II levels 12 h (12H) and 7 days (7D) after MCA:O/R; n⫽7 animals for each cohort at each time point. I, ischemic hemisphere; Non-I, non-ischemic hemisphere. : mean⫾standard deviation, ⫹ P⬍0.01, ⫹⫹ P⬍0.001 to the non-ischemic hemisphere with paired t-test. * P⬍0.001 to the vehicle rats, † P⬍0.001 to the olmesartan rats (0.01 mol/kg/h) with Bonferroni’s correction following two-way ANOVA.
pared with the vehicle rats at 7 days after MCA:O/R (F2,18⫽7.193, P⫽0.005 with one-way ANOVA, n⫽7 per cohort), and in MMP-9 and MT1-MMP both at 12 h (F2,18⫽17.850, P⬍0.001 and F2,18⫽5.159, P⫽0.017, n⫽7 per cohort; respectively) and at 7 days (F2,18⫽10.545, P⬍0.001 and F2,18⫽3.918, P⫽0.039, n⫽7 per cohort; respectively) after MCA:O/R in the ischemic hemispheres, but not in MMP-2 at 12 h after MCA:O/R (F2,18⫽0.442, P⫽0.650, n⫽7 per cohort).
DISCUSSION In our cerebral ischemia model, olmesartan treatment improved stroke index scores, infarct volumes, and cerebral edema at two dose levels: a high dose, which decreased blood pressures, and a low dose, which did not. Paralleling these effects on cerebral ischemia, olmesartan treatment at both dose levels reduced the reactive upregulation in brain angiotensin II, MMP-2, MMP-9, and MT1-MMP in the ischemic area. In the present study, we found that brain angiotensin II was upregulated 12 h after MCA:O/R in the ischemic region. Since plasma angiotensin II levels were around 40% of the antigen content of the non-ischemic hemispheres over the course of the experiment, cerebral edema that causes transudation of plasma angiotensin II cannot account for the increased levels in the ischemic hemispheres. Furthermore, brain angiotensin II was decreased with AT1 receptor blocker in a dose-dependent fashion in both the ischemic and the non-ischemic hemispheres. Conversely, plasma angiotensin II increased in the olmesartan-treated rats, as previously reported (Nishiyama et al., 2003). Since plasma angiotensin II levels were around 300% of the antigen content of non-ischemic and ischemic
hemisphere over the experiment, plasma transudation with cerebral edema cannot account for the decreases of angiotensin II in the olmesartan- (0.1 mol/kg/h) treated rats. The binding of angiotensin II to AT1 receptor initiates internalization of the complex in vascular smooth muscle cells (Anderson and Peach, 1994). Interestingly, AT1 receptor plays a role in the increased intrarenal angiotensin II levels observed in angiotensin II dependent hypertension (Nishiyama et al., 2003). The present study suggests that AT1 receptor-mediated angiotensin II augmentation from plasma or brain local angiotensin II synthesis may similarly occur in the brain. There have been various reports of the effects of peripheral administration of AT1 receptor blockers (Nishimura et al., 2000a; Wang et al., 2003). Several studies of pretreatment with AT1 receptor blockers in experimental cerebral ischemia have demonstrated that renin-angiotensin system inhibition decreased infarct volumes and improved neurological outcomes (Nishimura et al., 2000b; Yamakawa et al., 2003). In a previous study, we found slight blood pressure reduction along with increased cerebral edema and mortality following transient forebrain ischemia (Hosomi et al., 1999). Hence the blood pressure reduction with post-ischemic AT1 receptor blocker treatment in the present study does not account for the improved neurological behavior, infarct volume, and cerebral edema following cerebral ischemia. Recently, Engelhorn et al. (2004) demonstrated that chronic post-treatment with AT1 receptor blockers improved infarct sizes and neurological behavior after transient cerebral ischemia. Our results are in qualitative agreement: olmesartan post-treatment also improved neurological behavior and infarct volumes following MCA:O/R, even at a high dose that decreased sys-
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Fig. 6. Brain MMP-2, MMP-9, and MT1-MMP levels at 12 h (12H) and 7 days (D7) after MCA:O/R. I, ischemic hemisphere; Non-I, non-ischemic hemisphere. : mean⫾standard deviation, ⫹ P⬍0.05 to the non-ischemic hemisphere with paired t-test. * P⬍0.05, *** P⬍0.001 to the vehicle rats with Bonferroni’s correction following two-way ANOVA.
temic blood pressure. Recently, it has been reported that AT1 receptor inhibition reversed the pathological vascular hypertrophy, normalized endothelial nitric oxide synthase expression, and decreased intercellular adhesion molecule-1 expression and the number of infiltrating macrophages in cerebral vessels of hypertensive rats (Ando et al., 2004). These anti-inflammatory effects of AT1 receptor blocker may be an important mechanism in protecting against cerebral ischemia. I.v. infusion of angiotensin II increases cerebral edema and mortality following cerebral ischemia (Hosomi et al.,
1999). The deleterious effect on cerebral edema can be counteracted by treatment with an AT1 receptor blocker. Whether angiotensin II increases MMP expression through AT1 receptor stimulation or another mechanism is not definitively known. Angiotensin II induces MMP-2 and MMP-9 secretion from endothelial cells (Arenas et al., 2004). Treatment with an AT1 receptor blocker results in decreased MMP and mRNA levels in carotid plaques under hypertensive diastolic heart failure (Yoshida et al., 2003; Cipollone et al., 2004). The secretions of MMP-2, MMP-9, and MT1-MMP were not dependent on AT1 receptor stim-
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ulation, since olmesartan treatment could not decrease MMP-2, MMP-9 and MT1-MMP levels in the non-ischemic hemispheres. Interestingly, the over-expressions of brain MMP-2, MMP-9, and MT1-MMP were dependent on AT1 receptor stimulation following cerebral ischemia, since these were reduced with AT1 receptor blockage. In this regard, MMPs may play important roles in the interplay between angiotensin II and cerebral edema. Blood pressure reduction in the first 24 h after stroke onset is an independent risk factor for poor outcomes as assessed by Rankin or Barthel scores 3 months after acute ischemic stroke (Oliveira-Filho et al., 2003). The ACCESS study demonstrated a reduction in mortality in patients with acute ischemic stroke who received an AT1 receptor blocker for acute blood pressure treatment, despite the absence of a significant effect on blood pressures compared with placebo (Schrader et al., 2003). In the present study, olmesartan treatment at a low dose was able to reduce infarct volumes and cerebral edema without decreasing blood pressures. Olmesartan treatment at the high dose [an amount equivalent to clinical usage] demonstrated the same clinical benefits of reduced infarct volumes and cerebral ischemia, but did decrease blood pressures. In translation to the clinic, one would initially need to establish a dosage regimen for an AT1 receptor blocker that would not induce any reduction in blood pressure, prior to investigating its therapeutic potential in acute cerebral ischemia. Acknowledgment—This study was supported by Sankyo Co. Ltd. (Tokyo, Japan). We are grateful to Ms Anne C. Feng (The Scripps Research Institute, CA, USA) for her technical assistance.
