Background— Although lymphocyte recruitment and activation are associated with cerebral ischemia-reperfusion (I/R) injury, the contributions of specific lymphocyte subpopulations and lymphocyte-derived interferon-γ (IFN-γ) to stroke remain unknown. The objectives of this study were to define the contribution of specific populations of lymphocytes to the inflammatory and prothrombogenic responses elicited in the cerebral microvasculature by I/R and to investigate the role of T-cell–associated IFN-γ in the pathogenesis of ischemic stroke. Methods and Results— Middle cerebral artery occlusion was induced for 1 hour (followed by 4 or 24 hours of reperfusion) in wild-type mice and mice deficient in lymphocytes (Rag1 −/− ), CD4 + T cells, CD8 + T cells, B cells, or IFN-γ. Platelet and leukocyte adhesion was assessed in cortical venules with intravital video microscopy. Neurological deficit and infarct volume were determined 24 hours after reperfusion. Rag1 −/− , CD4 + T-cell −/− , CD8 + T-cell −/− , and IFN-γ −/− mice exhibited comparable significant reductions in I/R-induced leukocyte and platelet adhesion compared with wild-type mice exposed to I/R. Infarct volume was reduced and I/R-induced neurological deficit was improved in immunodeficient Rag1 −/− mice. These protective responses were reversed in Rag1 −/− mice reconstituted with either wild-type or, to a lesser extent, IFN-γ −/− splenocytes. B-cell–deficient mice failed to show improvement against ischemic stroke injury. Conclusions— These findings indicate that CD4 + and CD8 + T lymphocytes, but not B lymphocytes, contribute to the inflammatory and thrombogenic responses, brain injury, and neurological deficit associated with experimental stroke. Although IFN-γ plays a pivotal role in stroke-induced inflammatory responses, T lymphocytes appear to be a minor source of this cytokine.

Mice transgenic for antisense Notch and normal mice treated with inhibitors of the Notch-activating enzyme gamma-secretase showed reduced damage to brain cells and improved functional outcome in a model of focal ischemic stroke. Notch endangers neurons by modulating pathways that increase their vulnerability to apoptosis, and by activating microglial cells and stimulating the infiltration of proinflammatory leukocytes. These findings suggest that Notch signaling may be a therapeutic target for treatment of stroke and related neurodegenerative conditions.

Leukocytes are recruited into the cerebral microcirculation following an ischemic insult. The leukocyte-endothelial cell adhesion manifested within a few hours after ischemia (followed by reperfusion, I/R) largely reflects an infiltration of neutrophils, while other leukocyte populations appear to dominate the adhesive interactions with the vessel wall at 24 h of reperfusion. The influx of rolling and adherent leukocytes is accompanied by the recruitment of adherent platelets, which likely enhances the cytotoxic potential of the leukocytes to which they are attached. The recruitment of leukocytes and platelets in the postischemic brain is mediated by specific adhesion glycoproteins expressed by the activated blood cells and on cerebral microvascular endothelial cells. This process is also modulated by different signaling pathways (e.g., CD40/CD40L, Notch) and cytokines (e.g., RANTES) that are activated/released following I/R. Some of the known risk factors for cardiovascular disease, including hypercholesterolemia and obesity appear to exacerbate the leukocyte and platelet recruitment elicited by brain I/R. Although lymphocyte-endothelial cell and -platelet interactions in the postischemic cerebral microcirculation have not been evaluated to date, recent evidence in experimental animals implicate both CD4+ and CD8+ T-lymphocytes in the cerebral microvascular dysfunction, inflammation, and tissue injury associated with brain I/R. Evidence implicating regulatory T-cells as cerebroprotective modulators of the inflammatory and tissue injury responses to brain I/R support a continued focus on leukocytes as a target for therapeutic intervention in ischemic stroke.

