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Background and Purpose— Ischemic stroke is one of the leading causes of disability and death. The principal goal of acute stroke treatment is the recanalization of the occluded cerebral arteries, which is, however, only effective in a very narrow time window. Therefore, neuroprotective treatments that can be combined with recanalization strategies are needed. Calcium overload is one of the major triggers of neuronal cell death. We have previously shown that capacitative Ca 2+ entry, which is triggered by the depletion of intracellular calcium stores, contributes to ischemia-induced calcium influx in neurons, but the responsible Ca 2+ channel is not known. Methods— Here, we have generated mice lacking the calcium channel subunit Orai2 and analyzed them in experimental stroke. Results— Orai2-deficient mice were protected from ischemic neuronal death both during acute ischemia under vessel occlusion and during ischemia/reperfusion upon successful recanalization. Calcium signals induced by calcium store depletion or oxygen/glucose deprivation were significantly diminished in Orai2-deficient neurons demonstrating that Orai2 is a central mediator of neuronal capacitative Ca 2+ entry and is involved in calcium overload during ischemia. Conclusions— Our experimental data identify Orai2 as an attractive target for pharmaceutical intervention in acute stroke.

Supplemental Digital Content is available in the text., Background and Purpose— Acetylsalicylic acid and clopidogrel are the 2 main antithrombotic drugs for secondary prevention in patients with ischemic stroke (IS) without indication for anticoagulation. Because of their limited efficacy and potential side effects, novel antiplatelet agents are urgently needed. Cilostazol, a specific phosphodiesterase (PDE)-3 inhibitor, protected from IS in clinical studies comprising mainly Asian populations. Nevertheless, the detailed mechanistic role of PDE-3 inhibitors in IS pathophysiology is hardly understood. In this project, we analyzed the efficacy and pathophysiologic mechanisms of a novel and only recently described PDE-3 inhibitor (substance V) in a mouse model of focal cerebral ischemia. Methods— Focal cerebral ischemia was induced by transient middle cerebral artery occlusion in 6- to 8-week-old male C57Bl/6 wild-type mice receiving substance V or vehicle 1 hour after ischemia induction. Infarct volumes and functional outcomes were assessed between day 1 and day 7, and findings were validated by magnetic resonance imaging. Blood-brain barrier damage, as well as the extent of local inflammatory response and cell death, was determined. Results— Inhibition of PDE-3 by pharmacological blockade with substance V significantly reduced infarct volumes and improved neurological outcome on day 1 and 7 after experimental cerebral ischemia. Reduced blood-brain barrier damage, attenuated brain tissue inflammation, and decreased local cell death could be identified as potential mechanisms. PDE-3 inhibitor treatment did neither increase the number of intracerebral hemorrhages nor affect platelet function. Conclusions— The novel PDE-3 inhibitor substance V protected mice from IS independent from platelet function. Pharmaceutical inactivation of PDE-3 might become a promising therapeutic approach to combat IS via inhibition of thromboinflammatory mechanisms and stabilization of the blood-brain barrier.

Background and Purpose- Ischemic stroke is one of the leading causes of disability and death. The principal goal of acute stroke treatment is the recanalization of the occluded cerebral arteries, which is, however, only effective in a very narrow time window. Therefore, neuroprotective treatments that can be combined with recanalization strategies are needed. Calcium overload is one of the major triggers of neuronal cell death. We have previously shown that capacitative Ca

Background and Purpose— The selection of appropriate neurological scores and tests is crucial for the evaluation of stroke consequences. The validity and reliability of neurological deficit scores and tests has repeatedly been questioned in ischemic stroke models in the past. Methods— In 198 male mice exposed to transient intraluminal middle cerebral artery occlusion, we examined the validity and reliability of 11 neurological scores (Bederson score 0–3, Bederson score 0–4, Bederson score 0–5, modified neurological severity [0–14], subjective overall impression [0–10], or simple neurological tests: grip test, latency to move body length test, pole test, wire hanging test, negative geotaxis test, and elevated body swing test) in the acute stroke phase, that is, after 24 hours. Combinations of neurological scores or tests for predicting infarct volume were statistically analyzed. Results— Infarct volume was left skewed (median [Q1–Q3], 78.4 [54.8–101.3] mm 3 ). Among all tests, the Bederson (0–5; r=0.63, P P P P 3 ; r=0.33, P =0.027) and large (>50 mm 3 ; r=0.48, P Conclusions— Despite their simplicity, the Bederson (0–5) score, modified neurological severity score, and subjective overall impression have reasonable validity and reliability in the acute stroke phase. The Bederson (0–5) score equally distinguishes infarct volume in small and large infarcts. Visual Overview— An online visual overview is available for this article.