REFERENCES Anderson KM, Peach MJ (1994) Receptor binding and internalization of a unique biologically active angiotensin II-colloidal gold conjugate: morphological analysis of angiotensin II processing in isolated vascular strips. J Vasc Res 31:10 –17. Ando H, Zhou J, Macova M, Imboden H, Saavedra JM (2004) Angiotensin II AT1 receptor blockade reverses pathological hypertrophy and inflammation in brain microvessels of spontaneously hypertensive rats. Stroke 35:1726 –1731. Arenas IA, Xu Y, Lopez-Jaramillo P, Davidge ST (2004) Angiotensin II-induced MMP-2 release from endothelial cells is mediated by TNF-␣. Am J Physiol Cell Physiol 286:C779 –C784. Cipollone F, Fazia M, Iezzi A, Pini B, Cuccurullo C, Zucchelli M, de Cesare D, Ucchino S, Spigonardo F, De Luca M, Muraro R, Bei R, Bucci M, Cuccurullo F, Mezzetti A (2004) Blockade of the angiotensin II type 1 receptor stabilizes atherosclerotic plaques in humans by inhibiting prostaglandin E2-dependent matrix metalloproteinase activity. Circulation 109:1482–1488. Engelhorn T, Goerike S, Doerfler A, Okorn C, Forsting M, Heusch G, Schulz R (2004) The angiotensin II type 1-receptor blocker candesartan increases cerebral blood flow, reduces infarct size, and improves neurologic outcome after transient cerebral ischemia in rats. J Cereb Blood Flow Metab 24:467– 474. Gasche Y, Copin JC, Sugawara T, Fujimura M, Chan PH (2001) Matrix metalloproteinase inhibition prevents oxidative stress-associated blood-brain barrier disruption after transient focal cerebral ischemia. J Cereb Blood Flow Metab 21:1393–1400.
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Hamann GF, Okada Y, Fitridge R, del Zoppo GJ (1995) Microvascular basal lamina antigens disappear during cerebral ischemia and reperfusion. Stroke 26:2120 –2126. Hosomi N, Lucero J, Heo JH, Koziol JA, Copeland BR, del Zoppo GJ (2001) Rapid differential endogenous plasminogen activator expression after acute middle cerebral artery occlusion. Stroke 32:1341–1348. Hosomi N, Mizushige K, Kitadai M, Ohyama H, Ichihara SI, Takahashi T, Matsuo H (1999) Induced hypertension treatment to improve cerebral ischemic injury after transient forebrain ischemia. Brain Res 835:188 –196. Kim GW, Gasche Y, Grzeschik S, Copin JC, Maier CM, Chan PH (2003) Neurodegeneration in striatum induced by the mitochondrial toxin 3-nitropropionic acid: role of matrix metalloproteinase-9 in early blood-brain barrier disruption? J Neurosci 23:8733– 8742. Lin TN, He YY, Wu G, Khan M, Hsu CY (1993) Effect of brain edema on infarct volume in a focal cerebral ischemia model in rats. Stroke 24:117–121. McGraw CP (1977) Experimental cerebral infarction effects of pentobarbital in Mongolian gerbils. Arch Neurol 34:334 –336. Nishimura Y, Ito T, Hoe K, Saavedra JM (2000a) Chronic peripheral administration of the angiotensin II AT(1) receptor antagonist candesartan blocks brain AT(1) receptors. Brain Res 871:29 –38. Nishimura Y, Ito T, Saavedra JM (2000b) Angiotensin II AT(1) blockade normalizes cerebrovascular autoregulation and reduces cerebral ischemia in spontaneously hypertensive rats. Stroke 31: 2478 –2486. Nishiyama A, Seth DM, Navar LG (2002) Renal interstitial fluid concentrations of angiotensins I and II in anesthetized rats. Hypertension 39:129 –134. Nishiyama A, Seth DM, Navar LG (2003) Angiotensin II type 1 receptor-mediated augmentation of renal interstitial fluid angiotensin II in angiotensin II-induced hypertension. J Hypertens 21:1897–1903. Oliveira-Filho J, Silva SC, Trabuco CC, Pedreira BB, Sousa EU, Bacellar A (2003) Detrimental effect of blood pressure reduction in the first 24 hours of acute stroke onset. Neurology 61:1047–1051. Partridge CA, Jeffrey JJ, Malik AB (1993) A 96-kDa gelatinase induced by TNF-alpha contributes to increased microvascular endothelial permeability. Am J Physiol 265:L438 –L447. Re F, Zanetti A, Sironi M, Polentarutti N, Lanfrancone L, Dejana E, Colotta F (1994) Inhibition of anchorage-dependent cell spreading triggers apoptosis in cultured human endothelial cells. J Cell Biol 127:537–546. Rosenberg GA, Navratil M, Barone F, Feuerstein G (1996) Proteolytic cascade enzymes increase in focal cerebral ischemia in rat. J Cereb Blood Flow Metab 16:360 –366. Schrader J, Lüders S, Kulschewski A, Berger J, Zidek W, Treib J, Einhäupl K, Diener HC, Dominiak P (2003) The ACCESS study: evaluation of Acute Candesartan Cilexetil Therapy in Stroke Survivors. Stroke 34:1699 –1703. Wang JM, Tan J, Leenen FH (2003) Central nervous system blockade by peripheral administration of AT1 receptor blockers. J Cardiovasc Pharmacol 41:593–599. Yamakawa H, Jezova M, Ando H, Saavedra JM (2003) Normalization of endothelial and inducible nitric oxide synthase expression in brain microvessels of spontaneously hypertensive rats by angiotensin II AT1 receptor inhibition. J Cereb Blood Flow Metab 23:371–380. Yoshida J, Yamamoto K, Mano T, Sakata Y, Nishikawa N, Miwa T, Hori M, Masuyama T (2003) Angiotensin II type 1 and endothelin type A receptor antagonists modulate the extracellular matrix regulatory system differently in diastolic heart failure. J Hypertens 21:437–444. Zea-Longa E, Weinstein PR, Carlson S, Cummins R (1989) Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20:84 –91.
