Cerebral Venules Research Articles

Blood Cell-Derived RANTES Mediates Cerebral Microvascular Dysfunction, Inflammation, and Tissue Injury After Focal Ischemia–Reperfusion

Although chemokines have been implicated in cardiovascular diseases, few studies have addressed the role of these inflammatory mediators in ischemic stroke. This study tested the hypothesis that RANTES (CCL5; regulated on activation, normal T-cell expressed and secreted) mediates the cerebral microvascular dysfunction, inflammation, and tissue injury induced by brain ischemia and reperfusion. After 60-minute middle cerebral artery occlusion and reperfusion, the adhesion of leukocytes and platelets in cerebral venules, infarct volume, and blood-brain barrier permeability were measured in wild-type mice (WT), RANTES-deficient mice (RANTES(-/-)), WT mice transplanted with RANTES(-/-) bone marrow (RANTES>WT), and control bone marrow chimeras (WT>WT). The concentration of RANTES and several cytokines was also measured by enzyme-linked immunosorbent assay and a cytometric bead array. The enhanced leukocyte and platelet adhesion, increased blood-brain barrier permeability, and tissue infarction elicited in WT and WT>WT mice after middle cerebral artery occlusion and reperfusion were significantly blunted in RANTES(-/-) mice. Similar attenuation of the middle cerebral artery occlusion and reperfusion-induced responses were noted in RANTES>WT chimeras. Although RANTES deficiency did not alter the changes in tissue cytokine levels elicited by middle cerebral artery occlusion and reperfusion, plasma concentrations interleukin-6, interleukin-10, and interleukin-12 were all reduced. These findings implicate blood cell-derived RANTES in the microvascular, inflammatory, and tissue injury responses of the brain to ischemia and reperfusion.

Cerebral microvascular dysfunction in angiotensin II‐induced hypertension

Inflammation has been implicated in the pathogenesis of hypertension. Although hypertension is a major risk factor for ischemic stroke, and inflammation contributes to the pathogenesis of stroke, relatively little is known about the inflammatory effects of arterial hypertension in the cerebral microcirculation. The objective of this study was to determine whether angiotensin II (AngII)‐induced hypertension promotes blood cell‐endothelial cell adhesion in cerebral venules. Leukocyte and platelet adhesion were monitored in cerebral venules of C57Bl/6 mice via a cranial window. Hypertension was induced by subcutaneous implantation of an AngII‐loaded Alzet pump for a 2 wk period. Mice were placed on either a normal (ND) or high salt diet (HSD). AngII infusion induced a dose‐dependent (400–2000 ng/kg/min) increase in arterial pressure, which was higher in HSD mice. Mice on either ND or HSD exhibited an increased adhesion of both leukocytes and platelets at 600 ng/kg/min. Higher AngII doses did not elicit further increases in blood cell adhesion in ND mice, but further dose‐dependent increases were noted HSD mice. Blood brain barrier (BBB) permeability was increased by AngII in both ND and HSD mice. These findings indicate that the cerebral microcirculation assumes an inflammatory and pro‐thrombogenic phenotype in AngII‐induced hypertension, which may account for the accompanying BBB dysfunction. (HL26441)

Inflammatory and Injury Responses to Ischemic Stroke in Obese Mice

Although epidemiological studies reveal an increased incidence of obesity and an association between obesity and the prevalence/severity of ischemic stroke, little is known about the mechanisms that link obesity to ischemic stroke. This study tested the hypothesis that obesity exacerbates the cerebrovascular dysfunction and tissue injury induced by brain ischemia and reperfusion. The adhesion of leukocytes and platelets in cerebral venules, blood-brain barrier permeability, brain water content, and infarct volume were measured in wild-type, obese (ob/ob), and leptin-reconstituted ob/ob mice subjected to 30 minutes middle cerebral artery occlusion and reperfusion. Tissue and plasma cytokine levels were determined by cytometric bead array, and a role for monocyte chemoattractant protein-1 and interleukin-6 was assessed using blocking antibodies. Compared with wild-type mice, ob/ob exhibited larger increases in leukocyte and platelet adhesion, blood-brain barrier permeability, water content, and infarct volume after middle cerebral artery occlusion-reperfusion. Reconstitution of leptin in ob/ob mice tended to further enhance all reperfusion-induced responses. Ob/ob mice also exhibited higher plasma levels of monocyte chemoattractant protein-1 and interleukin-6 than wild-type mice. Immunoneutralization of monocyte chemoattractant protein-1, but not interleukin-6, reduced infarct volume in ob/ob mice. Obesity worsens the inflammatory and injury responses to middle cerebral artery occlusion and reperfusion by a mechanism independent of leptin deficiency. monocyte chemoattractant protein-1 appears to contribute to the exaggerated responses to ischemic stroke in obese mice.

