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

Mechanisms of ischaemic stroke damage

Brain inflammation contributes to ischaemic and reperfusion injury, and thus worsens outcome after stroke. This thesis aimed to identify and investigate some of the inflammatory mechanisms that occur after cerebral ischaemia-reperfusion, to further enhance our understanding of them, and to potentially target them for future ischaemic stroke therapies. This study primarily used C57Bl6/J mice, as well as genetically modified mice. The model of focal cerebral ischaemia utilised was the intraluminal filament-induced middle cerebral artery occlusion. Real-time PCR and Western blotting were used to examine mRNA and protein expression levels, respectively, in the brain, and immunofluorescence and immunohistochemistry were used to localise proteins and cells in the brain. T lymphocytes were isolated from the blood and spleen using Dynal negative isolation kits, and T lymphocyte-generated superoxide was measured using L-012-enhanced chemiluminescence. Chapters 3 and 4 provided the first evidence that the larger infarct volume in males versus females following cerebral ischaemia is reperfusion-dependent and may be due to greater neuro-inflammation and brain infiltration of Nox2-expressing CD3+ T lymphocytes in male mice. Moreover, this gender difference was found to be dependent on Nox2 expression. The study also demonstrated for the first time that Nox2-expressing circulating CD3+ T lymphocytes produce ~15-fold more superoxide after stroke, compared to CD3+ T lymphocytes from control mice. These findings raise the possibility that therapies to reduce CD3+ T lymphocyte infiltration and/or the production of superoxide from these cells in ischaemic stroke patients who receive recombinant t-PA, might be useful for reducing reperfusion injury. Chapter 5 confirmed and extended the above findings and demonstrated for the first time that circulating Nox2-containing CD4+ and CD8+ T lymphocytes generate substantially higher levels of superoxide after cerebral ischaemia-reperfusion compared with similar T lymphocyte subsets from control mice. Chapter 6 demonstrated that the mRNA expression of various chemokines and chemokine receptors, including the potent neutrophil chemoattractants, CXCR2, CXCL1 and CXCL2, are increased in the brain after ischaemia-reperfusion. Administration of the CXCR2 antagonist, SB 225002, reduced the expression of these chemokines in the brain, as well as the infiltration of neutrophils, but did not improve outcome at 72 h after ischaemia-reperfusion. These findings suggest that the infiltration of neutrophils do not contribute to ischaemia-reperfusion injury. Chapter 7 examined for the first time the role of the endogenous calcineurin inhibitor, Down syndrome candidate region 1 (DSCR1), on outcome following cerebral ischaemia-reperfusion. We found that the over-expression of DSCR1 improves neurological outcome, and reduces infarct and oedema volume. This protection was most likely through the inhibition of calcineurin in neurons and also potentially in T lymphocytes, and the subsequent reduction in pro-inflammatory mediators. Interestingly, we also found preliminary evidence that the deficiency of DSCR1 improves neurological outcome and reduces infarct and oedema volume. DSCR1-deficiency thus may also reduce calcineurin activity, however the precise mechanisms underlying this are still unclear. Overall, this thesis presents important findings on the role of inflammation in ischaemic injury following cerebral ischaemia-reperfusion, and supports the concept that strategies targeting inflammation in combination with recombinant t-PA may be a successful stroke therapy

