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Departments of 1Neurosurgery and 2Internal Medicine, National Hospital Organization Saitama Hospital, Wako-city, Japan; 3Department of Neurosurgery, Saitama City Hospital, Saitama City, Japan; 4Graduate School of Science and Technology, Keio University, Yokohama, Japan; and 5Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana
Submitted 8 June 2006 ; accepted in final form 20 December 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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P-selectin; cerebral ischemia; reactive oxygen species; reduced nicotinamide adenine dinucleotide phosphate oxidase
Specific receptor antagonists for the AT1 receptor have been used to demonstrate a role for this receptor in different vascular beds exposed to ischemic or oxidative stress. For example, it has recently been reported that the AT1 receptor antagonist candesartan inhibits the exaggerated oxidative stress and tissue injury elicited by focal cerebral ischemia in diabetic mice (21). It has also been shown that ischemic stroke results in an increased plasma level of ANG II (20). Whereas the mechanisms underlying the ischemic tissue injury mediated in the brain by AT1 receptors remain undefined, data derived from other tissues suggest a potential role for AT1-dependent inflammatory and thrombogenic responses. This contention is based on recent reports describing AT1-antagonist-mediated attenuation of platelet-leukocyte-endothelial cell interactions in intestinal venules exposed to either hypercholesterolemia or I/R (27, 28). Additional support for AT1 receptors as mediators of the inflammatory and prothrombogenic phenotype elicited by gut I/R was obtained in AT1-receptor-deficient mice, which also exhibit reduced reperfusion-induced leukocyte and platelet adhesion (29).
The major objectives of this study were to define the role of AT1 receptors in mediating the recruitment of leukocytes and platelets in the cerebral microvasculature induced by either systemic ANG II administration or brain I/R and to define the molecular mechanisms that underlie the AT1-receptor-dependent inflammatory and prothrombogenic phenotype. These objectives were achieved by applying the techniques of intravital microscopy and two-color imaging of the brain microcirculation for quantification of platelet-leukocyte-endothelial cell interactions and oxidative stress in postcapillary venules. Our findings are consistent with an AT1 receptor signaling mechanism that involves oxygen radical production and ultimately results in P-selectin- and P-selectin glycoprotein ligand 1 (PSGL-1)-mediated platelet-leukocyte-endothelial cell interactions in the cerebral microcirculation.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Experiments were performed using male C57Bl/6J mice weighing 21–25 g. The animals were anesthetized with an intraperitoneal injection of 
-chloralose (60 mg/kg) and urethane (600 mg/kg), whereas lidocaine (1%) was employed for local anesthesia. All mice were tracheostomized with a polyethylene catheter (PE-90 Intramedic; Clay Adams, Parsippany, NJ) and allowed to breathe spontaneously. The femoral artery and vein were cannulated with a polyethylene catheter (PE-10 Intramedic, Clay Adams) to monitor mean arterial blood pressure and sample arterial blood for blood gas analysis and for intravenous administration of labeled platelets and rhodamine-6G. Rectal temperature was monitored and maintained at 36.5°–37.5°C with an overhead heat lamp throughout the experimental procedure.
Brain Preparation and Bilateral Common Carotid Artery Occlusion
The head of each mouse was fixed in a plastic frame in a sphinx position. The skull bone was exposed by a midline skin incision, followed by a craniectomy (diameter, 2 mm) using a drill at the parietal bone (ANG II model) or the frontal bone [experiments of bilateral common carotid artery occlusion (BCCAO)]. The dura mater was not cut when platelets and leukocytes were observed. A 12-mm glass coverslip was placed over the craniectomy, and the space between the glass and dura mater was filled with artificial cerebrospinal fluid. The dura mater was cut when oxygen radical production was determined during superfusion with dihydrorhodamine (DHR)-123 in the presence or absence of ANG II, candesartan, and losartan in ANG II model and experiments of BCCAO, respectively. The cranial window was closed with dental cement and a 7-mm glass coverslip to prevent leak of cerebrospinal fluid (15).