(Accepted 26 March 2005)
ANGIOTENSIN TYPE 1 RECEPTOR BLOCKAGE IMPROVES ISCHEMIC INJURY FOLLOWING TRANSIENT FOCAL CEREBRAL ISCHEMIA N. HOSOMI,a* A. NISHIYAMA,b C. R. BAN,a T. NAYA,a T. TAKAHASHI,a M. KOHNOa AND J. A. KOZIOLc
I.v. infusion of angiotensin II following transient forebrain ischemia increases cerebral edema and mortality (Hosomi et al., 1999). Furthermore, prior angiotensin type 1 (AT1) receptor blockage in the rat brain improves neurological outcomes and infarct volumes in cerebral ischemia (Nishimura et al., 2000b; Yamakawa et al., 2003). Nevertheless, the tissue-related contributions of angiotensin II to cerebrovascular and neuronal injury during cerebral ischemia have not been detailed. Recently, Ando et al. (2004) showed that AT1 receptor blocker had an anti-inflammatory effect on cerebral vessels in hypertension. Angiotensin II has been reported to be a potent stimulator of the expression of matrix metalloproteinases (MMPs), which degrade the basal lamina constituents (Arenas et al., 2004). The potential of angiotensin II to increase cerebral edema can be mediated by the expression of MMPs. MMP-2 and MMP-9 have been reported to be upregulated in the ischemic area at different time points in acute stroke (Rosenberg et al., 1996). MMP-9 may contribute to increased vascular endothelial permeability through the degradation of extracellular matrix components (Partridge et al., 1993). We have hypothesized that the stimulation of MMP expression with angiotensin II may be associated with this mechanism, since basal lamina cleavage with MMPs is involved in the induction of cerebral microvascular permeability following cerebral ischemia (Gasche et al., 2001; Kim et al., 2003). The major goals of the present study were to ascertain whether brain angiotensin II is upregulated in our experimental model of focal cerebral ischemia, and to quantify the effects of olmesartan, an AT1 receptor blocker, on ischemic injury in the model.
a Second Department of Internal Medicine, Division of Stroke, Kagawa University School of Medicine, 1750-1 Ikenobe, Miki-cho, Kagawa 761-0793, Japan b Department of Pharmacology, Kagawa University School of Medicine, Kagawa 761-0793, Japan c
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA
Abstract—Following cerebral ischemia, i.v. infusion of angiotensin II increases cerebral edema and mortality. Angiotensin type 1 receptor blockage should therefore improve acute cerebral ischemia. Left middle cerebral artery occlusion (120 min) followed by reperfusion was performed with the thread method under halothane anesthesia in Sprague–Dawley rats. Olmesartan (angiotensin type 1 receptor blocker; 0.01 or 0.1 mol/ kg/h) was infused i.p. for 7 days following middle cerebral artery occlusion followed by reperfusion. Stroke index score, infarct volume, specific gravity, and brain angiotensin II and matrix metalloproteinases were quantified in the ischemic and non-ischemic hemispheres. Olmesartan treatment improved stroke index score, infarct volume, and cerebral edema in our cerebral ischemia model. In particular, stroke index score, infarct volume, and cerebral edema were reduced even with a low dose of olmesartan that did not decrease blood pressure. Paralleling these effects on cerebral ischemia, olmesartan treatment also reduced the reactive upregulation in brain angiotensin II, matrix metalloproteinase-2, matrix metalloproteinase-9, and membrane type 1-matrix metalloproteinase in the ischemic area. Angiotensin type 1 receptor stimulation may be one of the important factors that cause cerebral edema following cerebral ischemia, and that its inhibition may be of therapeutic advantage in cerebral ischemia. © 2005 Published by Elsevier Ltd on behalf of IBRO.
EXPERIMENTAL PROCEDURES
Key words: cerebral ischemia, cerebral edema, angiotensin II, matrix metalloproteinases.