Mechanisms of ischaemic stroke damage

Brain inflammation contributes to ischaemic and reperfusion injury, and thus worsens outcome after stroke. This thesis aimed to identify and investigate some of the inflammatory mechanisms that occur after cerebral ischaemia-reperfusion, to further enhance our understanding of them, and to potentially target them for future ischaemic stroke therapies. This study primarily used C57Bl6/J mice, as well as genetically modified mice. The model of focal cerebral ischaemia utilised was the intraluminal filament-induced middle cerebral artery occlusion. Real-time PCR and Western blotting were used to examine mRNA and protein expression levels, respectively, in the brain, and immunofluorescence and immunohistochemistry were used to localise proteins and cells in the brain. T lymphocytes were isolated from the blood and spleen using Dynal negative isolation kits, and T lymphocyte-generated superoxide was measured using L-012-enhanced chemiluminescence. Chapters 3 and 4 provided the first evidence that the larger infarct volume in males versus females following cerebral ischaemia is reperfusion-dependent and may be due to greater neuro-inflammation and brain infiltration of Nox2-expressing CD3+ T lymphocytes in male mice. Moreover, this gender difference was found to be dependent on Nox2 expression. The study also demonstrated for the first time that Nox2-expressing circulating CD3+ T lymphocytes produce ~15-fold more superoxide after stroke, compared to CD3+ T lymphocytes from control mice. These findings raise the possibility that therapies to reduce CD3+ T lymphocyte infiltration and/or the production of superoxide from these cells in ischaemic stroke patients who receive recombinant t-PA, might be useful for reducing reperfusion injury. Chapter 5 confirmed and extended the above findings and demonstrated for the first time that circulating Nox2-containing CD4+ and CD8+ T lymphocytes generate substantially higher levels of superoxide after cerebral ischaemia-reperfusion compared with similar T lymphocyte subsets from control mice. Chapter 6 demonstrated that the mRNA expression of various chemokines and chemokine receptors, including the potent neutrophil chemoattractants, CXCR2, CXCL1 and CXCL2, are increased in the brain after ischaemia-reperfusion. Administration of the CXCR2 antagonist, SB 225002, reduced the expression of these chemokines in the brain, as well as the infiltration of neutrophils, but did not improve outcome at 72 h after ischaemia-reperfusion. These findings suggest that the infiltration of neutrophils do not contribute to ischaemia-reperfusion injury. Chapter 7 examined for the first time the role of the endogenous calcineurin inhibitor, Down syndrome candidate region 1 (DSCR1), on outcome following cerebral ischaemia-reperfusion. We found that the over-expression of DSCR1 improves neurological outcome, and reduces infarct and oedema volume. This protection was most likely through the inhibition of calcineurin in neurons and also potentially in T lymphocytes, and the subsequent reduction in pro-inflammatory mediators. Interestingly, we also found preliminary evidence that the deficiency of DSCR1 improves neurological outcome and reduces infarct and oedema volume. DSCR1-deficiency thus may also reduce calcineurin activity, however the precise mechanisms underlying this are still unclear. Overall, this thesis presents important findings on the role of inflammation in ischaemic injury following cerebral ischaemia-reperfusion, and supports the concept that strategies targeting inflammation in combination with recombinant t-PA may be a successful stroke therapy

Selective Sphingosine 1-Phosphate Receptor 1 Agonist Is Protective Against Ischemia/Reperfusion in Mice

[Background and Purpose] Growing evidence supports that the immunomodulatory drug fingolimod is protective in stroke. Fingolimod binds to 4 of 5 sphingosine-1-phosphate (S1P) receptors and, among other actions, it induces lymphopenia. In this study, we investigated whether a selective S1P1 agonist is protective in experimental stroke. [Methods] Drug selectivity was studied in vitro in cells overexpressing the human S1P receptors. Mice (n=54) received different doses of LASW1238 (3 or 10 mg/kg), fingolimod (1 mg/kg), or the vehicle intraperitoneal, and lymphopenia was studied at different time points. After intraluminal middle cerebral artery occlusion for 45 minutes and immediately after reperfusion, mice (n=56) received the drug treatment. At 24 hours, a neurological test was performed and infarct volume was measured. Treatment and all the analyses were performed in a blind fashion. [Results] In vitro functional assays showed that LASW1238 is a selective agonist of the S1P1 receptor. At 10 mg/kg, this compound induced sustained lymphopenia in mice comparable with fingolimod, whereas at 3 mg/kg it induced short-lasting lymphopenia. After ischemia, both LASW1238 (10 mg/kg) and fingolimod reduced infarct volume, but only LASW1238 (10 mg/kg) showed statistically significant differences versus the vehicle. The neurological function and plasma cytokine levels were not different between groups. [Conclusions] The selective S1P1 agonist LASW1238 reduces infarct volume after ischemia/reperfusion in mice, but only when lymphopenia is sustained for at least 24 hours. S1P1 and lymphocytes are potential targets for drug treatment in stroke. Defining the best drug dosing regimens to control the extent and duration of lymphopenia is critical to achieve the desired effects.Peer Reviewe