In the ischemic groups, ligatures were placed around the carotid arteries for either 30 or 60 min, followed by removal of the ligatures (14). The area of brain tissue under intravital microscopic observation experiences >90% reduction in blood flow (assessed by laser-Doppler flowmetry). The experimental procedures described above were reviewed and approved by the Saitama Hospital Animal Care and Use Committee.
Platelet Preparation
Approximately 0.9 ml of blood was harvested from the carotid artery of an untreated donor mouse. Platelets were isolated and labeled with carboxyfluorescein diacetate, succinimidyl ester (CFDASE; Molecular Probes, Eugene, OR) as previously described (14). Manual blood cell counts yielded <0.05% leukocytes in the platelet suspension. In each recipient mouse, 100 x 106 platelets (
30 min after final suspension) were infused over 5 min using a Harvard Apparatus (Econoflo, Holliston, MA) infusion pump, yielding 10% of the total platelet count. The platelets were allowed to circulate for a period of 5 min before being recorded (14). We have previously demonstrated using flow cytometric measurements of platelet P-selectin and GPIIb/IIIa that the isolation procedure produces minimal activation of platelets (32, 34). This assumption is supported by the minimal levels of platelet adhesion observed in the microcirculation of the brain (see RESULTS) and other tissues (32, 34).
Intravital Fluorescence Microscopy
An upright Sankei microscope equipped with a 3CCD camera (JK-TU52H, Toshiba, Japan) for cell adhesion experiments and a black and white CCD camera (model C2400–08, Hamamatsu Photonics, Hamamatsu, Japan) for DHR oxidation experiments as well as a xenon lamp were used to observe the cerebral microcirculation. With water immersion x20 and x40 objectives, the magnifications on the video screen (Sony, Tokyo, Japan) were x1,100 and x2,200, respectively. The images were collected on a video recorder (Sony) equipped with a video timer (VTG-10, FOR-A). Imaging for CFDASE, which forms the stable fluorochrome carboxyfluorescein succinimidyl ester (absorption peak, 492 nm; emission peak, 518 nm) after reaction with intracellular esterases, and rhodamine-6G (absorption peak of 525 nm and emission peak of 555 nm) required a filter block (Nikon, FITC/tetramethylrhodamine isothiocyanate C-FL, an excitation filter for 470–490 nm/540–570 nm; a dichroic mirror for 470–495 nm/540–570 nm and 495–550 nm/570–660 nm; and a barrier filter of 500–535 nm/580–625 nm). During the DHR oxidation experiments, another filter block (Nikon, G-2A, an excitation filter for 510–560 nm; a dichroic mirror for 575 nm and 590 nm; and a barrier filter of 590 nm) was used.
Video Analysis
The randomly selected venular segments evaluated for platelet and/or leukocyte adhesion were 30–40 µm in diameter and at least 100 µm in length. Platelets were classified according to the quality or duration of their interaction with the venular wall as either free-flowing, rolling, or adherent. Rolling platelets and leukocytes were defined as cells crossing the 100-µm venular segment at a velocity that is significantly lower than the center line velocity; their numbers are expressed as cells per 30 seconds per square millimeter. Diameter of the analyzed venules were between 25 and 40 µm. Adherent platelets and leukocytes were stationary for >2 s and >30 s, respectively. Total adherent platelets are divided into platelets directly bound to adherent leukocytes and platelets bound directly to endothelial cell. Rolling and adherent cells were expressed as the number of cells per square millimeter of venular surface, calculated from diameter and length, with cylindrical vessel shape assumed.