Activation of the annexin 1 counter‐regulatory circuit affords protection in the mouse brain microcirculation

The purpose of this study was to investigate the role of the homeostatic antiinflammatory axis centered on annexin 1 (AnxA1) in cerebral microvascular dysfunction and tissue injury associated with middle cerebral artery (MCA) occlusion and reperfusion. Intravital fluorescence microscopy was used to visualize the mouse cerebral microcirculation: AnxA1 null mice exhibited more white blood cell adhesion in cerebral venules than their wild-type counterparts, and this was accompanied by a larger cerebral infarct vol and worse neurological score. All parameters were rescued by delivery of human recombinant AnxA1. To further explore these findings using pharmacological tools, the effect of a short AnxA1 peptidomimetic was tested. When given during the reperfusion phase, peptide Ac2-26 produced similar cerebroprotection, which was associated with a marked attenuation of cell adhesion and markers of inflammation as measured in tissue homogenates. The pharmacological effects of peptide Ac2-26 occurred via receptors of the formyl-peptide receptor (FPR) family, most likely FPR-rs2, as deduced by displacement assays with transfected cells and in vivo experiments with transgenic mice and receptor antagonists. Our findings indicate that the endogenous antiinflammatory circuit centered on AnxA1 produces significant cerebral protection, and that these properties might have therapeutic potential for stroke treatment.

Angiotensin II type 1 receptor signaling contributes to platelet-leukocyte-endothelial cell interactions in the cerebral microvasculature

Angiotensin II type 1 (AT(1)) receptor signaling has been implicated in cerebral microvascular alterations associated with ischemia, diabetes mellitus, hypercholesterolemia, and atherosclerosis. Platelets, which express AT(1) receptors, also appear to contribute to the thrombogenic and inflammatory responses that are elicited by these pathological conditions. This study assesses the role of AT(1) receptor activation on platelet-leukocyte-endothelial cell interactions elicited in cerebral microvasculature by ischemia and reperfusion. Intravital microscopy was used to monitor the adhesion of platelets and leukocytes that were labeled with different fluorochromes, whereas dihydrorhodamine-123 was used to quantify oxygen radical production in cerebral surface of mice that were either treated with the AT(1) receptor agonist Val-angiotensin II (ANG II) or subjected to bilateral common carotid artery occlusion (BCCAO) followed by reperfusion. ANG II elicited a dose- and time- dependent increase in platelet-leukocyte-endothelial cell interactions in cerebral venules that included rolling platelets, adherent platelets on the leukocytes and the endothelial cells, rolling leukocytes, and adherent leukocytes. All of these interactions were attenuated by treatment with either P-selectin or P-selectin glycoprotein ligand 1 (PSGL-1) antibody. The AT(1) receptor antagonist candesartan and losartan as well as diphenyleneiodonium, an inhibitor of flavoproteins including NAD(P)H oxidase, significantly reduced the platelet-leukocyte-endothelial cell interactions elicited by either ANG II administration or BCCAO/reperfusion. The increased oxygen radical generation elicited by BCCAO/reperfusion was also attenuated by candesartan. These findings are consistent with an AT(1) receptor signaling mechanism, which involves oxygen radical production and ultimately results in P-selectin- and PSGL-1-mediated platelet-leukocyte-endothelial cell interactions in the cerebral microcirculation.