Inflammation has been implicated in the pathogenesis of ischemic stroke and the recruitment of inflammatory cells appears to exacerbate ischemic brain injury. Since leukocyte-endothelial cell adhesion is a rate-determining step in the recruitment of leukocytes into post-ischemic brain tissue, much attention has been devoted to defining the contribution of different adhesion molecules, expressed either on leukocytes or endothelial cells, to the leukocyte recruitment process. Similarly, a large effort has been made to determine whether interference with leukocyte-endothelial cell adhesion protects the brain against ischemic tissue injury. These efforts have revealed an important contribution of β2-integrins (CD11/CD18), ICAM-1, and P-selectin in the recruitment of leukocytes as well as platelets in the post-ischemic cerebral microvasculature. Immunoblockade or genetic deletion of these adhesion molecules has been shown to reduce infarct volume, edema, behavioral deficits, and/or mortality in different animal models of ischemic stroke. Anti-adhesion agents also appear to widen the therapeutic window for thrombolytic therapy in these experimental models. Emerging evidence on the role of signaling pathways (eg, CD40/CD40L, Notch-1) and immune cells in the regulation of ischemia-reperfusion induced leukocyte recruitment in the cerebral microvasculature offer novel targets for controlling inflammation in stroke. The few clinical trials assessing anti-adhesion therapy in ischemic stroke have all failed to show efficacy. It remains to be determined whether inflammation in general and leukocyte adhesion in particular represent useful targets for therapeutic intervention in stroke patients.

Background and Purpose-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. Methods-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 Ϫ/Ϫ ), WT mice transplanted with RANTES Ϫ/Ϫ bone marrow (RANTESϾWT), and control bone marrow chimeras (WTϾWT). The concentration of RANTES and several cytokines was also measured by enzyme-linked immunosorbent assay and a cytometric bead array. Results-The enhanced leukocyte and platelet adhesion, increased blood-brain barrier permeability, and tissue infarction elicited in WT and WTϾWT mice after middle cerebral artery occlusion and reperfusion were significantly blunted in RANTES Ϫ/Ϫ mice. Similar attenuation of the middle cerebral artery occlusion and reperfusion-induced responses were noted in RANTESϾWT 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. Conclusions-These findings implicate blood cell-derived RANTES in the microvascular, inflammatory, and tissue injury responses of the brain to ischemia and reperfusion.

Background and Purpose-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. Methods-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. Results-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. Conclusions-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.

Background-CD40/CD40 ligand (CD40L) signaling contributes to proinflammatory and prothrombogenic responses in the vasculature. CD40/CD40L expression is elevated in patients after a transient ischemic attack or stroke. The purpose of this study was to investigate the role of CD40/CD40L signaling in cerebral microvascular dysfunction and tissue injury response to middle cerebral artery occlusion (MCAO) and reperfusion. Methods and Results-Intravital fluorescence microscopy was used to visualize the cerebral microcirculation of wild-type (WT), CD40-deficient, and CD40L-deficient mice subjected to 1-hour MCAO and 4-hour reperfusion. The adhesion of platelets and of leukocytes and vascular permeability were measured in postcapillary venules after 4-hour and 1-hour reperfusions, respectively. Cerebral infarct volume was analyzed 24 hours after reperfusion. Platelet and leukocyte adhesion was elevated and blood/brain barrier function was compromised by MCAO in WT mice. Blood cell recruitment and increased permeability were blunted in both CD40-deficient and CD40L-deficient mice. Infarct volume was also reduced in CD40-and CD40L-deficient mice compared with WT mice. Conclusions-Our findings indicate that CD40/CD40L signaling contributes to inflammatory and prothrombogenic responses and brain infarction induced by MCAO and reperfusion. The CD40/CD40L dyad may play a significant pathogenic role in the acute phase of ischemic stroke.

Background and Purpose-Increased blood-brain barrier (BBB) permeability, brain edema, and hemorrhage are important consequences of cerebral venous sinus thrombosis (CVST). The objective of this study was to define the role of the protein C pathway in the BBB permeability and edema elicited by experimental CVST. The role of neutrophil recruitment was also evaluated. Methods-Edema, BBB permeability, leukocyte-endothelial cell adhesion (LECA) and inflammatory cytokine levels were monitored in a murine model of CVST. The role of activated protein C (APC) was assessed in wild type mice (WT) receiving APC neutralizing antibody and in endothelial protein C receptor overexpressing mice (EPCR-tg). Neutrophil involvement was evaluated using an anti-CD18 antibody (Ab) and antineutrophil serum. Results-Brain edema and increases in BBB permeability and LECA were noted 48 hours after CVST. APC immunoblockade exacerbated these responses, while EPCR-tg exhibited blunted responses, as did WT treated with either antineutrophil serum or the CD18 Ab. Conclusions-The