Ischemic depolarizing events, such as repetitive spontaneous periinfarct spreading depolarizations (PIDs), expand the infarct size after experimental middle cerebral artery (MCA) occlusion. This worsening may result from increased metabolic demand, exacerbating the mismatch between cerebral blood flow (CBF) and metabolism. Here, we present data showing that anoxic depolarization (AD) and PIDs caused vasoconstriction and abruptly reduced CBF in the ischemic cortex in a distal MCA occlusion model in mice. This reduction in CBF during AD increased the area of cortex with 20% or less residual CBF by 140%. With each subsequent PID, this area expanded by an additional 19%. Drugs that are known to inhibit cortical spreading depression (CSD), such as N-methyl-D-aspartate receptor antagonists MK-801 and 7-chlorokynurenic acid, and r-1 receptor agonists dextromethorphan and carbetapentane, did not reduce the frequency of PIDs, but did diminish the severity of episodic hypoperfusions, and prevented the expansion of severely hypoperfused cortex, thus improving CBF during 90 mins of acute focal ischemia. In contrast, AMPA receptor antagonist NBQX, which does not inhibit CSD, did not impact the deterioration in CBF. When measured 24 h after distal MCA occlusion, infarct size was reduced by MK-801, but not by NBQX. Our results suggest that AD and PIDs expand the CBF deficit, and by so doing negatively impact lesion development in ischemic mouse brain. Mitigating the vasoconstrictive neurovascular coupling during intense ischemic depolarizations may provide a novel hemodynamic mechanism of neuroprotection by inhibitors of CSD.

Objective The sphingosine-1-phosphate receptor agonist fingolimod (FTY720), that has shown efficacy in advanced multiple sclerosis clinical trials, decreases reperfusion injury in heart, liver and kidney. We therefore tested the therapeutic effects of fingolimod in several rodent models of focal cerebral ischemia. To assess the translational significance of these findings, we asked whether fingolimod improved long-term behavioral outcomes, whether delayed treatment was still effective, and whether neuroprotection can be obtained in a second species. Methods We used rodent models of middle cerebral artery occlusion and cell culture models of neurotoxicity and inflammation to examine the therapeutic potential and mechanisms of neuroprotection by fingolimod. Results In a transient mouse model, fingolimod reduced infarct size, neurological deficit, edema and the number of dying cells in the core and periinfarct area. Neuroprotection was accompanied by decreased inflammation, as fingolimod-treated mice had fewer activated neutrophils, microglia/macrophages, and ICAM-1-positive blood vessels. Fingolimod-treated mice showed a smaller infarct and performed better in behavioral tests up to 15 days after ischemia. Reduced infarct was observed in a permanent model even when mice were treated 4 hours after ischemic onset. Fingolimod also decreased infarct size in a rat model of focal ischemia. Fingolimod did not protect primary neurons against glutamate excitotoxicity or hydrogen peroxide, but decreased ICAM-1 expression in brain endothelial cells stimulated by TNFalpha. Interpretation These findings suggest that anti-inflammatory mechanisms, and possibly vasculo-protection, rather than direct effects on neurons, underlie the beneficial effects of fingolimod after stroke. S1P receptors are a highly promising target in stroke treatment.

We studied unique cerebral blood flow (CBF) responses to cortical spreading depression in mice using a novel two-dimensional CBF imaging technique, laser speckle flowmetry. Cortical spreading depression caused a triphasic CBF response in both rat and mouse cortex. In rats, mild initial hypoperfusion (approximately 75% of baseline) was followed by a transient hyperemia reaching approximately 220% of baseline. In mice, the initial hypoperfusion was pronounced (40-50% of baseline), and the anticipated hyperemic phase barely reached baseline. The duration of hypoperfusion significantly correlated with the duration of the DC shift. As a possible explanation for the pronounced hypoperfusion, mouse cerebral vessels showed enhanced resistance to relaxation by acetylcholine (3 M) after K +-induced preconstriction (20, 40,