Recruitment of bone marrow stromal cells in the cerebral microvasculature after focal ischemia‐reperfusion is mediated by selectins

The therapeutic potential of bone marrow stromal cells (BMSC) has been demonstrated in different models of stroke. While it is well known that BMSC migrate and home to the site of brain injury, the mechanism underlying this cell recruitment is not known. We hypothesize that after stroke, cerebrovascular endothelium expresses adhesion molecules that allow BMSC to recognize and home to the injury site. Stroke was induced in C57BL/6 mice by middle cerebral artery occlusion (MCAo) for 1 h, followed by 24 h reperfusion (MCAo/R). BMSC from H-2Kb-tsA58 mice were fluorescently labeled and administered (24 h after stroke) i.v. with or without an E- or P-selectin blocking antibody. BMSC were observed in cortical vessels using intravital microscopy. Significant adhesion (76±5 cells/mm2) of BMSC was observed in venules after MCAo/R, while a low number of adherent BMSC was detected in sham mice (16±4 cells/mm2). E- and P-selectin antibodies blocked the adhesion of BMSC in cerebral venules after stroke (36 ±4 & 10±3 cells/mm2 respectively). These findings indicate that cerebral endothelium assume a pro-adhesive phenotype following MCAO/R that favors the recruitment of BMSC into the infarct area. This adhesive interface between the blood and endothelial cell surface represents an important therapeutic target for enhancement of stem cell delivery to sites of ischemic infarction following stroke. (HL26441)

Obesity and the cerebral microvascular responses to ischemic stroke

Obesity is a well-documented and modifiable risk factor for ischemic stroke. We tested the hypothesis that obesity exacerbates the microvascular dysfunction and brain damage induced by ischemia-reperfusion (I/R). Middle cerebral artery occlusion (30 min) and reperfusion was induced in wild type (WT) and leptin-deficient ob/ob mice. The adhesion of leukocytes (LA) and platelets (PA) in postischemic cerebral venules was monitored after 4 hrs of reperfusion by intravital microscopy. Infarct volume was estimated from brain slices (obtained 24 hrs after reperfusion) using the TTC staining method. Blood-brain barrier dysfunction was quantified using the Evans blue (EB) method. Brain water (WC) content was determined as (wet weight–dry weight)/wet weight (%). I/R elicited significant increases in LA (116 ± 82 vs 308 ± 202 cells/mm2) and PA in cerebral venules of both WT and ob/ob mice, however the responses were exaggerated in ob/ob mice. A similar pattern of I/R-induced increases in infarct volume (16.4 ± 6.0 vs 27.6 ± 3.2 %), EB extravasation (0.013 ± 0.0082 vs 0.031 ± 0.012), and WC (79.8 ± 0.62 vs 81.3 ± 0.31%) was noted in WT and ob/ob mice, with the latter group exhibiting more profound changes. These findings indicate that the microvascular dysfunction and brain injury after I/R is exaggerated in ob/ob mice and that obesity leads to greater morbidity following ischemic stroke. (HL26441)

Hypoxia/Reoxygenation Induced Blood Cell Adhesion in Cerebral Venules of Sickle Cell Transgenic (β S ) Mice: The Two Faces of eNOS

Oxidative stress has been implicated in blood cell-vessel wall interactions of βSmice. Intravital fluorescence microscopy and bone marrow (BM) transplantation were used to examine the influence of different superoxide- and nitric oxide-directed interventions on leukocyte and platelet adhesion in cerebral venules of βS mice. Two h hypoxia and 4 h reoxygenation elicited intense leukocyte and platelet adhesion responses in βS mice that were attenuated by genetic deficiency of vascular gp91phox or overexpression of vascular CuZnSOD; allopurinol treatment was without effect. Treatment with L-NAME or genetic deficiency of vascular eNOS also decreased cell adhesion responses whereas treatment with DETA-NO or genetic overexpression of vascular eNOS exacerbated the cell adhesion responses; genetic deficiency of vascular iNOS was without effect. Sepiapterin, similar to PEG-SOD, effectively reduced the enhanced cell adhesion responses in βS mice. In conclusion, vascular NADPH oxidase and eNOS are major contributors to the pro-inflammatory and pro-thrombogenic phenotype assumed by the cerebral microvasculature of βS mice. These findings suggest that the inflammatory environment created by βS BM leads to NADPH oxidase activation in endothelial cells as well as the functional inactivation of vascular eNOS enzyme, and possibly includes roles for peroxynitrite formation and/or eNOS uncoupling. (HL26441, P01-HL55552)

