J. Vivancos

Statin treatment withdrawal in ischemic stroke A controlled randomized study

Background: Pretreatment with statins has been shown to reduce brain injury in cerebral ischemia. In this controlled randomized study, we investigated the influence of statin pretreatment and its withdrawal on the outcome of acute ischemic stroke patients. Methods: From 215 patients admitted within 24 hours of a hemispheric ischemic stroke, 89 patients on chronic statin treatment were randomly assigned either to statin withdrawal for the first 3 days after admission (n = 46) or to immediately receive atorvastatin 20 mg/day (n = 43). The primary outcome event was death or dependency (modified Rankin Scale [mRS] score > 2) at 3 months. Early neurologic deterioration (END) and infarct volume at days 4 to 7 were secondary outcome variables. In a secondary analysis, outcome variables were compared with the nonrandomized patients without previous statin therapy (n = 126). Results: Patients with statin withdrawal showed a higher frequency of mRS score > 2 at the end of follow-up (60.0% vs 39.0%; p = 0.043), END (65.2% vs 20.9%; p < 0.0001), and greater infarct volume (74 [45, 126] vs 26 [12, 70] mL; p = 0.002) compared with the non–statin-withdrawal group. Statin withdrawal was associated with a 4.66 (1.46 to 14.91)–fold increase in the risk of death or dependency, a 8.67 (3.05 to 24.63)–fold increase in the risk of END, and an increase in mean infarct volume of 37.63 mL (SE 10.01; p < 0.001) after adjusting for age and baseline stroke severity. Compared with patients without previous treatment with statins, statin withdrawal was associated with a 19.01 (1.96 to 184.09)–fold increase in the risk of END and an increase in mean infarct volume of 43.51 mL (SE 21.91; p = 0.048). Conclusion: 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.

The Prognostic Value of Capillary Glucose Levels in Acute Stroke The GLycemia in Acute Stroke (GLIAS) Study

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 >2) 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; P<0.01) 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 ≥155 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.

Ischemic Preconditioning Reveals that GLT1/EAAT2 Glutamate Transporter is a Novel PPARγ Target Gene Involved in Neuroprotection

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)γ, known for its antidiabetic and antiinflammatory properties, is involved in glutamate transport. First, we found that the PPARγ 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 μmol/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 PPARγ transcriptional activity in neurons and astrocytes (122.1% ± 8.1% and 158.6% ± 22.6% of control PPARγ transcriptional activity, n = 6, P < 0.05). Second, the PPARγ agonist rosiglitazone increased both GLT-1/EAAT2 mRNA and protein expression and [3H]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 μmol/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 PPARγ leading to neuroprotection by increasing glutamate uptake.

Cannabinoid Type 2 Receptor Activation Downregulates Stroke-Induced Classic and Alternative Brain Macrophage/Microglial Activation Concomitant to Neuroprotection

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)-1&bgr;, IL-6, tumor necrosis factor &agr; (TNF-&agr;), monocyte chemoattractant protein–1 (MCP-1), macrophage inflammatory peptide (MIP) –1&agr;, RANTES, inducible nitric oxide synthase (iNOS), cyclooxygenase-2, IL-4, IL-10, transforming growth factor &bgr; (TGF-&bgr;), 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-&agr;, MCP-1, MIP-1&agr;, RANTES, and iNOS) and alternative mediators/markers (IL-10, TGF-&bgr;, 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.

Serum Cellular Fibronectin and Matrix Metalloproteinase-9 as Screening Biomarkers for the Prediction of Parenchymal Hematoma After Thrombolytic Therapy in Acute Ischemic Stroke: A Multicenter Confirmatory Study

Background and Purpose— Plasma levels of cellular fibronectin (c-Fn) ≥3.6 μg/mL and of matrix metalloproteinase-9 (MMP-9) ≥140 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 P<0.001). The sensitivity, specificity, and positive and negative predictive values for PH by c-Fn levels ≥3.6 μg/mL were 100%, 60%, 20%, and 100%, respectively, whereas corresponding values were 92%, 74%, 26%, and 99% for MMP-9 levels ≥140 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.

Rosiglitazone and 15.deoxy-Δ12,14-prostaglandin J2 cause potent neuroprotection after experimental stroke through noncompletely overlapping mechanisms