The Tg2576 transgenic mouse model of human cerebral amyloid angiopathy is characterized by age-dependent cerebrovascular deposition of amyloid-beta (Abeta) starting from 9 months of age and progressively worsening to involve most pial arterioles by 18 months; soluble Abeta levels are elevated long before vascular deposition takes place in this model. It has been suggested that elevated soluble Abeta levels alone are sufficient to impair cerebral blood flow (CBF) regulation thereby contributing to the early progression of Alzheimer's disease. Using laser speckle flowmetry through an intact skull, we studied the impact of elevated soluble Abeta levels and vascular Abeta deposition on a wide range of CBF responses to evaluate vasodilation and vasoconstriction in young or aged Tg2576 mice. Nineteen-month-old Tg2576 with severe vascular Abeta deposits showed an attenuated hyperaemic response during hypercapnia and whisker stimulation compared to wild-type littermates. The anticipated increase in CBF due to isoflurane anaesthesia was also suppressed, as were the typical hypoperfusion responses during cortical spreading depression and alpha-chloralose anaesthesia. The responses of 8-month-old Tg2576 with elevated soluble Abeta levels, but without vascular Abeta deposition, did not differ from age-matched controls. In conclusion, our data suggest that vascular Abeta deposition is associated with impaired vasodilator as well as vasoconstrictor responses to a wide range of stimuli. These responses do not differ from controls when studied non-invasively prior to vascular Abeta deposition, thus challenging the view that elevated soluble Abeta levels are sufficient to cause cerebrovascular dysfunction.

Background Migraine is an independent risk factor for stroke. Mechanisms underlying this association are unclear. Familial hemiplegic migraine (FHM), a migraine subtype that also carries an increased stroke risk, is a useful model for common migraine phenotypes because of shared aura and headache features, trigger factors, and underlying glutamatergic mechanisms. Methods and Results Here, we show that FHM type 1 (FHM1) mutations in CaV2.1 voltage-gated Ca2+ channels render the brain more vulnerable to ischemic stroke. Compared to wild-type, two FHM1 mutant mouse strains developed earlier onset of anoxic depolarization and more frequent peri-infarct depolarizations, associated with rapid expansion of infarct core on diffusion-weighted MRI and larger perfusion deficits on laser speckle flowmetry. Cerebral blood flow required for tissue survival was higher in the mutants, leading to infarction with milder ischemia. As a result, mutants developed larger infarcts and worse neurological outcomes after stroke, which were selectively attenuated by a glutamate receptor antagonist. Conclusions We propose that enhanced susceptibility to ischemic depolarizations akin to spreading depression predisposes migraineurs to infarction during mild ischemic events, thereby increasing the stroke risk.

Background-Remnant lipoprotein particles (RLPs), products of lipolytic degradation of triglyceride-rich lipoprotein derived from VLDL, exert atherogenesis. In this study, we observed how RLPs induced cytotoxicity in human umbilical vein endothelial cells (HUVECs) and cilostazol prevented cell death. Methods and Results-RLPs were isolated from the plasma of hyperlipidemic patients by use of an immunoaffinity gel mixture of anti-apolipoprotein A-1 and anti-apolipoprotein B-100 monoclonal antibodies. RLPs (50 g/mL) significantly increased superoxide formation in HUVECs associated with elevated gp91phox mRNA and protein expression and Rac1 translocation, accompanied by increased production of tumor necrosis factor (TNF)-␣ and interleukin-1␤, DNA fragmentation, and cell death. Cilostazol (1 to 100 mol/L) significantly suppressed not only NAD(P)H oxidase-dependent superoxide production but also TNF-␣ and interleukin-1␤ release and restored viability. RLPs activated a lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), which was not inhibited by cilostazol. Treatment of HUVECs with monoclonal antibody for LOX-1 attenuated RLP-mediated production of superoxide, TNF-␣, and interleukin-1␤ and DNA fragmentation. Conclusions-RLPs stimulated NAD(P)H oxidase-dependent superoxide formation and induction of cytokines inHUVECs via activation of LOX-1, consequently leading to reduction in cell viability with DNA fragmentation, and cilostazol exerts a cell-protective effect by suppressing these variables.