Critical role of endothelial cell-derived nitric oxide synthase in sickle cell disease-induced microvascular dysfunction

Superoxide, which can limit nitric oxide bioavailability, has been implicated in blood cell–vessel wall interactions observed in sickle cell transgenic (β S) mice. Here we report that nonselective chemical inhibition of nitric oxide synthase isoforms dramatically reduces the enhanced leukocyte and platelet adhesion normally observed in cerebral venules of β S mice. Although genetic deficiency of vascular wall inducible nitric oxide synthase does not alter adhesion responses in β S mice, a significant attenuation is noted in β S mice with vascular wall endothelial nitric oxide synthase (eNOS) deficiency, while the adhesion responses are exacerbated when eNOS is overexpressed in microvessels. The eNOS-mediated enhancement of blood cell adhesion is reversible by pretreatment with sepiapterin (which generates the eNOS cofactor tetrahydrobiopterin) or polyethyleneglycol–superoxide dismutase, implicating a role for eNOS-dependent superoxide production. These findings suggest that an imbalance between eNOS-derived nitric oxide and superoxide, both generated by the vessel wall, is critical to the proinflammatory and prothrombogenic phenotype that is assumed by the microvasculature in sickle cell disease.

Contribution of vascular adhesion protein-1 (VAP-1) to post-ischemic cerebral venular leukocyte adhesion and infiltration in diabetic, ovariectomized (OVX) and estrogen-treated female rats

We examined the role of VAP-1 in the increased leukocyte adhesion seen in cerebral venules during the reperfusion period following transient forebrain ischemia (TFI). We used a model associated with a high level of post-ischemic leukocyte adhesion and infiltration — diabetic (6–8 wks post-streptozotocin) OVX females given 1 week of estrogen replacement therapy (ERT). The behavior of rhodamine 6G-labeled leukocytes in pial venules was monitored through a closed cranial window, using intravital microscopy and digital videometry. The rats were observed over 10 h reperfusion after 20 min TFI. We compared rats treated (at 0 h or 6 h reperfusion) with saline or the novel and selective VAP-1 blocker, LJP-1207 (30 mg/kg iv). Since results in the 2 control groups were indistinguishable, the data was pooled (n=6). In controls, increased neutrophil adherence was observed within 1 h of reperfusion onset. The intravascular accumulation of adherent leukocytes was seen to gradually rise up to the point where neutrophil infiltration commenced (at >6 h, in 83% of controls — fig. 1). Not counting extravasated cells, neutrophil adhesion reached 17% of the viewed venular area at 6–10 h (fig. 2). In the rats treated with LJP-1207 at the onset of reperfusion (0 h, n=5), limited neutrophil adhesion, and no infiltration, was observed over the entire 10 h period of post-ischemic observation (fig. 1), with the level of adhesion rising only to 5% of the venular area (fig. 2). In rats treated at 6 h reperfusion (n=4), the pattern of neutrophil adhesion was similar to that of the control group up to 6 h, but subsequent infiltration was much more limited vs controls (fig. 1), and the level of adhesion actually declined (from 14% to 9%) when going from 6–10 h reperfusion (fig. 2). These findings indicate that VAP-1, presumably on cerebral vascular endothelium, plays a major role in the process of leukocyte adhesion, as well as infiltration, in diabetic OVX females provided with chronic ERT.

