Nox2 Oxidase Activity Accounts for the Oxidative Stress and Vasomotor Dysfunction in Mouse Cerebral Arteries following Ischemic Stroke

Background and Purpose: Post-ischemic oxidative stress and vasomotor dysfunction in cerebral arteries may increase the likelihood of cognitive impairment and secondary stroke. However, the underlying mechanisms of post-stroke vascular abnormalities, as distinct from those causing primary brain injury, are poorly understood. We tested whether augmented superoxide-dependent dysfunction occurs in the mouse cerebral circulation following ischemia-reperfusion, and evaluated the role of Nox2 oxidase. Methods: Cerebral ischemia was induced in male C57Bl6/J wild-type (WT) and Nox2-deficient (Nox2 -/-) mice by middle cerebral artery occlusion (MCAO; 0.5 h), followed by reperfusion (23.5 h). Superoxide production by MCA was measured by L-012-enhanced chemiluminescence. Nitric oxide (NO) function was assessed in cannulated and pressurized MCA via the vasoconstrictor response to N ω-nitro-L-arginine methyl ester (L-NAME; 100 μmol/L). Expression of Nox2, the nitration marker 3-nitrotyrosine, and leukocyte marker CD45 was assessed in cerebral arteries by Western blotting. Results: Following ischemia-reperfusion, superoxide production was markedly increased in the MCA of WT, but not Nox2 -/- mice. In WT mice, L-NAME-induced constriction was reduced by ~50% in ischemic MCA, whereas ischemia-reperfusion had no effect on responses to L-NAME in vessels from Nox2 -/- mice. In ischemic MCA from WT mice, expression of Nox2 and 3-nitrotyrosine were ~1.4-fold higher than in the contralateral MCA, or in ischemic or contralateral vessels from Nox2 -/- mice. Vascular CD45 levels were unchanged by ischemia-reperfusion. Conclusions: Excessive superoxide production, impaired NO function and nitrosative stress occur in mouse cerebral arteries after ischemia-reperfusion. These abnormalities appear to be exclusively due to increased activity of vascular Nox2 oxidase

T Cells Prevent Hemorrhagic Transformation in Ischemic Stroke by P-Selectin Binding

Objective Hemorrhagic transformation is a serious complication of ischemic stroke after recanalization therapies. This study aims to identify mechanisms underlying hemorrhagic transformation after cerebral ischemia/reperfusion. Approach and Results We used wild-type mice and Selplg(-/-) and Fut7(-/-) mice defective in P-selectin binding and lymphopenic Rag2(-/-) mice. We induced 30-minute or 45-minute ischemia by intraluminal occlusion of the middle cerebral artery and assessed hemorrhagic transformation at 48 hours with a hemorrhage grading score, histological means, brain hemoglobin content, or magnetic resonance imaging. We depleted platelets and adoptively transferred T cells of the different genotypes to lymphopenic mice. Interactions of T cells with platelets in blood were studied by flow cytometry and image stream technology. We show that platelet depletion increased the bleeding risk only after large infarcts. Lymphopenia predisposed to hemorrhagic transformation after severe stroke, and adoptive transfer of T cells prevented hemorrhagic transformation in lymphopenic mice. CD4(+) memory T cells were the subset of T cells binding P-selectin and platelets through functional P-selectin glycoprotein ligand-1. Mice defective in P-selectin binding had a higher hemorrhagic score than wild-type mice. Adoptive transfer of T cells defective in P-selectin binding into lymphopenic mice did not prevent hemorrhagic transformation. Conclusions The study identifies lymphopenia as a previously unrecognized risk factor for secondary hemorrhagic transformation in mice after severe ischemic stroke. T cells prevent hemorrhagic transformation by their capacity to bind platelets through P-selectin. The results highlight the role of T cells in bridging immunity and hemostasis in ischemic stroke

Importance of T lymphocytes in brain injury, immunodeficiency, and recovery after cerebral ischemia

Following an ischemic stroke, T lymphocytes become activated, infiltrate the brain, and appear to release cytokines and reactive oxygen species to contribute to early inflammation and brain injury. However, some subsets of T lymphocytes may be beneficial even in the early stages after a stroke, and recent evidence suggests that T lymphocytes can also contribute to the repair and regeneration of the brain at later stages. In the hours to days after stroke, T-lymphocyte numbers are then reduced in the blood and in secondary lymphoid organs as part of a ‘stroke-induced immunodeficiency syndrome,’ which is mediated by hyperactivity of the sympathetic nervous system and the hypothalamic–pituitary–adrenal axis, resulting in increased risk of infectious complications. Whether or not poststroke T-lymphocyte activation occurs via an antigen-independent process, as opposed to a classical antigen-dependent process, is still controversial. Although considerable recent progress has been made, a better understanding of the roles of the different T-lymphocyte subpopulations and their temporal profile of damage versus repair will help to clarify whether T-lymphocyte targeting may be a viable poststroke therapy for clinical use

Effect of cerebral ischemia-reperfusion on vascular superoxide production.

<p>Basal (A and B) and phorbol-12, 13-dibutyrate (10 µmol/L; PDB: Nox2 activator)-stimulated (C and D) superoxide production by middle cerebral arteries (MCA) as measured by 100 µmol/L L-012-enhanced chemiluminescence. <b>A.</b> Contralateral and ischemic MCA from wild-type mice 24 h after MCA occlusion, and pooled left and right MCA from sham-operated wild-type mice. <b>B.</b> Contralateral and ischemic MCA from Nox2-deficient (Nox2^-/-) mice 24 h after MCAO. <b>C.</b> Contralateral and ischemic MCA from wild-type mice 24 h after MCA occlusion (MCAO), and pooled left and right MCA from sham operated wild-type mice. <b>D.</b> Contralateral and ischemic MCA from Nox2^-/- 24 h after MCAO. All results are expressed as 10³ counts/mg of dry tissue weight and given as mean±SEM (A and C, n = 9 for both groups of MCAO; n = 7 for sham; B and D, n = 6 for both groups). *<i>P</i>&lt;0.05 vs. contralateral MCA (one way ANOVA with a Bonferroni mutiple comparison post hoc test).</p

Effect of ischemia-reperfusion on CD45 expression in cerebral arteries.