Background and Purpose— Despite the medical and socioeconomic effect of ischemic stroke and extensive preclinical research, treatment options for ischemic stroke are limited. We recently identified and characterized essential steps of thrombus formation in stroke and demonstrated that inhibition of the platelet glycoprotein (GP) receptors Ib and VI, but not IIb/IIIa, protects young and healthy mice from ischemic neurodegeneration. Whether these findings translate to the clinic remains unclear. Considering that the typical stroke patient is elderly with comorbidity, we aimed to analyze the efficacy and safety of novel preclinical antithrombotics in adult and comorbid mice with acute experimental stroke. Methods— We subjected adult, healthy, atherosclerotic ( Ldlr −/− ), diabetic (streptozotocin treated), and hypertensive ( RenTgMK ) mice to a 60-minute transient middle cerebral artery occlusion. Animals were pretreated with anti-GPVI antibodies or treated 1 hour after stroke induction with anti-GPIb or anti-GPIIb/IIIa antigen–binding fragments, respectively. Isotype treatment served as control. Twenty-four hours after transient middle cerebral artery occlusion, we visually assessed the intracerebral hemorrhage rate and measured infarct volumes (using 2,3,5-triphenyltetrazolium chloride–stained brain slices) and functional outcome (using Bederson and grip-test scores). Results— GPIb and GPVI inhibition protected the mice from ischemic stroke without increasing bleeding complications. In contrast, GPIIb/IIIa inhibition was not protective but increased the intracerebral hemorrhage rate. Conclusions— Inhibition of early steps of thrombus formation protects adult and comorbid mice from ischemic stroke. The use of clinically meaningful mouse strains might improve the translation of preclinical stroke research to the clinic.

Background and Purpose— T lymphocytes have recently been identified as key mediators of tissue damage in ischemic stroke. The interaction between very late antigen-4 (VLA-4) and vascular adhesion molecule-1 is crucial for the transvascular egress of T lymphocytes, and inhibition of this interaction by specific antibodies is a powerful strategy to combat autoimmune neuroinflammation. However, whether pharmacological blocking of T-lymphocyte trafficking is also protective during brain ischemia is still unclear. We investigated the efficacy of a monoclonal antibody directed against VLA-4 in mouse models of ischemic stroke. Methods— Transient and permanent middle cerebral artery occlusion was induced in male C57Bl/6 mice. Animals treated with a monoclonal anti-CD49d antibody (300 μg) 24 hours before or 3 hours after the onset of cerebral ischemia and stroke outcome, including infarct size, functional status, and mortality, were assessed between day 1 and day 7. The numbers of immune cells invading the ischemic brain were determined by immunocytochemistry and flow cytometry. Results— Blocking of VLA-4 significantly reduced the invasion of T lymphocytes and neutrophils on day 5 after middle cerebral artery occlusion and inhibited the upregulation of vascular adhesion molecule-1. However, the anti-CD49d antibody failed to influence stroke outcome positively irrespective of the model or the time point investigated. Conclusions— Pharmacological inhibition of the VLA-4/vascular adhesion molecule-1 axis in experimental stroke was ineffective in our hands. Our results cast doubt on the effectiveness of anti-CD49d as a stroke treatment. Further translational studies should be performed before testing anti–VLA-4 antibodies in patients with stroke.

Background and Purpose— Lymphocytes are important players in the pathophysiology of acute ischemic stroke. The interaction of lymphocytes with endothelial cells and platelets, termed thrombo-inflammation, fosters microvascular dysfunction and secondary infarct growth. FTY720, a sphingosine-1-phosphate receptor modulator, blocks the egress of lymphocytes from lymphoid organs and has been shown to reduce ischemic neurodegeneration; however, the underlying mechanisms are unclear. We investigated the mode of FTY720 action in models of cerebral ischemia. Methods— Transient middle cerebral artery occlusion (tMCAO) was induced in wild-type and lymphocyte-deficient Rag1 −/− mice treated with FTY720 (1 mg/kg) or vehicle immediately before reperfusion. Stroke outcome was assessed 24 hours later. Immune cells in the blood and brain were counted by flow cytometry. The integrity of the blood–brain barrier was analyzed using Evans Blue dye. Thrombus formation was determined by immunohistochemistry and Western blot, and was correlated with cerebral perfusion. Results— FTY720 significantly reduced stroke size and improved functional outcome in wild-type mice on day 1 and day 3 after transient middle cerebral artery occlusion. This protective effect was lost in lymphocyte-deficient Rag1 −/− mice and in cultured neurons subjected to hypoxia. Less lymphocytes were present in the cerebral vasculature of FTY720-treated wild-type mice, which in turn reduced thrombosis and increased cerebral perfusion. In contrast, FTY720 was unable to prevent blood–brain barrier breakdown and transendothelial immune cell trafficking after transient middle cerebral artery occlusion. Conclusions— Induction of lymphocytopenia and concomitant reduction of microvascular thrombosis are key modes of FTY720 action in stroke. In contrast, our findings in Rag1 −/− mice and cultured neurons argue against direct neuroprotective effects of FTY720.