Prolonged therapeutic window associated with pharmacologic blockade of vascular adhesion protein-1 (VAP-1)-related post-ischemic leukocyte adhesion in diabetic, ovariectomized (OVX) female rats given chronic estrogen replacement

Endothelial VAP-1 facilitates post-ischemic leukocyte adhesion and infiltration in cerebral venules (abstract-this meeting). Evidence suggests that this role of VAP-1 relates, in part, to its function as an amine oxidase and the formation of H2O2 and aldehydes. In the present study, we examined whether treatment with a specific inhibitor of the enzymatic function of VAP-1 (LJP-1207) was capable of reducing the neuropathology accompanying transient forebrain ischemia (TFI). We used a model associated with a high level of post-ischemic leukocyte adhesion and infiltration—diabetic (6–8 wks post-streptozotocin) OVX female rats given 1 week of estrogen replacement therapy (ERT). The rats were allowed to recover over 3 days following 20 min TFI. We compared rats treated, either at the onset or at 6 h reperfusion, with saline or LJP-1207 (30 mg/kg iv). Neurologic function was scored each day over the 3-day reperfusion period. The daily scores could range from 0 (no dysfunction) to 18 (severe dysfunction). In controls (n=10), the 3-day score was 13 2; while in the VAP-1-inhibited rats, the scores were 2 1 (LJP-1207 treatment @0 h reperfusion [n=7]) and 6 1 (LJP-1207 treatment at 6 h reperfusion [n=5]) (see figure 1). Both treatment groups were statistically different from control. Findings from our laboratory have shown that leukocyte infiltration, in diabetic OVX females given ERT, commences at >6 h reperfusion (Stroke 35: 1974–8, 2004), and that this transformation is largely prevented by LJP-1207 (abstract-this meeting). These findings, therefore, indicate that VAP-1-mediated post-TFI leukocyte adhesion/infiltration, in diabetic OVX females given chronic ERT, contributes substantially to neuropathology. One intriguing implication of these findings is that preventing leukocyte infiltration may provide a substantial measure of neuroprotection. This could explain the finding of LJP-1207 having at least a 6-hour therapeutic window, in this model.

Endothelial cell NADPH oxidase mediates the cerebral microvascular dysfunction in sickle cell transgenic mice

Although blood cell-endothelial cell adhesion and oxidative stress have been implicated in the pathogenesis of sickle cell disease (SCD), the nature of the linkage between these vascular responses in SCD remains unclear. The objective of this study was to determine whether superoxide derived from endothelial cell-associated NADPH oxidase mediates the leukocyte-endothelial (L/E) and platelet-endothelial cell (P/E) adhesion that is observed in the cerebral microvasculature of sickle cell transgenic (betaS) mice. Intravital fluorescence microscopy was used to monitor L/E and P/E adhesion in brain postcapillary venules of wild-type (WT), SOD1 transgenic (SOD1-TgN), and gp91phox (NADPH oxidase)-deficient mice that were transplanted with bone marrow from betaS mice. Hypoxia/reoxygenation (H/R) yielded intense P/E and L/E adhesion responses in cerebral venules of betaS/WT chimeras that were significantly attenuated in both betaS/SOD1-TgN, and betaS/gp91phox-/- chimeras. Pretreatment of betaS/WT chimeras with the iron-chelator desferroxamine blunted the blood cell-endothelial cell adhesion responses to H/R, whereas pretreatment with the xanthine oxidase inhibitor allopurinol had no effect. These findings suggest that superoxide derived from endothelial cell NADPH-oxidase and catalytically active iron contribute to the proinflammatory and prothrombogenic responses associated with sickle cell disease.