One hundred twenty-two male Sprague–Dawley (SD) rats were obtained from Clea Japan, Inc. (Tokyo, Japan). Our experiments were conducted when the animals were 6 –7 weeks old (weights 180 –250 g). The experimental procedures were approved by the Institutional Animal Care and Use Committee in Kagawa University School of Medicine and were in accordance with the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health. The protocol was planned with the care to minimize the number of animals used and their suffering. Focal cerebral ischemia and reperfusion in the SD rats were induced according to procedures described by Zea-Longa et al. (1989). Briefly, each animal was anesthetized with 4% halothane and spontaneously respired with 1% halothane in a 2:1 N2O/O2 mixture, with the use of a facemask. Under the operating microscope, a 30-mm segment of 4-0 ethylon monofilament, its tip rounded by heating, was gently introduced from the external carotid artery into the internal carotid artery lumen until a slight resistance was felt and the skin incision closed. The rats were reanesthetized and the filament was withdrawn 120 min after induction of ischemia. Animals
Cerebral ischemia is responsible for the loss of microvascular integrity, a consequence of the loss of basal lamina matrix ligands. The major basal lamina constituents decrease roughly in parallel during experimental middle cerebral artery occlusion (MCA:O) followed by reperfusion (MCA:O/R) (Hamann et al., 1995). This disruption of normal cell-matrix adhesion has been associated with cell death, edema, and loss of cell viability (Re et al., 1994). *Corresponding author. Tel: ⫹81-87-891-2150; fax: ⫹81-87-891-2152. E-mail address: [email protected] (N. Hosomi). Abbreviations: AT1, angiotensin type 1; MCA:O, middle cerebral artery occlusion; MCA:O/R, middle cerebral artery occlusion followed by reperfusion; MMP, matrix metalloproteinase; MT, membrane type; SD, Sprague–Dawley. 0306-4522/05$30.00⫹0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2005.03.054
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were then returned to their cages and closely monitored until they recovered from anesthesia. In sham-operated rats, the external carotid artery was surgically prepared for insertion of the filament, but the filament was not inserted. Rectal temperature was monitored continuously and maintained between 36.5 °C and 37.5 °C throughout the anesthesia via a feedback-regulated heating pad placed underneath the rat. Fourteen animals died within 4 h after reperfusion following MCA:O. These were considered operation failures and excluded from all analyses. Animals that survived longer than 4 h after reperfusion had fairly constant significant cerebral infarction with 2,3,5-triphenyltetrazolium chloride staining.
Experimental protocols This study consisted of three parts, each involving the administration of olmesartan or its vehicle as a continuous i.p. injection via an osmotic mini-pump. In this regard, an osmotic mini-pump (Model No. 201, ALZET, Cupertino, CA, USA) was filled with the vehicle (2.5% NaHCO3) or olmesartan (AT1 receptor blocker; Sankyo Co., Tokyo, Japan) and was inserted into the abdomen of experimental animals, and the wound was sutured. The first part of this study was a preliminary dose-finding experiment. Olmesartan at 0.001, 0.01, 0.1, and 1 mol/kg/h and its vehicle as control were administered continuously for 7 days in different groups of the non-ischemic SD rats (n⫽5 per group). During the experimental period, blood pressures were monitored using the tail cuff method. The experiment was terminated 7 days after the osmotic mini-pump implantation under halothane anesthesia by left ventricular transcardiac perfusion of the cranial structures with chilled (4 °C) saline perfusate containing heparin (200 IU/l), nitroprusside (1 mg/l), and bovine serum albumin (50 g/l). Brain perfusion was regarded as complete by the visual absence of blood and the absence of blood elements on microscopic inspection. The removed brains were quickly frozen in 2-methylbutane/dry ice for subsequent quantification of tissue angiotensin II. In the second part of this study, osmotic mini-pumps were implanted just after reperfusion following MCA:O, and olmesartan at 0.01 or 0.1 mol/kg/h or its vehicle was administered continuously from the onset of reperfusion (n⫽12 animals per group). Again, blood pressures were monitored during the experimental period using the tail cuff method. Stroke index scores were evaluated 7 days after MCA:O/R. The experiment was then terminated with decapitation under halothane anesthesia, and infarct volumes (n⫽5 animals per group) and specific gravity measurements (n⫽7 animals per group) were made. In the last part of this study, olmesartan at 0.01 or 0.1 mol/ kg/h or its vehicle was administered as described in the second part of this study. The experiments were terminated 12 h or 7 days after MCA:O/R under halothane anesthesia by left ventricular transcardiac perfusion as described above. The removed brains were quickly frozen in 2-methylbutane/dry ice for subsequent quantification of brain angiotensin II or MMP-2, MMP-9, and membrane type (MT)1-MMP (n⫽7 for each time point, respectively). Plasma was collected at the termination of the experiment.
Stroke index score To determine neurological deficits, neurological examinations were performed at 7 days after reperfusion in the second experiment, as mentioned above. The animals were evaluated for a decrease in alertness and movement, ptosis, cocked head, circling behavior, splayed or rotated limbs, seizures, and tremor. Each symptom was assigned a numerical weight, which was used in calculating the stroke index score (McGraw, 1977). The stroke index was weighted as hair roughed up or tremor: 1, obtunded: 1, paucity of movements: 1, head cocked: 3, eye fixed open: 3, ptosis: 2, a splayed out hind limb: 3, extreme rotation of hind: 3, circling behavior: 3, seizures: 2, rolling seizure: 3, extreme weak-
ness: 6, and death: grand sum⫹3. The maximum score was 34, in deceased animals.
Infarct volume Each brain was cut into 2-mm-thick coronal slices with a rodent brain matrix. The extent of ischemic infarction was revealed by reaction with a 2% solution of 2,3,5-triphenyltetrazolium chloride (Sigma Chemical, St. Louis, MO, USA) for 20 –30 min. The border between infarct and non-infarct tissue was outlined with an image analysis system (Image 1.3: National Institute of Mental Health, Bethesda, MD, USA), and the area of infarction was measured by subtracting the area of the non-lesioned ipsilateral hemisphere from that of the contralateral side (Lin et al., 1993). The volume of infarction was calculated by integration of the lesion areas.