In a separate group of mice, the oxidant-sensitive fluorescent probe DHR-123 (10 µmol/l; Molecular Probes) was dissolved in artificial cerebrospinal fluid and superfused on the brain surface (the dura mater was cut to allow for direct exposure) in the closed cranial window. Brain tissue was exposed to DHR for 15 min, followed by suffusion with plain artificial cerebrospinal fluid for 5 min. DHR oxidation was visualized and quantified by using previously described procedures (15). DHR fluorescence intensities were monitored in three regions (outside of the vessels) of brain surface that were randomly selected rectangular areas (50 x 50 µm) in each mouse.
Experimental Protocols
After labeled platelets were infused into the recipient mouse, 50 µl of 0.02% rhodamine-6G (Sigma Chemicals, St. Louis, MO) were administered intravenously, followed by a continuous infusion (2 ml/h) of the fluorochrome at the same concentration for 5–10 min. The adhesive interactions of both platelets and leukocytes were monitored and recorded in five randomly selected venular segments.
Protocol 1: angiotensin II dose-dependent adhesion responses. Wild-type mice were assigned to one of four experimental groups: 1) 24 h after intraperitoneal injection of Val-ANG II (the AT1 receptor agonist, Sigma; 2.5 x 10–3, x 10–1, and x 10 nmol/0.5 ml PBS); 2) 4 h after intraperitoneal injection of Val-ANG II (2.5 x 10 nmol/0.5 ml PBS); 3) immediately after 60-min superfusion of Val-ANG II dissolved in artificial cerebrospinal fluid (50 µM, 15 ml/h); and 4) 24 h following sham surgery and intraperitoneal injection of 0.5 ml PBS.
Protocol 2: determinants of ANG II-mediated blood cell adhesion. In mice receiving Val-ANG II (as per group in protocol 1; 2.5 x 10 nmol), a monoclonal antibody directed against either P-selectin (RB40.34, 2 mg/kg) or PSGL-1 (2PH1, 2 mg/kg) was administered intravenously 30 min before quantification of blood cell adhesion (15). In three additional groups, mice were pretreated twice (1 h before the intraperitoneal injection of Val-ANG II and 1 h before the quantification of blood cell adhesion) with either the AT1-receptor antagonist candesartan (1 mg/kg, Takeda Chemical, Tokyo, Japan) or losartan (10 mg/kg, LKT Laboratories) or with diphenyleneiodonium (DPI, 2.5 mg/kg, Sigma), an inhibitor of flavoproteins including NAD(P)H oxidase.
Protocol 3: BCCAO and reperfusion. In an additional four groups, mice were exposed to 60 min of BCCAO followed by 4 h of reperfusion before quantification of platelet and leukocyte adhesion. In two groups, candesartan (1 mg/kg) or losartan (10 mg/kg) was administered 1 h before BCCAO and 1 h before quantification of blood cell adhesion. In a second group, DPI (2.5 mg/kg) was administered instead of candesartan or losartan. The fifth group was treated with PBS.
Protocol 4: DHR oxidation. DHR oxidation was monitored in four groups: 1) 24 h after intraperitoneal injection of Val-ANG II (2.5 x 10 nmol/0.5 ml saline); 2) 30 min after reperfusion following 30 min BCCAO; 3) candesartan (10 mg/kg) treatment of mice exposed to protocol 2; and 4) 24 h following sham surgery and intraperitoneal injection of 0.5 ml PBS.
Statistics
Data were analyzed using an analysis of variance and Fisher post hoc test. The data are reported as means ± SE. Statistical significance was set at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Angiotensin II induced the recruitment of both rolling and adherent leukocytes and platelets in venules of the brain surface. CFDASE-labeled (green) platelets adhered directly onto adherent rhodamine-6G-labeled (red) leukocytes or endothelial cells. Some free-flowing platelets were observed to suddenly bind to adherent leukocytes. Some of these platelets rolled on the leukocyte, and some adhered and then detached from the leukocyte, whereas others adhered firmly on leukocytes or onto endothelial cells. A few platelets adhered to other adherent platelets, and a few platelet-bearing leukocytes were seen rolling on endothelial cells, which were just observed but not counted because the numbers were few. These phenomena were clearly observed using two-color imaging of blood cells in the cerebral microcirculation (Fig. 1).