Normobaric hyperoxia is under investigation as a treatment for acute ischaemic stroke. In experimental models, normobaric hyperoxia reduces cerebral ischaemic injury and improves functional outcome. The mechanisms of neuroprotection are still debated because, (i) inhalation of 100% O2 does not significantly increase total blood O2 content; (ii) it is not known whether normobaric hyperoxia increases O2 delivery to the severely ischaemic cortex because of its short diffusion distance; and (iii) hyperoxia may reduce collateral cerebral blood flow (CBF) to ischaemic penumbra because it can cause vasoconstriction. We addressed these issues using real-time two-dimensional multispectral reflectance imaging and laser speckle flowmetry to simultaneously and non-invasively determine the impact of normobaric hyperoxia on CBF and oxygenation in ischaemic cortex. Ischaemia was induced by distal middle cerebral artery occlusion (dMCAO) in normoxic (30% inhaled O2, arterial pO2 134 +/- 9 mmHg), or hyperoxic mice (100% inhaled O2 starting 15 min after dMCAO, arterial pO2 312 +/- 10 mmHg). Post-ischaemic normobaric hyperoxia caused an immediate and progressive increase in oxyhaemoglobin (oxyHb) concentration, nearly doubling it in ischaemic core within 60 min. In addition, hyperoxia improved CBF so that the area of cortex with < or =20% residual CBF was decreased by 45% 60 min after dMCAO. Furthermore, hyperoxia reduced the frequency of peri-infarct depolarizations (PIDs) by more than 60%, and diminished their deleterious effects on CBF and metabolic load. Consistent with these findings, infarct size was reduced by 45% in the hyperoxia group 2 days after 75 min transient dMCAO. Our data show that normobaric hyperoxia increases tissue O2 delivery, and that novel mechanisms such as CBF augmentation, and suppression of PIDs may afford neuroprotection during hyperoxia.

Background-Adiponectin is a fat-derived plasma protein that has beneficial actions on cardiovascular disorders. A low level of plasma adiponectin is associated with increased mortality after ischemic stroke; however, the causal role of adiponectin in ischemic stroke is unknown. Methods and Results-To explore the role of adiponectin in the development of acute cerebral injury, we subjected adiponectin-deficient (APN-KO) and wild-type (WT) mice to 1 hour of middle cerebral artery occlusion followed by 23 hours of reperfusion. APN-KO mice exhibited enlarged brain infarction and increased neurological deficits after ischemia-reperfusion compared with WT mice. Conversely, adenovirus-mediated supplementation of adiponectin significantly reduced cerebral infarct size in WT and APN-KO mice. APN-KO mice showed decreased cerebral blood flow during ischemia by laser speckle flowmetry methods. Adiponectin colocalized within the cerebral vascular endothelium under transient ischemic conditions by immunohistochemical analysis. Phosphorylation of endothelial nitric oxide synthase in ischemic brain tissues and the production of nitric oxide metabolites in plasma were attenuated in APN-KO mice compared with WT mice. Adenovirus-mediated administration of adiponectin stimulated endothelial nitric oxide synthase phosphorylation and nitric oxide metabolites during cerebral ischemia in both WT and APN-KO mice. Neuronal nitric oxide synthase expression during ischemia did not differ between WT and APN-KO mice. Adenovirusmediated delivery of adiponectin did not affect brain infarction in mice deficient in endothelial nitric oxide synthase. Conclusions-These data provide causal evidence that adiponectin exerts a cerebroprotective action through an endothelial nitric oxide synthase-dependent mechanism. Adiponectin could represent a molecular target for the prevention of ischemic stroke.