Cerebral specific gravity Cerebral specific gravity, an index marker of cerebral edema, was determined at the cortex and caudate putamen by microgravimetry as described previously (Hosomi et al., 1999).
Brain and plasma angiotensin II Brain and plasma angiotensin II levels were measured by radioimmunoassay, as previously described (Nishiyama et al., 2002).
Brain MMP-2, MMP-9, MT1-MMP Brain was minced and homogenized in lysis buffer (50 mmol/l Tris–HCl [pH 7.6], 1.5 mmol/l NaCl, 0.5 mmol/l CaCl2, 1 mol/l ZnCl2, 1.0% Triton X-100, 0.01% Brij 35, and 0.25% Triton X-100) on ice and centrifuged at 4 °C for 20 min at 9000 r.p.m. Supernatants were divided into aliquots and stored at ⫺80 °C. Preliminary experiments determined the optimal conditions for homogenization and activity extraction. MMP-2, MMP-9, and MT1-MMP in brain extract were determined by commercially available activity assay system kits (MMP-2, MMP-9, and MT1-MMP; Amersham Biosciences UK Limited; Buckinghamshire, UK). Assays were performed according to the manufacturer’s instructions. Concentrations were calculated in nanograms per milligram protein. The linearity ranges for the assays were 0.19 –3 ng/ml in MMP-2, 0.125– 4 ng/ml in MMP-9, and 0.125– 4 ng/ml in MT1-MMP. Both latent and active forms of the MMPs are detected in the assays. The assays are applicable to different species, but not to other MMPs. The protein contents of tissue and plasma samples were determined according to the Bradford method with bovine serum albumin as the standard (Hosomi et al., 2001). The reproducibility of the angiotensin II, MMP-2, MMP-9, and MT1-MMP assays was reasonably good: coefficient of variation of angiotensin II, MMP-2, MMP-9, and MT1-MMP measurements was 8.8%, 9.2%, 9.6%, and 8.2% respectively.
Statistical analysis Summary statistics are expressed as the mean⫾standard deviation. Differences in changes in blood pressure among groups were examined using repeated measures analysis of variance (ANOVA). Differences in blood pressure at each time point, infarct volume, and specific gravity among groups were examined using one way-ANOVA. Differences in brain angiotensin II, MMP-2, MMP-9, and MT1-MMP among groups were examined using two way-ANOVA. When the overall ANOVA P-value was ⬍0.05, Bonferroni’s correction for multiple comparisons was then used to assess individual group differences. Overall differences in stroke index scores among groups were examined using the KruskalWallis test, followed by Mann-Whitney U tests for individual comparisons. Differences between the ischemic and non-ischemic hemispheres were examined using paired t-tests.
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Fig. 1. Time courses of mean blood pressure in vehicle rats olmesartan rats at two dose levels (0.01 and 0.1 and mol/kg/h); n⫽12 rats per cohort. O-1, 1 h after MCA:O; O-2, 2 h after MCA:O; Pre, preliminary; R-1h, 1 h after MCA:O/R; R-4h, 4 h after MCA:O/R; R-1d, 1 day after MCA:O/R; R-4d, 4 days after MCA:O/R; R-7d, 7 days after MCA:O/R. ‡ P⬍0.001 to the vehicle and olmesartan (0.01 mol/kg/h) rats with Bonferroni’s correction following repeated measure ANOVA.
RESULTS In the dose-finding experiment, mean blood pressures decreased significantly in the 0.1 and 1 mol/kg/h cohorts (F4,20⫽7.760, P⬍0.001 with repeated measure-ANOVA, P⬍0.005 with Bonferroni’s correction, n⫽5; respectively), but did not decrease in the 0.01 and 0.001 mol/kg/h cohorts. Brain angiotensin II was decreased in a dosedependent manner relative to olmesartan infusion dose (F4,20⫽10.294, P⬍0.001 with one way-ANOVA, n⫽5 per cohort). In subsequent experiments examining the effects of olmesartan on cerebral ischemic injury, we therefore chose the doses of olmesartan in 0.1 mol/kg/h, the minimum dose to decrease blood pressure, and 0.01 mol/ kg/h, the maximal dose not to decrease blood pressure. In the first MCA:O/R experiments, mean blood pressure did not decrease in the olmesartan (0.01 mol/kg/h) rats through the experimental period. Mean blood pressure started to decrease in the olmesartan (0.1 mol/kg/h) rats from 1 h after MCA:O/R, reached to the significantly lower level at 4 h after MCA:O/R compared with the vehicle and olmesartan (0.01 mol/kg/h) rats, and was stable afterward through the experimental period (F2,33⫽10.512, P⬍0.001 with repeated measure-ANOVA, P⬍0.001 with Bonferroni’s correction, n⫽12; respectively; Fig. 1). Stroke index scores at 7 days after MCA:O/R were significantly decreased in both olmesartan (0.01 and 0.1 mol/kg/h) rats (P⬍0.001 with Mann-Whitney U test, n⫽12; respectively; Fig. 2). There was no significant difference in stroke index score between the olmesartan (0.01 and 0.1 mol/kg/h) rats. Infarct volumes at 7 days after MCA:O/R were significantly reduced in both olmesartan (0.01 and 0.1 mol/kg/h) rats (F2,12⫽8.110, P⬍0.01 with one way-ANOVA, P⫽0.012 and 0.002 with Bonferroni’s correction, n⫽5; respectively; Fig. 3). There was no significant difference in infarct volume between the olmesartan (0.01 and 0.1 mol/kg/h) rats. In all three cohorts, specific
gravity, a marker of cerebral edema, significantly decreased in both the cortex and the caudate putamen in the infarct areas compared with the non-ischemic hemispheres (P⬍0.01 or 0.001 with paired t-test, n⫽7; Fig. 4). When comparing cohorts, specific gravity was significantly higher at both olmesartan doses (0.01 and 0.1 mol/kg/h) in the infarct areas of both the cortex (F2,18⫽6.307, P⬍0.01 with one way-ANOVA, P⫽0.010 and 0.004 with Bonferroni’s correction, n⫽7; respectively) and the caudate putamen (F2,18⫽9.795, P⬍0.005 with one way-
Fig. 2. Stroke index score of vehicle rats and olmesartan rats (0.01 and 0.1 mol/kg/h) 7 days after MCA:O/R (n⫽12 per cohort). The bar showed the median value. * P⬍0.001 to the vehicle rats with MannWhitney U test following Kruskal-Wallis test.