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The dose- and time-dependent effects of ANG II on leukocyte and platelet rolling and adhesion are shown in Fig. 2. Intraperitoneal administration of ANG II (10 x 2.5 nmol) elicited the recruitment of both rolling and adherent leukocytes and platelets. However, 4-h ANG II administration or superfusion of ANG II did not result in the adhesion of either leukocytes or platelets. Approximately 40% of platelet adhesion observed 24 h after administration of ANG II resulted from direct platelet-endothelial cell interactions, with the remaining 60% of adherent platelets being bound to adherent leukocytes (Fig. 3).
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No leukocytes and platelets were observed to either roll or adhere in cerebral arterioles. In contrast with the leukocyte and platelet interactions described above, the numbers of rolling and adherent leukocytes and platelets in venules were unaffected by 60 min after superfusion of ANG II, when compared with values obtained from a sham experiment.
Effects of Treatment With MAb Against P-Selectin or PSGL-1
The ANG II-induced recruitment of rolling leukocytes was significantly attenuated in P-selectin- or PSGL-1 MAb-treated mice when compared with the responses noted in untreated mice receiving the same dose of ANG II (Fig. 4). The number of adherent leukocytes after P-selectin or PSGL-1 MAb treatment remained elevated above the level detected in sham experiments, although adherent platelets on leukocytes were not detected in P-selectin MAb-treated mice, and they were attenuated significantly by the PSGL-1 MAb. Both the rolling and adherence of platelets were reduced significantly in the P-selectin- or PSGL-1 MAb-treated mice, especially platelets adhering to leukocytes.
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Rolling and adherent leukocytes and platelets were reduced significantly in mice receiving the AT1-receptor antagonist candesartan (1 mg/kg) or receiving losartan (10 mg/kg) except rolling platelets (Fig. 5). In DPI-treated mice, rolling and adherent platelets as well as rolling leukocytes and platelet-bearing adherent leukocytes were significantly reduced. However, total adherent leukocytes were not significantly altered.
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The increased number of rolling and adherent leukocytes and platelets induced by cerebral ischemia and reperfusion was significantly reduced in mice treated with either candesartan, losartan, or DPI (Fig. 6). Although the number of total adherent leukocytes tended to be reduced, this did not reach statistical significance in the losartan-treated mice.
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DHR oxidation, a measure of oxidative stress, was significantly increased in the cerebral microcirculation of mice subjected to BCCAO/R but not in mice receiving ANG II. Candesartan treatment significantly reduced the DHR oxidation elicited by BCCAO/R (Fig. 7).
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Arterial blood pH (7.279 ± 0.006), PO2 (110.6 ± 2.1 mmHg), and PCO2 (42.5 ± 0.9 mmHg) (obtained after platelet-leukocyte adhesion measurements) did not differ between the various experimental groups. Figure 8 illustrates that Val-ANG II elicited a dose-dependent increase in blood pressure, although a significant change was achieved at 2.5 x 10 nmol. The increased blood pressure was significantly attenuated by pretreatment with either candesartan, losartan, or DPI. Candesartan, losartan, and DPI also attenuated blood pressure in mice subjected to BCCAO/R.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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A signaling pathway for blood cell recruitment that has received much more attention in vascular beds other than in the brain is the renin-angiotensin system. For example, it has been shown that topical application of ANG II elicits leukocyte-endothelial interactions in mesenteric venules through engagement of AT1 receptors (30). Similarly, it has been reported that the leukocyte and platelet adhesion induced in venules of intestine and skeletal muscle by either hypercholesterolemia (27) or I/R (29) is attenuated in mice that are either treated with an AT1 receptor antagonist or that are genetically deficient in the receptor. The results of the present study provide the first evidence for ANG II-mediated adhesion of leukocytes and platelets in the cerebral microcirculation. Whereas superfusion of ANG II onto the brain surface does not elicit blood cell adhesion (presumably due to inaccessibility of the peptide to the intravascular compartment), as observed in