Thrombus and encapsulated hematoma in cerebral cavernous malformations

Thrombi, encapsulated hematomas, and granulation tissue are frequently seen in cerebral cavernous malformations (CCMs). We investigated the role that these histological changes play in repeated hemorrhages in CCMs as well as lesion growth, examining specimens of CCMs surgically harvested from 20 patients. The immunohistochemical study included thrombomodulin (TM) and endothelial cell protein C receptor (EPCR), which are important regulators of blood coagulation. Thick capsules, which contained blood degradation product, were seen in cases with encapsulated hematomas. Clusters of sinusoidal vessels were found outside of these thick capsules. Granulation tissue with inflammatory infiltrates and capillaries was seen in 4 cases with non-capsulated hematomas. Organizing thrombi were seen in sinusoidal vessels in 15 out of 20 cases. Factor VIII-related antigen staining demonstrated numerous capillaries in and around organizing thrombi and within the thickened vessel walls as well as in both the inner and outer sides of the hematoma capsule. TM and EPCR were positive in the endothelial cells of these capillaries, whereas they were negative in those of capillaries in the brain surrounding the lesions. Our study suggests that thrombosed sinusoidal blood vessels could gradually expand by repeated bleeding from numerous capillaries inside the wall and become encapsulated hematomas, and capillaries outside the thickened vessel wall could become sinusoidal blood vessels. Thrombosis within cerebral venules could be one of the causal factors of CCMs.

Obesity Exacerbates Sepsis‐Induced Inflammation and Microvascular Dysfunction in Mouse Brain

Obese patients with sepsis have higher morbidity and mortality than lean counterparts, but the mechanisms involved are unknown. The authors examined the inflammatory and thrombogenic responses of the cerebral microvasculature to sepsis induced by cecal ligation and perforation in obese and lean wild-type mice. Leukocyte and platelet adhesion in cerebral microvasculature and behavioral responses were measured in wild-type and obese mice 4 h postperforation. P-selectin expression in different vascular beds was assessed 6 h postperforation. The effects of immunoblockade of P-selectin, ICAM-1, and CD18 on leukocyte and platelet recruitment were evaluated in obese septic animals. Cerebral venules of obese and wild-type mice assumed a proinflammatory and prothrombogenic phenotype 4 h post-perforation, with greatly exaggerated responses in obese mice compared to the lean counterparts. These enhanced responses were attenuated by blocking P-selectin, CD18, or ICAM-1. Obese mice also exhibited a more profound behavioral deficit after sepsis, which appears to be unrelated to the recruitment of leukocytes and platelets. Cecal ligation and perforation-induced P-selectin expression was greater in obese mice compared with lean counterparts. These findings suggest that the increased morbidity to sepsis in obesity may result from exaggerated microvascular inflammatory and thrombogenic responses that include the activation of endothelial cells with subsequent expression of adhesion molecules, such as P-selectin.

Diapedesis of mononuclear cells across cerebral venules during experimental autoimmune encephalomyelitis leaves tight junctions intact

Diapedesis of leukocytes across endothelial barriers is generally believed to require the opening of endothelial tight junctions. At the blood-brain barrier (BBB), endothelial cells are interconnected by complex tight junctions. Here, we show by serial section conventional electron microscopy that during experimental autoimmune encephalomyelitis mononuclear cells traverse cerebral microvessels by a transcellular pathway, leaving the endothelial tight junctions intact. Cerebral endothelial cells were found to form filopodia-like membrane protrusions on their luminal aspect, thus embracing the mononuclear cells and forming cup-like structures, and eventually pores, through which the traversing cell could reach the abluminal side. At the abluminal side endothelial cell protrusions surrounding a migrating inflammatory cell were found to be progressively lined with basal lamina, suggesting a change from luminal to abluminal membrane characteristics of endothelial cell membranes during inflammatory cell diapedesis. Morphological evidence for the involvement of tight junctions in the diapedesis of mononuclear cells across the inflamed BBB could not be obtained in any case. Taken together, the presence of morphologically intact tight junctions and our novel finding of the presence of a basal lamina on both sides of abluminal endothelial cell protrusions surrounding migrating inflammatory cells suggests that during experimental autoimmune encephalomyelitis diapedesis of mononuclear cells occurs via a transendothelial process.

Contributions of LFA-1 and Mac-1 to brain injury and microvascular dysfunction induced by transient middle cerebral artery occlusion.