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Fig. 3. Infarct volumes of vehicle rats and olmesartan rats (0.01 and 0.1 mol/kg/h) 7 days after MCA:O/R (n⫽5 per cohort). : mean⫾standard deviation, * P⬍0.05, ** P⬍0.01 to the vehicle rats with Bonferroni’s correction following one-way ANOVA.
ANOVA, P⫽0.007 and P⬍0.001 with Bonferroni’s correction, n⫽7; respectively). In the vehicle rats, brain angiotensin II increased 12 h after MCA:O/R in the ischemic hemisphere compared with the non-ischemic hemisphere (P⬍0.001 with paired t-test, n⫽7; Fig. 5), and decreased to the similar level in the non-ischemic hemisphere 7 days after MCA:O/R. In the
olmesartan (0.01 mol/kg/h) rats, angiotensin II was elevated 12 h after MCA:O/R in the ischemic hemispheres relative to the non-ischemic hemispheres (P⫽0.008 with paired t-test, n⫽7), and decreased to the similar level in the non-ischemic hemisphere 7 days after MCA:O/R. There were significant dose-dependent declines in angiotensin II relative to the vehicle in both hemispheres at 12 h after MCA:O/R, maintained out to 7 days after MCA:O/R (F2,36⫽446.566, P⬍0.001 with two-way ANOVA, n⫽7 per cohort at each time point). Plasma angiotensin II was 12.6⫾2.4 fmol/g protein and did not change over the time course of the experiment in the vehicle rats. In the olmesartan (0.01 and 0.1 mol/kg/h) rats, plasma angiotensin II increased to 18.4⫾3.1 and 31.4⫾2.4 fmol/g protein at 12 h after reperfusion (F2,18⫽102.9, P⬍0.001 with one-way ANOVA, n⫽7 per cohort). In the vehicle rats, brain MMP-2 significantly increased in the ischemic hemispheres at 7 days after MCA:O/R (P⫽0.012 with paired t-test, n⫽7; Fig. 6) but not at 12 h after MCA:O/R compared with the non-ischemic hemispheres. There were significant increases in MMP-9 and MT1-MMP at 12 h after MCA:O/R (P⫽0.017 and 0.022 with paired t-test, n⫽7; respectively) but not at 7 days after MCA:O/R. In olmesartan rats at dose 0.01 and 0.1 mol/ kg/h, there was no significant elevation in MMP-2, MMP-9 or MT1-MMP in the ischemic hemispheres relative to the non-ischemic hemispheres, at either 12 h or 7 days after MCA:O/R, except MMP-2 at 7 days after MCA:O/R in the olmesartan (0.01 mol/kg/h) rats (P⬍0.05 with paired t-test, n⫽7). Lastly, there were significant dose-dependent reductions in MMP-2 levels in the olmesartan rats com-
Fig. 4. Specific gravity in the cortex and caudate putamen of vehicle rats and olmesartan rats (0.01 and 0.1 mol/kg/h) 7 days after MCA:O/R (n⫽7 per cohort). White circle: non-ischemic hemisphere (Non-I); Black circle: ischemic hemisphere (I); : mean⫾standard deviation, ⫹ P⬍0.01, ⫹⫹ P⬍0.001 to the non-ischemic hemisphere with paired t-test. * P⬍0.05, ** P⬍0.01, *** P⬍0.001 to the vehicle rats with Bonferroni’s correction following one-way ANOVA.
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Fig. 5. Brain angiotensin II levels 12 h (12H) and 7 days (7D) after MCA:O/R; n⫽7 animals for each cohort at each time point. I, ischemic hemisphere; Non-I, non-ischemic hemisphere. : mean⫾standard deviation, ⫹ P⬍0.01, ⫹⫹ P⬍0.001 to the non-ischemic hemisphere with paired t-test. * P⬍0.001 to the vehicle rats, † P⬍0.001 to the olmesartan rats (0.01 mol/kg/h) with Bonferroni’s correction following two-way ANOVA.
pared with the vehicle rats at 7 days after MCA:O/R (F2,18⫽7.193, P⫽0.005 with one-way ANOVA, n⫽7 per cohort), and in MMP-9 and MT1-MMP both at 12 h (F2,18⫽17.850, P⬍0.001 and F2,18⫽5.159, P⫽0.017, n⫽7 per cohort; respectively) and at 7 days (F2,18⫽10.545, P⬍0.001 and F2,18⫽3.918, P⫽0.039, n⫽7 per cohort; respectively) after MCA:O/R in the ischemic hemispheres, but not in MMP-2 at 12 h after MCA:O/R (F2,18⫽0.442, P⫽0.650, n⫽7 per cohort).