mesenteric venules (30), a dose-dependent increase in adhesion was noted in cerebral venules 24 h after intraperitoneal ANG II administration. Also unlike in the mesenteric circulation, where ANG II mediates leukocyte adhesion in both arterioles and venules (2), cerebral arterioles were unresponsive to the proadhesive effects of ANG II. However, like the mesenteric circulation, we found that the proadhesive effects of ANG II in the brain microcirculation are dependent on the AT1 receptor, inasmuch as candesartan and losartan effectively blocked the recruitment of both platelets and leukocytes. The dependency of ANG II-induced blood cell adhesion on the AT1 receptor is consistent with previous reports that describe the expression of these receptors in the brain (20, 24) as well as a functional consequence of AT1 receptor blockade in the brain microcirculation (21). Whereas it is tempting to speculate from our data that the different adhesion responses of brain arterioles and venules to ANG II stimulation reflect comparable differences in the density of AT1 receptors, our previous work on hypercholesterolemia-induced blood cell adhesion indicates that AT1 receptors on circulating blood cells also contribute to the leukocyte (but not platelet) recruitment response (28).
We (16) have previously reported that ischemic stroke results in the accumulation of both leukocytes and platelets in cerebral venules. Whereas AT1 receptors have been implicated in reperfusion-induced leukocyte (31) and platelet adhesion in postischemic venules of the intestine, such a role for the AT1 receptor has been demonstrated in other regional vascular beds exposed to ischemia and reperfusion. This study provides the first evidence that AT1 receptors are involved in the recruitment of blood cells into the postischemic cerebral vasculature. AT1 receptor agonist Val-ANG II elicited a dose- and time-dependent increase in platelet-leukocyte-endothelial cell interactions, and I/R also elicited the same inflammatory responses, which were significantly reduced by two AT1 receptor inhibitors, candesartan and losartan. These results stress that AT1 receptor signaling is important for the I/R injury and may be important for other pathological conditions with which ANG II is associated. Our findings are consistent with reports describing increased circulating levels of ANG II after ischemic stroke (20) and suggest that the ANG II engages with AT1 receptors expressed on endothelial cells, leukocytes, and/or platelets. Our results may also explain the mechanism by which AT1 receptor antagonist affords protection against the cerebral infarction following ischemic stroke (9, 10, 19, 21, 23, 26, 33, 37).
The adhesion molecules that mediate leukocyte and platelet adhesion in the cerebral microcirculation appear to be model dependent. However, P-selectin:PSGL-1 interactions have been implicated as mediators of the adhesion associated with several models of human disease, including hypercholesterolemia (15) and ischemic stroke (14, 17). The results of the present study add further support for P-selectin:PSGL-1 interactions as mediators of both the leukocyte and platelet adhesion elicited by ischemic stroke. In addition, we provide novel data demonstrating that these adhesion molecules also mediate the platelet and leukocyte adhesive interactions induced by ANG II in cerebral venules. Our use of two-color imaging provides clear evidence that binding of platelets to leukocytes and binding of platelets to the venular wall are both dependent on P-selectin and PSGL-1. The observation that P-selectin mediates ANG II-induced leukocyte adhesion in venules is consistent with a report by Alvarez el al. (2), who demonstrated a similar role for P-selectin in mesenteric venules exposed to ANG II. Our observation that P-selectin:PSGL-1 interactions play a major role in mediating the blood cell adhesion responses in the brain elicited by both ANG II and I/R is consistent with the finding that AT1-receptor activation also contributes to the blood cell adhesion induced by the two stimuli. Taken together, the results of this study are consistent with a mechanism wherein ischemic stroke elicits the engagement/activation of AT1 receptors on endothelial cells, platelets, and/or leukocytes, which, in turn, results in an increased expression of P-selectin on the surface of cerebral endothelial cells and circulating platelets. The P-selectin on these cells then can engage with its counter-receptor PSGL-1 that is constitutively expressed on leukocytes, thereby resulting in the leukocyte-endothelial cell adhesion and platelet-leukocyte adhesion that is observed in postischemic cerebral venules.
