Background and Purpose-Neutrophils have been traditionally recognized as major mediators of a deleterious inflammatory response in acute ischemic stroke, but their potential as a therapeutic target remains unexplored. Recent evidence indicates that neutrophils may acquire different phenotypes and contribute to resolution of inflammation through the release of anti-inflammatory mediators. Thus, similar to M2 macrophages, neutrophils have been proposed to shift toward an N2 phenotype, a polarization that is peroxisome proliferator-activated receptor-γ dependent in macrophages. We hypothesize that peroxisome proliferator-activated receptor-γ activation with rosiglitazone induces changes in neutrophilic mobilization and phenotype that might influence stroke outcome. Methods-Brain sections and cell suspensions were prepared from mice exposed to permanent distal middle cerebral artery occlusion. Double immunostaining with stereological counting of brain sections and flow-cytometry analysis of brain cell suspensions were performed. Results-Rosiglitazone accelerated neutrophil infiltration to the ischemic core, concomitantly to neuroprotection. Some neutrophils (≈31%) expressed M2 markers, namely Ym1 and CD206 (mannose receptor). After treatment with the peroxisome proliferator-activated receptor-γ agonist rosiglitazone, most neutrophils (≈77%) acquired an N2 phenotype. Interestingly, rosiglitazone increased neutrophil engulfment by microglia/macrophages, a clearance that preferentially affected the N2 subset. Conclusions-We present the first evidence of neutrophil reprogramming toward an N2 phenotype in brain inflammation, which can be modulated by activation of the peroxisome proliferator-activated receptor-γ nuclear receptor. We also show that N2 polarization is associated with an increased neutrophil clearance, thus suggesting that this switch is a crucial event for resolution of inflammation that may participate in neuroprotection. (Stroke. 2013;44:3498-3508.)

Statin withdrawal is associated with increased risk of death or dependency at 90 days. Hence, this treatment should be continued in the acute phase of ischemic stroke.

Background and Purpose-Evidence is accumulating regarding the prognostic influence of hyperglycemia in patients with acute ischemic stroke. However, the level associated with poor outcome is unknown. Our objectives were to establish the capillary glucose threshold with the highest predictive accuracy of poor outcome and to evaluate its hypothetical value in influencing functional outcome by adjusting for other well-known prognostic factors in acute stroke. Methods-The authors conducted a multicenter, prospective, and observational cohort study of 476 patients with ischemic stroke within less than 24 hours from stroke onset. Capillary finger-prick glucose and stroke severity were determined on admission and 3 times a day during the first 48 hours. Poor outcome (modified Rankin Scale Ͼ2) was evaluated at 3 months. Results-The receiver operating characteristic curves showed the predictive value of maximum capillary glucose at any time within the first 48 hours with an area under the curve of 0.656 (95% CI, 0.592 to 0.720; PϽ0.01) and pointed to 155 mg/dL as the optimal cutoff level for poor outcome at 3 months (53% sensitivity; 73% specificity). This point was associated with a 2.7-fold increase (95% CI, 1.42 to 5.24) in the odds of poor outcome after adjustment for age, diabetes, capillary glucose on admission, infarct volume, and baseline stroke severity and with a 3-fold increase in the risk of death at 3 months (hazard ratio, 3.80; 95% CI, 1.79 to 8.10). Conclusions-Hyperglycemia Ն155 mg/dL at any time within the first 48 hours from stroke onset, and not only the isolated value of admission glycemia, is associated with poor outcome independently of stroke severity, infarct volume, diabetes, or age. (Stroke. 2009;40:562-568.)