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

Dendritic cells in brain diseases

Dendritic cells (DCs) are professional antigen presenting cells that constantly survey the environment acting as sentinels of the immune system, including in the CNS. DCs are strategically located near the cerebrospinal fluid, but they can potentiallymigrate to draining cervical lymph nodes either triggering immunogenic T cell responses or displaying tolerogenic functions. Under physiological conditions, the presence of DCs in the brain parenchyma is minimal but their numbers increase in neuroinflammation. Although DCs belong to a distinct immune cell lineage, they show various phenotypes and share certain common markers with monocytes, macrophages, and microglia. All these cells can expressmajor histocompatibility complex class II, and acquire similar morphologies hampering their precise identification. Neuroinflammation is increasingly recognized in many brain disorders; here we review the literature reporting DCs in the inflamed brain in disease conditions and corresponding animal models of multiple sclerosis, stroke, brain tumors, Alzheimer's disease, Parkinson's disease, and epilepsy. This article is part of a Special Issue entitled: Neuro Inflammation edited by Helga E. de Vries and Markus Schwaninger.Authors receive financial support by the European Community (FP7-PEOPLE-2013-ITN-n°07962, supporting M.G.; the ERA-NET program PRI-PIMNEU-2011-1342; and FP7-HEALTH-F2-2011-n°278850); the Werner-Otto-Stiftung (17/86, supporting S.B.); the University of Hamburg (NWF-15/07); and the Spanish Ministry of Economy MINECO (SAF2014-56279)Peer Reviewe

CD69 plays a beneficial role in ischemic stroke potentially via the modulation of leukocyte recruitment and secondary microthrombosis

Trabajo presentado en el Brain & BrainPET, celebrado en Canadá, del 27 al 30 de junio de 2015[Objectives] Expression of CD69 is a hallmark of lymphocyte activation and the majority of research on CD69 has focused on its effects on the immune system. Several lines of evidence support that inflammatory and immune responses are involved in stroke brain damage. The aim of this study was to examine whether CD69 plays a role in stroke outcome and to investigate the underlying mechanisms.[Methods] Cerebral ischemia was produced by 45-min intraluminal middle cerebral artery occlusion (tMCAo) followed by reperfusion in male CD69 KO (n=56) and Wt (n=68) mice. In addition, permanent distal MCAo (pMCAo) was induced in male CD69 KO (n=28) and Wt (n=41) mice and in Rag2-/- CD69-/- (n=27) and Rag2-/- CD69+/+ (n=31) mice. Neurological impairment was assessed and brain infarct and edema volume were measured using MRI (T2 maps). Flow cytometry was used to measure changes in immune cell populations in the brain, spleen, cervical lymph nodes (CLNs) and the blood. PCR was performed to measure changes in inflammatory gene expression in the brain. Circulating von Willebrand factor (vWF) levels (ELISA) and function (Collagen Binding Assay) were studied in plasma, and fibrin(ogen) deposition was also studied in cerebral blood vessels (Western Blot). In other mice, we performed the tail-bleeding test. Animal work was carried out in compliance with Spanish law and with approval of the Ethics Committee (CEEA) of the University of Barcelona. Results: CD69 KO mice had a significantly larger infarct volume compared to Wt mice at 24 and 72h after tMCAo (P<0.05). Also, CD69 deficiency increased infarct volume 24h after pMCAo (P<0.05). Following tMCAo, CD69 KO mice were more functionally impaired using a neurological score and tape test than the Wt mice. 96h after tMCAo, CD69 KO mice had a greater percentage of T cells (CD45hi CD3+) in the CLNs, spleen and brain compared to WT mice and less B cells (CD45+ CD45R+) in the CLNs. There was also a greater number of neutrophils (CD11b+ Ly6G+) in the brain. To test whether the absence of CD69 in lymphocytes was responsible for the observed effects, we carried out pMCAo in lymphocyte deficient Rag2-/- mice. Rag2-/- mice showed smaller infarct volumes than the Wt mice (P<0.001) and CD69 deficiency in Rag2-/- mice increased infarct volume (P<0.001) demonstrating that the absence of CD69 in cells other than lymphocytes was playing a role. In addition to lymphocytes, CD69 is expressed in platelets, which led us to hypothesize that CD69 deficiency might affect thrombosis. Preliminary findings in the tail-bleeding test showed a trend for less total bleeding time and less rebleeds in the CD69 KO mice compared to the Wt mice. In the plasma, the concentration and activity of vWF was higher 6h after pMCAo in CD69 KO mice compared to Wt mice (P<0.05). In cerebral blood vessels, CD69 deficiency enhanced vWF expression and fibrin(ogen) deposition after ischemia.[Conclusions] Our results suggest that CD69 may play a beneficial role in cerebral ischemia by regulating leukocyte recruitment and secondary local microthrombosis.Supported by the Spanish Ministry of Economy (SAF2011-30492).Peer Reviewe