DISCUSSION In our cerebral ischemia model, olmesartan treatment improved stroke index scores, infarct volumes, and cerebral edema at two dose levels: a high dose, which decreased blood pressures, and a low dose, which did not. Paralleling these effects on cerebral ischemia, olmesartan treatment at both dose levels reduced the reactive upregulation in brain angiotensin II, MMP-2, MMP-9, and MT1-MMP in the ischemic area. In the present study, we found that brain angiotensin II was upregulated 12 h after MCA:O/R in the ischemic region. Since plasma angiotensin II levels were around 40% of the antigen content of the non-ischemic hemispheres over the course of the experiment, cerebral edema that causes transudation of plasma angiotensin II cannot account for the increased levels in the ischemic hemispheres. Furthermore, brain angiotensin II was decreased with AT1 receptor blocker in a dose-dependent fashion in both the ischemic and the non-ischemic hemispheres. Conversely, plasma angiotensin II increased in the olmesartan-treated rats, as previously reported (Nishiyama et al., 2003). Since plasma angiotensin II levels were around 300% of the antigen content of non-ischemic and ischemic
hemisphere over the experiment, plasma transudation with cerebral edema cannot account for the decreases of angiotensin II in the olmesartan- (0.1 mol/kg/h) treated rats. The binding of angiotensin II to AT1 receptor initiates internalization of the complex in vascular smooth muscle cells (Anderson and Peach, 1994). Interestingly, AT1 receptor plays a role in the increased intrarenal angiotensin II levels observed in angiotensin II dependent hypertension (Nishiyama et al., 2003). The present study suggests that AT1 receptor-mediated angiotensin II augmentation from plasma or brain local angiotensin II synthesis may similarly occur in the brain. There have been various reports of the effects of peripheral administration of AT1 receptor blockers (Nishimura et al., 2000a; Wang et al., 2003). Several studies of pretreatment with AT1 receptor blockers in experimental cerebral ischemia have demonstrated that renin-angiotensin system inhibition decreased infarct volumes and improved neurological outcomes (Nishimura et al., 2000b; Yamakawa et al., 2003). In a previous study, we found slight blood pressure reduction along with increased cerebral edema and mortality following transient forebrain ischemia (Hosomi et al., 1999). Hence the blood pressure reduction with post-ischemic AT1 receptor blocker treatment in the present study does not account for the improved neurological behavior, infarct volume, and cerebral edema following cerebral ischemia. Recently, Engelhorn et al. (2004) demonstrated that chronic post-treatment with AT1 receptor blockers improved infarct sizes and neurological behavior after transient cerebral ischemia. Our results are in qualitative agreement: olmesartan post-treatment also improved neurological behavior and infarct volumes following MCA:O/R, even at a high dose that decreased sys-
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Fig. 6. Brain MMP-2, MMP-9, and MT1-MMP levels at 12 h (12H) and 7 days (D7) after MCA:O/R. I, ischemic hemisphere; Non-I, non-ischemic hemisphere. : mean⫾standard deviation, ⫹ P⬍0.05 to the non-ischemic hemisphere with paired t-test. * P⬍0.05, *** P⬍0.001 to the vehicle rats with Bonferroni’s correction following two-way ANOVA.
temic blood pressure. Recently, it has been reported that AT1 receptor inhibition reversed the pathological vascular hypertrophy, normalized endothelial nitric oxide synthase expression, and decreased intercellular adhesion molecule-1 expression and the number of infiltrating macrophages in cerebral vessels of hypertensive rats (Ando et al., 2004). These anti-inflammatory effects of AT1 receptor blocker may be an important mechanism in protecting against cerebral ischemia. I.v. infusion of angiotensin II increases cerebral edema and mortality following cerebral ischemia (Hosomi et al.,
1999). The deleterious effect on cerebral edema can be counteracted by treatment with an AT1 receptor blocker. Whether angiotensin II increases MMP expression through AT1 receptor stimulation or another mechanism is not definitively known. Angiotensin II induces MMP-2 and MMP-9 secretion from endothelial cells (Arenas et al., 2004). Treatment with an AT1 receptor blocker results in decreased MMP and mRNA levels in carotid plaques under hypertensive diastolic heart failure (Yoshida et al., 2003; Cipollone et al., 2004). The secretions of MMP-2, MMP-9, and MT1-MMP were not dependent on AT1 receptor stim-
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ulation, since olmesartan treatment could not decrease MMP-2, MMP-9 and MT1-MMP levels in the non-ischemic hemispheres. Interestingly, the over-expressions of brain MMP-2, MMP-9, and MT1-MMP were dependent on AT1 receptor stimulation following cerebral ischemia, since these were reduced with AT1 receptor blockage. In this regard, MMPs may play important roles in the interplay between angiotensin II and cerebral edema. Blood pressure reduction in the first 24 h after stroke onset is an independent risk factor for poor outcomes as assessed by Rankin or Barthel scores 3 months after acute ischemic stroke (Oliveira-Filho et al., 2003). The ACCESS study demonstrated a reduction in mortality in patients with acute ischemic stroke who received an AT1 receptor blocker for acute blood pressure treatment, despite the absence of a significant effect on blood pressures compared with placebo (Schrader et al., 2003). In the present study, olmesartan treatment at a low dose was able to reduce infarct volumes and cerebral edema without decreasing blood pressures. Olmesartan treatment at the high dose [an amount equivalent to clinical usage] demonstrated the same clinical benefits of reduced infarct volumes and cerebral ischemia, but did decrease blood pressures. In translation to the clinic, one would initially need to establish a dosage regimen for an AT1 receptor blocker that would not induce any reduction in blood pressure, prior to investigating its therapeutic potential in acute cerebral ischemia. Acknowledgment—This study was supported by Sankyo Co. Ltd. (Tokyo, Japan). We are grateful to Ms Anne C. Feng (The Scripps Research Institute, CA, USA) for her technical assistance.