Background and Purpose— Ischemic stroke continues to be one of the main causes of death worldwide. Inflammation accounts for a large part of damage in this pathology. The cannabinoid type 2 receptor (CB2R) has been proposed to have neuroprotective properties in neurological diseases. Therefore, our aim was to determine the effects of the activation of CB2R on infarct outcome and on ischemia-induced brain expression of classic and alternative markers of macrophage/microglial activation. Methods— Swiss wild-type and CB2R knockout male mice were subjected to a permanent middle cerebral artery occlusion. Mice were treated with either a CB2R agonist (JWH-133), with or without a CB2R antagonist (SR144528) or vehicle. Infarct outcome was determined by measuring infarct volume and neurological outcome. An additional group of animals was used to assess mRNA and protein expression of CB2R, interleukin (IL)-1β, IL-6, tumor necrosis factor α (TNF-α), monocyte chemoattractant protein–1 (MCP-1), macrophage inflammatory peptide (MIP) –1α, RANTES, inducible nitric oxide synthase (iNOS), cyclooxygenase-2, IL-4, IL-10, transforming growth factor β (TGF-β), arginase I, and Ym1. Results— Administration of JWH-133 significantly improved infarct outcome, as shown by a reduction in brain infarction and neurological impairment. This effect was reversed by the CB2R antagonist and was absent in CB2R knockout mice. Concomitantly, administration of JWH-133 led to a lower intensity of Iba1+ microglia/macrophages and a decrease in middle cerebral artery occlusion–induced gene expression of both classic (IL-6, TNF-α, MCP-1, MIP-1α, RANTES, and iNOS) and alternative mediators/markers (IL-10, TGF-β, and Ym1) of microglial/macrophage activation after permanent middle cerebral artery occlusion. Conclusions— The inhibitory effect of CB2R on the activation of different subpopulations of microglia/macrophages may account for the protective effect of the selective CB2R agonist JWH-133 after stroke.

Background and Purpose-Plasma levels of cellular fibronectin (c-Fn) Ն3.6 g/mL and of matrix metalloproteinase-9 (MMP-9) Ն140 ng/mL have been associated with parenchymal hematoma (PH) after treatment with tissue-type plasminogen activator (t-PA) in patients with acute ischemic stroke. In this prospective study, we sought to validate the predictive capacity of the preestablished cutoff values of these biomarkers for PH in a larger series of patients. Methods-We studied 134 patients treated with t-PA within 3 hours from symptom onset according to the SITS-MOST criteria (median time to infusion, 152 minutes; median National Institutes of Health Stroke Scale score, 14) in 4 university hospitals. Hemorrhagic transformation was classified according to the European-Australasian Acute Stroke Study II definitions on computed tomography scans performed 24 to 36 hours after treatment. Relevant hemorrhagic transformation was defined as hemorrhagic infarction type 2 or any PH. Serum c-Fn and MMP-9 levels were determined by an ELISA om blood samples obtained before treatment. Results-Cranial computed tomography showed hemorrhagic transformation in 27 patients (20%), hemorrhagic infarction in 15 (type 2 in 8 patients), and PH in 12 patients (symptomatic in 4). Serum c-Fn and MMP-9 concentrations at baseline were significantly higher in patients with relevant hemorrhagic transformation and PH than in those without (all PϽ0.001). The sensitivity, specificity, and positive and negative predictive values for PH by c-Fn levels Ն3.6 g/mL were 100%, 60%, 20%, and 100%, respectively, whereas corresponding values were 92%, 74%, 26%, and 99% for MMP-9 levels Ն140 ng/mL. When both biomarkers were at levels above the cutoff points, specificity increased to 87% and the positive predictive value increased to 41%. Conclusions-This prospective study confirmed the high sensitivity and negative predictive value, with retained good specificity, of c-Fn and MMP-9 for the prediction of PH in patients treated with t-PA. Development of faster analytic methods will prove the applicability of these biomarkers in routine clinical practice.