Stroke triggers an inflammatory cascade which contributes to a delayed cerebral damage, thus implying that antiinflammmatory strategies might be useful in the treatment of acute ischaemic stroke. Since two unrelated peroxisome proliferator-activated receptor-γ (PPARγ) agonists, the thiazolidinedione rosiglitazone (RSG) and the cyclopentenone prostaglandin 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), have been shown to possess antiinflammatory properties, we have tested their neuroprotective effects in experimental stroke. Rosiglitazone or 15d-PGJ2 were administered to rats 10 mins or 2 h after permanent middle cerebral artery occlusion (MCAO). Stroke outcome was evaluated by determination of infarct volume and assesment of neurological scores. Brains were collected for protein expression, gene array analyses and gene shift assays. Our results show that both compounds decrease MCAO-induced infarct size and improve neurological scores. At late times, the two compounds converge in the inhibition of MCAO-induced brain expression of inducible NO synthase and the matrix metalloproteinase 9. Interestingly, at early times, complementary DNA microarrays and gene shift assays show that different mechanisms are recruited. Analysis of early nuclear p65 and late cytosolic IκBα protein levels shows that both compounds inhibit nuclear factor-κB signalling, although at different levels. All these results suggest both PPARγ-dependent and independent pathways, and might be useful to design both therapeutic strategies and prognostic markers for stroke.

The Prediction of Malignant Cerebral Infarction by Molecular Brain Barrier Disruption Markers

Background and Purpose— Space-occupying brain edema is a life-threatening complication in patients with large hemispheric stroke. The aim of the study was to determine whether molecular markers of endothelial damage may help to predict secondary brain edema and, secondly, to identify patients who could benefit from aggressive therapies such as decompressive hemicraniectomy or hypothermia. Methods— We studied 40 consecutive patients with malignant middle cerebral artery (MCA) infarction and 35 controls with massive MCA infarctions <70 years of age and matched by stroke severity on admission. Cranial computed tomography (CT) was performed at entry and repeated between days 4 and 7, or earlier if there was neurological worsening. Malignant MCA (m-MCA) infarction was diagnosed when follow-up CT detected a more than two-thirds space-occupying MCA infarction with midline shift, compression of the basal cisterns, and neurological deterioration. Plasma concentrations of glutamate, glycine, γ-aminobutyric acid, interleukin-6 (IL-6), IL-10, tumor necrosis factor-α, matrix metalloproteinase-9 (MMP-9), and cellular-fibronectin (c-Fn) were determined in blood samples obtained at admission. Results— Mean time from stroke onset to blood sampling was 6.3±4.8 in m-MCA and 7.7±6.0 hours in the control group (P=0.63). Baseline characteristics were comparable in both groups. c-Fn and MMP-9 levels were significantly higher in patients with m-MCA than in controls (all P<0.001). c-Fn >16.6 &mgr;g/mL had the highest sensitivity (90%), specificity (100%), and negative and positive predictive values (89% and 100%, respectively) for the prediction of m-MCA infarction. Conclusions— A plasma c-Fn concentration >16.6 &mgr;g/mL at admission is associated with the development of m-MCA infarction with high sensitivity and specificity, suggesting that c-Fn might be useful in therapeutic decision making.

Evolution of Cognitive Impairment After Stroke and Risk Factors for Delayed Progression

Background and Purpose— Cognitive decline occurs in ≈30% of stroke patients. Acute risk factors have been identified, but long-term risk has not been examined in large samples. The purpose of this research was to determine factors associated with the progression of cognitive impairment after stroke. Methods— Consecutive stroke patients (193) without previous dementia were assessed 3 months after stroke with an extensive neuropsychological battery and diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders Fourth Edition criteria and the Clinical Dementia Rating as normal (139), cognitive decline without dementia (18), or dementia (18 mild, 10 moderate, and 8 severe). After a 24-month follow-up, they were classified as stable, progressing, or improving, according to change in Clinical Dementia Rating score. The determinants of progression of cognitive decline were ascertained by logistic regression analysis of all clinical, neuroimaging, and complementary data. Results— Cognitive status at 24 months was stable in most cases (151; 78.2%), decline progressed in 27 (14%; 6 demented and 21 nondemented), and improved in 15 (7.8%; 7 demented and 8 nondemented). Seven nondemented patients became demented at 24 months, and 5 demented became nondemented. The age (odds ratio [OR], 1.05; 95% CI, 1.01 to 1.1), mental decline before stroke (OR, 1.14; 95% CI, 1.02 to 1.27), number of prescribed drugs (OR, 1.34; 95% CI, 1.05 to 1.72), diastolic blood pressure on admission (OR, 0.96; 95% CI, 0.93 to 0.99), and episodes of hypotension during admission (OR, 7.61; 95% CI, 1.11 to 52.1) were significantly associated with cognitive deterioration. Conclusions— Cognition is rather stable for 2 years after stroke. Both progression and improvement of cognitive impairment are frequent in demented patients. Age, previous cognitive decline, polypharmacy, and hypotension during admission are risk factors for progression.

A polymorphism in the EAAT2 promoter is associated with higher glutamate concentrations and higher frequency of progressing stroke

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.

N2 Neutrophils, Novel Players in Brain Inflammation After Stroke Modulation by the PPARγ Agonist Rosiglitazone

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-&ggr; dependent in macrophages. We hypothesize that peroxisome proliferator-activated receptor-&ggr; 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-&ggr; 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-&ggr; 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.