Although the beta2-integrins have been implicated in the pathogenesis of cerebral ischemia-reperfusion (I/R) injury, the relative contributions of the alpha-subunits to the pathogenesis of ischemic stroke remains unclear. The objective of this study was to determine whether and how genetic deficiency of either lymphocyte function-associated antigen-1 (LFA-1) or macrophage-1 (Mac-1) alters the blood cell-endothelial cell interactions, tissue injury, and organ dysfunction in the mouse brain exposed to focal I/R. Middle cerebral artery occlusion was induced for 1 h (followed by either 4 or 24 h of reperfusion) in wild-type mice and in mice with null mutations for either LFA-1 or Mac-1. Neurological deficit and infarct volume were monitored for 24 h after reperfusion. Platelet- and leukocyte-vessel wall adhesive interactions were monitored in cortical venules by intravital microscopy. Mice with null mutations for LFA-1 or Mac-1 exhibited significant reductions in infarct volume. This was associated with a significant improvement in the I/R-induced neurological deficit. Leukocyte adhesion in cerebral venules did not differ between wild-type and mutant mice at 4 h after reperfusion. However, after 24 h of reperfusion, leukocyte adhesion was reduced in both LFA-1- and Mac-1-deficient mice compared with their wild-type counterparts. Platelet adhesion was also reduced at both 4 and 24 h after reperfusion in the LFA-1- and Mac-1-deficient mice. These findings indicate that both alpha-subunits of the beta2-integrins contribute to the brain injury and blood cell-vessel wall interactions that are associated with transient focal cerebral ischemia.

Estrogen replacement treatment in diabetic ovariectomized female rats potentiates postischemic leukocyte adhesion in cerebral venules.

Chronic 17beta-estradiol (E2) replacement therapy in ovariectomized (OVX) female rats reduces leukocyte adhesion and brain damage after transient forebrain ischemia. Recently, we found that E2 treatment in diabetic OVX females was associated with enhanced postischemic neuropathology. We tested the hypothesis that in chronically hyperglycemic diabetic OVX females, chronic E2 replacement potentiates post-transient forebrain ischemia leukocyte adhesion. Pial venules were observed through closed cranial windows. Adherence of rhodamine 6G-tagged leukocytes was monitored before and 10 hours after transient forebrain ischemia (20 minutes right common carotid artery occlusion plus hemorrhagic hypotension) in intact, untreated OVX and E2-treated OVX females rendered diabetic via streptozotocin. Leukocyte adhesion was quantitated as the percentage venular area occupied by adherent leukocytes. At 2 hours after transient forebrain ischemia, a similar low level of leukocyte adhesion was seen in the 3 groups (<3% of the venular area). Starting at approximately 4 hours after ischemia, leukocyte adhesion in the E2-treated OVX females rose to significantly higher levels compared with the other groups. Relative to the 2-hour value, the level of adhesion at 10 hours was 12.5-fold, 4-fold, and 5-fold greater in the E2-treated OVX, OVX, and intact groups, respectively. Leukocyte extravasation (beginning after 6 hours of reperfusion) was observed in a majority (64%) of the E2-treated animals, with limited or no extravasation seen in the intact or OVX groups. These results suggest that factors associated with diabetes and chronic hyperglycemia convert E2 from a counterinflammatory to a proinflammatory substance in an ischemic setting.

Hypothermia attenuates the vasodilator effects of dexmedetomidine on pial vessels in rabbits in vivo.