<p>Western blots (n = 2) showing CD45 levels in sham, contralateral (Contra) and ischemic (Isch) middle cerebral artery homogenates from wild-type mice at 24 h after MCA occlusion. Spleen homogenates were used as positive controls. MCA homogenates from 5 mice were pooled in order to obtain sufficient protein levels.</p

Effect of cerebral ischemia-reperfusion on vascular Nox2 expression.

<p>Representative Western blots (top) showing protein expression of Nox2 in contralateral (Contra) and ischemic (Isch) middle cerebral artery homogenates from wild-type and Nox2-deficient (Nox2^-/-) mice at 24 h after MCA occlusion. Also shown is a summary of immunoreactive band intensity (bottom). Values are expressed as relative intensity normalized to β-actin intensity, and are given as mean±SEM (n = 4 for all groups). *<i>P</i>&lt;0.05 vs. contralateral (one sample <i>t</i>-test).</p

Effect of cerebral ischemia-reperfusion on vascular 3-nitrotyrosine levels.

<p> Representative Western blots (A) showing expression of 3-nitrotyrosine in contralateral (Contra) and ischemic (Isch) middle cerebral artery homogenates from wild-type and Nox2-deficient (Nox2^-/-) mice at 24 h after MCA occlusion. Also shown is a summary of immunoreactive band intensity (bottom). Values are expressed as relative intensity normalized to β-actin intensity, and are given as mean±SEM (n = 5 for all groups). *<i>P</i>&lt;0.05 vs contralateral (one-way ANOVA with a Bonferroni multiple comparison post-hoc test).</p

Effect of cerebral ischemia-reperfusion on vascular function.

<p>Vasoconstrictor responses of isolated contralateral and ischemic middle cerebral arteries (MCA) from wild-type (A) and Nox2-deficient (Nox2^-/-; B) mice to L-NAME (100 µmol/L) 24 h after MCA occlusion. Also shown are vasoconstrictor responses of contralateral and ischemic MCA from wild-type mice to phenylephrine (PE: 1 nmol/L-1 µmol/L; C) and U46619 (1 nmol/L-1 µmol/L; D). Results are expressed as% change in intraluminal diameter and given as mean±SEM (A, n = 9 for contralateral, n = 10 for ischemic; B, n = 6 for contralateral, n = 6 for ischemic; C, n = 6 for contralateral, n = 4 for ischemic; D, n = 7 for contralateral, n = 6 for ischemic). *<i>P</i>&lt;0.05 vs. contralateral MCA (A: unpaired <i>t</i> test; C: two-way ANOVA with a Bonferroni multiple comparison post-hoc test).</p

Effect of middle cerebral artery occlusion on cerebral blood flow and infarct volume.

<p>Regional cerebral blood flow (rCBF; n = 7 for sham, n = 60 for wild-type and n = 22 for Nox2^-/-; A) and infarct volumes at 24 h following middle cerebral artery occlusion (MCAO; B) in wild-type and Nox2-deficient (Nox2^-/-) mice. rCBF was measured during MCAO and during reperfusion (30 min). Representative (n = 15 for wild type and n = 7 for Nox2^-/-) coronal brain sections are shown for wild-type and Nox2^-/- mice at 24 h after MCAO with the infarct area outlined in white (B, top). All results are presented as mean±SEM. *<i>P</i>&lt;0.05 vs. wild-type (unpaired <i>t</i> test).</p

Neuroprotective effect of an angiotensin receptor type 2 agonist following cerebral ischemia in vitro and in vivo

Intracerebral administration of the angiotensin II type 2 receptor (AT2R) agonist, CGP42112, is neuroprotective in a rat model of ischemic stroke. To explore further its possible cellular target(s) and therapeutic utility, we firstly examined whether CGP42112 may exert direct protective effects on primary neurons following glucose deprivation in vitro. Secondly, we tested whether CGP42112 is effective when administered systemically in a mouse model of cerebral ischemia.Primary cortical neurons were cultured from E17 C57Bl6 mouse embryos for 9 d, exposed to glucose deprivation for 24 h alone or with drug treatments, and percent cell survival assessed using trypan blue exclusion. Ischemic stroke was induced in adult male C57Bl6 mice by middle cerebral artery occlusion for 30 min, followed by reperfusion for 23.5 h. Neurological assessment was performed and then mice were euthanized and infarct and edema volume were analysed.During glucose deprivation, CGP42112 (1x10-8 M and 1x10-7 M) reduced cell death by ~30%, an effect that was prevented by the AT2R antagonist, PD123319 (1x10-6 M). Neuroprotection by CGP42112 was lost at a higher concentration (1x10-6 M) but was unmasked by co-application with the AT1R antagonist, candesartan (1x10-7 M). By contrast, Compound 21 (1x10-8 M to 1x10-6 M), a second AT2R agonist, had no effect on neuronal survival. Mice treated with CGP42112 (1 mg/kg i.p.) after cerebral ischemia had improved functional outcomes over vehicle-treated mice as well as reduced total and cortical infarct volumes.These results indicate that CGP42112 can directly protect neurons from ischemia-like injury in vitro via activation of AT2Rs, an effect opposed by AT1R activation at high concentrations. Furthermore, systemic administration of CGP42112 can reduce functional deficits and infarct volume following cerebral ischemia in vivo