Background and Purpose-The goal of the present study was to examine a series of putative risk factors of poststroke dementia (PSD), especially those factors usually associated with cerebrovascular disease and degenerative dementia, in a series of 251 consecutive unselected stroke patients. Methods-A standard protocol was prospectively applied at admission and 3 months after stroke; this protocol included clinical, functional, and cognitive assessments, hemogram and serum biochemistry, ECG and CT exams, apolipoprotein E and angiotensin-converting enzyme genotype, and neuropsychological examination. After a neuropsychological examination and an interview with a relative, the following diagnostic criteria were used: the Diagnostic and Statistical Manual of Mental Disorders (DSM)-IV for dementia after stroke, DSM-III-R for previous dementia and dementia stage, and Association Internationale pour la Recherche et l'Enseignement en Neurologie (NINDS-AIREN) for vascular dementia. Results-Seventy-five cases (30%) demonstrated dementia at 3-month follow up; 25 of them (10%) had demonstrated dementia before the stroke. Dementia was unrelated to type (ischemic/hemorrhagic) or location of stroke, vascular factors (hypertension, diabetes, ischemic heart disease, or hypercholesterolemia), apolipoprotein E or angiotensinconverting enzyme genotype, and serum homocysteine. Age (odds ratio [OR] 1.1, 95% CI 1.03 to 1.2), previous nephropathy (OR 6.1, 95% CI 1.5 to 24.3), atrial fibrillation (OR 4.4, 95% CI 1.4 to 13.9), low Canadian Neurological Scale score at discharge (OR 0.5, 95% CI 0.4 to 0.6), and previous mental decline assessed by the shortened Spanish version of the Informant Questionnaire on Cognitive Decline in the Elderly (SS-IQCODE; OR 1.2, 95% CI 1.1 to 1.4) were the correlates of dementia in logistic regression analyses. The same risks factors were found when cases with previous dementia and with hemorrhagic stroke were excluded. Conclusions-Dementia is frequent after ischemic or hemorrhagic stroke. Age, nephropathy, atrial fibrillation, previous mental decline, and stroke severity independently contribute to the risk.

Excessive levels of extracellular glutamate in the nervous system are excitotoxic and lead to neuronal death. Glutamate transport, mainly by glutamate transporter GLT1/EAAT2, is the only mechanism for maintaining extracellular glutamate concentrations below excitotoxic levels in the central nervous system. We recently showed that neuroprotection after experimental ischemic preconditioning (IPC) involves, at least partly, the upregulation of the GLT1/EAAT2 glutamate transporter in astrocytes, but the mechanisms were unknown. Thus, we decided to explore whether activation of the nuclear receptor peroxisome proliferator-activated receptor (PPAR) gamma, known for its antidiabetic and antiinflammatory properties, is involved in glutamate transport. First, we found that the PPARgamma antagonist T0070907 inhibits both IPC-induced tolerance and reduction of glutamate release after lethal oxygen-glucose deprivation (OGD) (70.1%+/-3.4% versus 97.7%+/-5.2% of OGD-induced lactate dehydrogenase (LDH) release and 61.8%+/-5.9% versus 85.9%+/-7.9% of OGD-induced glutamate release in IPC and IPC+T0070907 1 mumol/L, respectively, n=6 to 12, P<0.05), as well as IPC-induced astrocytic GLT-1 overexpression. IPC also caused an increase in nuclear PPARgamma transcriptional activity in neurons and astrocytes (122.1%+/-8.1% and 158.6%+/-22.6% of control PPARgamma transcriptional activity, n=6, P<0.05). Second, the PPARgamma agonist rosiglitazone increased both GLT-1/EAAT2 mRNA and protein expression and [(3)H]glutamate uptake, and reduced OGD-induced cell death and glutamate release (76.3%+/-7.9% and 65.5%+/-15.1% of OGD-induced LDH and glutamate release in rosiglitazone 1 mumol/l, respectively, n=6 to 12, P<0.05). Finally, we have identified six putative PPAR response elements (PPREs) in the GLT1/EAAT2 promoter and, consistently, rosiglitazone increased fourfold GLT1/EAAT2 promoter activity. All these data show that the GLT1/EAAT2 glutamate transporter is a target gene of PPARgamma leading to neuroprotection by increasing glutamate uptake.

It remains unclear why some individuals are susceptible to excitotoxicity after stroke. A possible explanation is impaired glutamate uptake. We have found a highly prevalent polymorphism in the promoter of the glutamate transporter EAAT2 gene that abolishes a putative regulatory site for activator protein–2 (AP-2) and creates a new consensus binding site for the repressor transcription factor GC-binding factor 2 (GCF2). The mutant genotype is associated with increased plasma glutamate concentrations and with a higher frequency of early neurological worsening in human stroke. After transfection into astrocytes, the mutant promoter was not activated by AP-2 and was effectively repressed by GCF2, and its activity in the presence of GCF2 was reduced when compared with the AP-2–cotransfected wild-type promoter. We also show that GCF2 is expressed in ischemic rat brain, suggesting that decreased glutamate uptake occurs in individuals carrying the mutation after stroke. These findings may explain individual susceptibility to excitotoxic damage after stroke as well as the failure of glutamate antagonists in those patients without this polymorphism.