REFERENCES Anderson KM, Peach MJ (1994) Receptor binding and internalization of a unique biologically active angiotensin II-colloidal gold conjugate: morphological analysis of angiotensin II processing in isolated vascular strips. J Vasc Res 31:10 –17. Ando H, Zhou J, Macova M, Imboden H, Saavedra JM (2004) Angiotensin II AT1 receptor blockade reverses pathological hypertrophy and inflammation in brain microvessels of spontaneously hypertensive rats. Stroke 35:1726 –1731. Arenas IA, Xu Y, Lopez-Jaramillo P, Davidge ST (2004) Angiotensin II-induced MMP-2 release from endothelial cells is mediated by TNF-␣. Am J Physiol Cell Physiol 286:C779 –C784. Cipollone F, Fazia M, Iezzi A, Pini B, Cuccurullo C, Zucchelli M, de Cesare D, Ucchino S, Spigonardo F, De Luca M, Muraro R, Bei R, Bucci M, Cuccurullo F, Mezzetti A (2004) Blockade of the angiotensin II type 1 receptor stabilizes atherosclerotic plaques in humans by inhibiting prostaglandin E2-dependent matrix metalloproteinase activity. Circulation 109:1482–1488. Engelhorn T, Goerike S, Doerfler A, Okorn C, Forsting M, Heusch G, Schulz R (2004) The angiotensin II type 1-receptor blocker candesartan increases cerebral blood flow, reduces infarct size, and improves neurologic outcome after transient cerebral ischemia in rats. J Cereb Blood Flow Metab 24:467– 474. Gasche Y, Copin JC, Sugawara T, Fujimura M, Chan PH (2001) Matrix metalloproteinase inhibition prevents oxidative stress-associated blood-brain barrier disruption after transient focal cerebral ischemia. J Cereb Blood Flow Metab 21:1393–1400.
231
Hamann GF, Okada Y, Fitridge R, del Zoppo GJ (1995) Microvascular basal lamina antigens disappear during cerebral ischemia and reperfusion. Stroke 26:2120 –2126. Hosomi N, Lucero J, Heo JH, Koziol JA, Copeland BR, del Zoppo GJ (2001) Rapid differential endogenous plasminogen activator expression after acute middle cerebral artery occlusion. Stroke 32:1341–1348. Hosomi N, Mizushige K, Kitadai M, Ohyama H, Ichihara SI, Takahashi T, Matsuo H (1999) Induced hypertension treatment to improve cerebral ischemic injury after transient forebrain ischemia. Brain Res 835:188 –196. Kim GW, Gasche Y, Grzeschik S, Copin JC, Maier CM, Chan PH (2003) Neurodegeneration in striatum induced by the mitochondrial toxin 3-nitropropionic acid: role of matrix metalloproteinase-9 in early blood-brain barrier disruption? J Neurosci 23:8733– 8742. Lin TN, He YY, Wu G, Khan M, Hsu CY (1993) Effect of brain edema on infarct volume in a focal cerebral ischemia model in rats. Stroke 24:117–121. McGraw CP (1977) Experimental cerebral infarction effects of pentobarbital in Mongolian gerbils. Arch Neurol 34:334 –336. Nishimura Y, Ito T, Hoe K, Saavedra JM (2000a) Chronic peripheral administration of the angiotensin II AT(1) receptor antagonist candesartan blocks brain AT(1) receptors. Brain Res 871:29 –38. Nishimura Y, Ito T, Saavedra JM (2000b) Angiotensin II AT(1) blockade normalizes cerebrovascular autoregulation and reduces cerebral ischemia in spontaneously hypertensive rats. Stroke 31: 2478 –2486. Nishiyama A, Seth DM, Navar LG (2002) Renal interstitial fluid concentrations of angiotensins I and II in anesthetized rats. Hypertension 39:129 –134. Nishiyama A, Seth DM, Navar LG (2003) Angiotensin II type 1 receptor-mediated augmentation of renal interstitial fluid angiotensin II in angiotensin II-induced hypertension. J Hypertens 21:1897–1903. Oliveira-Filho J, Silva SC, Trabuco CC, Pedreira BB, Sousa EU, Bacellar A (2003) Detrimental effect of blood pressure reduction in the first 24 hours of acute stroke onset. Neurology 61:1047–1051. Partridge CA, Jeffrey JJ, Malik AB (1993) A 96-kDa gelatinase induced by TNF-alpha contributes to increased microvascular endothelial permeability. Am J Physiol 265:L438 –L447. Re F, Zanetti A, Sironi M, Polentarutti N, Lanfrancone L, Dejana E, Colotta F (1994) Inhibition of anchorage-dependent cell spreading triggers apoptosis in cultured human endothelial cells. J Cell Biol 127:537–546. Rosenberg GA, Navratil M, Barone F, Feuerstein G (1996) Proteolytic cascade enzymes increase in focal cerebral ischemia in rat. J Cereb Blood Flow Metab 16:360 –366. Schrader J, Lüders S, Kulschewski A, Berger J, Zidek W, Treib J, Einhäupl K, Diener HC, Dominiak P (2003) The ACCESS study: evaluation of Acute Candesartan Cilexetil Therapy in Stroke Survivors. Stroke 34:1699 –1703. Wang JM, Tan J, Leenen FH (2003) Central nervous system blockade by peripheral administration of AT1 receptor blockers. J Cardiovasc Pharmacol 41:593–599. Yamakawa H, Jezova M, Ando H, Saavedra JM (2003) Normalization of endothelial and inducible nitric oxide synthase expression in brain microvessels of spontaneously hypertensive rats by angiotensin II AT1 receptor inhibition. J Cereb Blood Flow Metab 23:371–380. Yoshida J, Yamamoto K, Mano T, Sakata Y, Nishikawa N, Miwa T, Hori M, Masuyama T (2003) Angiotensin II type 1 and endothelin type A receptor antagonists modulate the extracellular matrix regulatory system differently in diastolic heart failure. J Hypertens 21:437–444. Zea-Longa E, Weinstein PR, Carlson S, Cummins R (1989) Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20:84 –91.
(Accepted 26 March 2005)