There is a large body of evidence that implicates oxidative stress in the inflammatory responses that result from activation of the AT1 receptor on vascular endothelial cells (25). Endothelial cells exposed to ANG II exhibit an accelerated rate of production of reactive oxygen metabolites, which is linked to activation of endothelial NAD(P)H oxidase (6). AT1 receptor antagonists block the increased ROS production elicited by ANG II, which indicates that engagement of the AT1 receptor by ANG II on endothelial cells results in the activation of NADPH oxidase and the subsequent accelerated formation of ROS. The production of ROS by platelets is also enhanced by ANG II, a response that is blocked by either an AT1 receptor antagonist or DPI, which inhibits flavoproteins such as NADPH oxidase (11). The results of our study indicate that, whereas ANG II does not induce an oxidative stress outside of cerebral venules (measured by DHR oxidation), ischemic stroke does produce a detectable increase in ROS production. DPI was effective in attenuating the I/R-induced production in ROS, and it also significantly attenuated the blood cell adhesion responses elicited by I/R. This agent also was quite effective in blunting the adhesion responses induced by ANG II. The protective effects of DPI may reflect an action of this drug on NADPH oxidase or it may relate to inhibition of a different flavoprotein. There are no known NADPH oxidase inhibitors with high specificity currently available. Since ANG II did not produce an oxidative stress, it is possible that the peptide is exerting its pro-oxidant effects in the brain largely through an action on platelet, which can also produce ROS via its own NADPH oxidase. This might explain why DPI was effective in attenuating blood cell adhesion in both the ANG II and ischemic stroke models of inflammation.
A likely role for NADPH oxidase-derived ROS in the recruitment of blood cells into cerebral venules following exposure to either I/R or ANG II is upregulation of P-selectin on endothelial cells and/or platelets. ROS are known to induce the mobilization of the preformed storage pool of P-selectin from granules in endothelial cells (Weibel-Palade bodies) and platelets (-granules) and to stimulate the synthesis of new P-selectin in endothelial cells (13, 35). If ANG II enhances ROS production in these cells via AT1 receptor engagement and ROS promote P-selectin expression, then the data obtained in this study would be consistent with a model in which blood cell adhesion is attenuated by treatment with either an AT1 receptor antagonist, blocking MAbs to P-selectin or PSGL-1, and an inhibitor of NAD(P)H oxidase (DPI).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-induced leukocyte recruitment in the brain microvasculature. Circ Res 87: 1141–1148, 2000.This article has been cited by other articles:
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  S. Chen, G. Li, W. Zhang, J. Wang, C. D. Sigmund, J. E. Olson, and Y. Chen Ischemia-induced brain damage is enhanced in human renin and angiotensinogen double-transgenic mice Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2009; 297(5): R1526 - R1531. [Abstract] [Full Text] [PDF]
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 S. Terao, G. Yilmaz, K. Y. Stokes, J. Russell, M. Ishikawa, T. Kawase, and D. N. Granger Blood Cell-Derived RANTES Mediates Cerebral Microvascular Dysfunction, Inflammation, and Tissue Injury After Focal Ischemia-Reperfusion Stroke, September 1, 2008; 39(9): 2560 - 2570. [Abstract] [Full Text] [PDF]
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S. Terao, G. Yilmaz, K. Y. Stokes, M. Ishikawa, T. Kawase, and D. N. Granger Inflammatory and Injury Responses to Ischemic Stroke in Obese Mice Stroke, March 1, 2008; 39(3): 943 - 950. [Abstract] [Full Text] [PDF]
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