Studies have indicated that mild to moderate hypothermia or dexmedetomidine may have neuroprotective properties in animal models. In this study, we investigated the effects of hypothermia on dexmedetomidine-induced responses in cerebral vessels in anesthetized rabbits by using the cranial-window preparation. After instrumentation under pentobarbital anesthesia, 12 rabbits were assigned to 1 of 2 equal groups: normothermic (nasopharyngeal and intrawindow temperature, 38.5 degrees C-39.5 degrees C) or hypothermic (33.0 degrees C-34.0 degrees C). Each rabbit received three different concentrations (10(-7), 10(-5), and 10(-3) M) of dexmedetomidine under the window, and cerebral pial vessel diameters were measured in a sequential manner. In the normothermic group, dexmedetomidine induced a significant concentration-dependent dilation in both large and small arterioles. In the hypothermia group, dexmedetomidine produced a U-shaped dose-response in both large and small cerebral arterioles (concentration-related vasoconstriction at 10(-7) and 10(-5) M, but vasodilation at 10(-3) M). In cerebral venules, a similar pattern of results was obtained, but changes were generally smaller than in arterioles. In conclusion, topically applied dexmedetomidine induces concentration-dependent dilation in cerebral arterioles in normothermic rabbits anesthetized with pentobarbital, but mild to moderate hypothermia attenuates these responses, with smaller dexmedetomidine concentrations causing vasoconstriction. In normothermic rabbits anesthetized with pentobarbital, topically applied dexmedetomidine induces a concentration-dependent dilation in both large and small cerebral arterioles, but mild to moderate hypothermia attenuates these responses.

Endothelial cell P-selectin mediates a proinflammatory and prothrombogenic phenotype in cerebral venules of sickle cell transgenic mice.

Whereas the adhesion of leukocytes and erythrocytes to vascular endothelium has been implicated in the vasooclusive events associated with sickle cell disease, the role of platelet-vessel wall interactions in this process remains undefined. The objectives of this study were to: 1) determine whether the adhesion of platelets and leukocytes in cerebral venules differs between sickle cell transgenic (betaS) mice and their wild-type (WT) counterparts (C57Bl/6) under both resting and posthypoxic conditions, and 2) define the contributions of P-selectin to these adhesion processes. Animals were anesthetized, and platelet and leukocyte interactions with endothelial cells of cerebral postcapillary venules were monitored and quantified using intravital fluorescence microscopy in WT, betaS, and chimeric mice produced by transplanting bone marrow from WT or betaS mice into WT or P-selectin-deficient (P-sel(-/-)) mice. Platelet and leukocyte adhesion to endothelial cells in both unstimulated and posthypoxic betaS mice were significantly elevated over WT levels. Chimeric mice involving bone marrow transfer from betaS mice to P-sel(-/-) mice exhibited a profound attenuation of both platelet and leukocyte adhesion compared with betaS bone marrow transfer to WT mice. These findings indicate that betaS mice assume both an inflammatory and prothrombogenic phenotype, with endothelial cell P-selectin playing a major role in mediating these microvascular responses.

Cerebral microvascular responses to hypercholesterolemia: roles of NADPH oxidase and P-selectin.

Although hypercholesterolemia is widely accepted as a major risk factor for coronary artery and peripheral vascular diseases, its role in the pathogenesis of stroke is controversial. The objectives of this study were to determine how hypercholesterolemia affects the cerebral microcirculation under resting conditions and after ischemia-reperfusion (I/R). Platelet- and leukocyte-endothelial cell interactions and oxidant production (using the oxidant-sensitive fluorochrome dihydrorhodamine-123) were monitored by intravital videomicroscopy in the cerebral microvasculature of mice placed on either a normal (ND) or cholesterol-enriched diet (HCD). Platelets labeled with carboxyfluorescein diacetate succinimidyl ester (CFDASE) and leukocytes labeled with rhodamine 6G were seen to roll and firmly adhere, with a corresponding increase in oxidant production, in venules of mice on HCD, but not ND. Immunoneutralization of P-selectin attenuated the platelet- and leukocyte-endothelial cell interactions and the enhanced oxidant production associated with HCD. A GPIIb/IIIa blocking antibody did not alter the blood cell-vessel wall interactions to HCD. Mice deficient in the NADPH oxidase subunit gp91(phox) exhibited significantly blunted platelet and leukocyte recruitment responses to HCD. Focal I/R also elicited inflammatory and prothrombogenic responses in cerebral venules and these were exaggerated in mice on HCD. These results implicate an oxidant-dependent, P-selectin-mediated mechanism in the blood cell-vessel wall interactions induced by hypercholesterolemia in the brain and demonstrate that the deleterious effects of I/R on the brain are exacerbated by this cardiovascular risk factor.