Role of the S1P pathway and inhibition by fingolimod in preventing hemorrhagic transformation after stroke

Hemorrhagic transformation (HT) is a complication of severe ischemic stroke after revascularization. Patients with low platelet counts do not receive reperfusion therapies due to high risk of HT. The immunomodulatory drug fingolimod attenuated HT after tissue plasminogen activator in a thromboembolic stroke model, but the underlying mechanism is unknown. Fingolimod acts on several sphingosine-1-phosphate (S1P) receptors, prevents lymphocyte trafficking to inflamed tissues, and affects brain and vascular cells. This study aimed to investigate changes in S1P-signaling in response to brain ischemia/reperfusion and the effects of the S1P receptor modulator fingolimod on HT. We studied brain expression of S1P signaling components, S1P concentration, and immune cell infiltration after ischemia/reperfusion in mice. We administered fingolimod after ischemia to wild-type mice, lymphocyte-deficient Rag2 mice, and mice with low platelet counts. Ischemia increased S1P-generating enzyme SphK1 mRNA, S1P concentration, and S1P receptor-1 (S1P1) T-cells in the brain. Fingolimod prevented lymphocyte infiltration, and attenuated the severity of HT in Rag2 mice but it was ineffective under thrombocytopenia. Fingolimod prevented β-catenin degradation but not Evans blue extravasation. Ischemia/reperfusion upregulates brain S1P signaling pathway, and fingolimod exerts local effects that attenuate HT. Although fingolimod seems to act on the brain tissue, it did not prevent blood-brain barrier leakage.Supported by a grant from the Spanish Ministerio de Economía y Competitividad (SAF2017-87459-R to AMP), and a grant FIS (PI15/00430 to AC) co-financed by the Instituto de Salud Carlos III (ISCIII)-Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER).We are grateful to David Bellido from the Centres Científics i Tecnològics of the University of Barcelona for the mass spectrometry analyses. We thank the Pla Estratègic de Recerca i Innovació en Salut (PERIS) program of the Health Department of the Generalitat de Catalunya for supporting FMM, the Centre de Recerca Biomèdica Cellex, Barcelona, where part of this work was performed, and the Centres de Recerca de Catalunya (CERCA) Programme of the Generalitat de Catalunya for supporting the Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS)

Role of the S1P pathway and inhibition by fingolimod in preventing hemorrhagic transformation after stroke

Hemorrhagic transformation (HT) is a complication of severe ischemic stroke after revascularization. Patients with low platelet counts do not receive reperfusion therapies due to high risk of HT. The immunomodulatory drug fingolimod attenuated HT after tissue plasminogen activator in a thromboembolic stroke model, but the underlying mechanism is unknown. Fingolimod acts on several sphingosine-1-phosphate (S1P) receptors, prevents lymphocyte trafficking to inflamed tissues, and affects brain and vascular cells. This study aimed to investigate changes in S1P-signaling in response to brain ischemia/reperfusion and the effects of the S1P receptor modulator fingolimod on HT. We studied brain expression of S1P signaling components, S1P concentration, and immune cell infiltration after ischemia/reperfusion in mice. We administered fingolimod after ischemia to wild-type mice, lymphocyte-deficient Rag2−/− mice, and mice with low platelet counts. Ischemia increased S1P-generating enzyme SphK1 mRNA, S1P concentration, and S1P receptor-1 (S1P1)+ T-cells in the brain. Fingolimod prevented lymphocyte infiltration, and attenuated the severity of HT in Rag2−/− mice but it was ineffective under thrombocytopenia. Fingolimod prevented β-catenin degradation but not Evans blue extravasation. Ischemia/reperfusion upregulates brain S1P signaling pathway, and fingolimod exerts local effects that attenuate HT. Although fingolimod seems to act on the brain tissue, it did not prevent blood-brain barrier leakage