David Brea

The Increase of Circulating Endothelial Progenitor Cells After Acute Ischemic Stroke Is Associated With Good Outcome

Background and Purpose— Increased circulating endothelial progenitor cells (EPC) have been associated with a low cardiovascular risk and may be involved in endothelial cell regeneration. The present study was designed to evaluate the prognostic value of EPC in acute ischemic stroke. Methods— Forty-eight patients with a first-ever nonlacunar ischemic stroke were prospectively included in the study within 12 hours of symptoms onset. Stroke severity was evaluated by the National Institutes of Health Stroke Scale, and functional outcome was assessed at 3 months by the modified Rankin Scale (mRS). Infarct volume growth between admission and days 4 to 7 was measured on multiparametric MRI. EPC colonies were defined as early outgrowth colony-forming unit-endothelial cell (CFU-EC). The increment of CFU-EC was quantified during the first week and defined as the absolute difference between the number of CFU-EC at day 7 and admission. The influence of CFU-EC increase on good functional outcome (mRS ≤2) and infarct growth was analyzed by logistic regression and linear models. Results— Patients with good outcome (n=25) showed a higher CFU-EC increment during the first week (median [quartiles], 23 [11, 36] versus −3 [−7, 1], P<0.0001) compared with patients with poor outcome. CFU-EC increment ≥4 during the first week was associated with good functional outcome at 3 months (odds ratio, 30.7; 95% CI, 2.4 to 375.7; P=0.004) after adjustment for baseline stroke severity, ischemic volume and thrombolytic treatment. For each unit increase in the CFU-EC the mean reduction in the growth of infarct volume was 0.39 (0.03 to 0.76) mL (P=0.033). Conclusions— The increase of circulating EPC after acute ischemic stroke is associated with good functional outcome and reduced infarct growth. These findings suggest that EPC might participate in neurorepair after ischemic stroke.

SUBCLINICAL KERATOCONUS AND INFLAMMATORY MOLECULES FROM TEARS.

Background/aims: Tissue degradation in corneal thinning disorders, such as keratoconus (KC), involves the expression of inflammatory mediators. The purpose of this study was to determine the levels of proinflammatory cytokines and matrix metalloproteinase 9 (MMP-9) in tears from both eyes of unilateral keratoconus (KC) patients. Methods: Thirty patients diagnosed as having asymmetrical KC (30 KC eyes, and 30 subclinical KC eyes) and 20 normal control subjects (one eye) were studied in a prospective, cross-sectional study. Keratoconus screening programmes were performed on these participants. Ten microlitres of tears was collected from each eye. The concentrations of cytokines (interleukin-6 (IL-6) and tumour necrosis factor α (TNF-α)) and MMP-9 were measured by ELISA. Results: Mean values for IL-6 levels were similar in KC and subclinical KC samples (5.5 (4.9 to 6.9) vs 5.7 (4.5 to 6.2) pg/ml, p = 0.131), but significantly higher in relation to the control group (2.2 (1.0 to 4.1) pg/ml, p<0.0001). Significant differences were found in TNF-α levels between KC and subclinical KC eyes (5.4 (4.1 to 6.8) vs 4.8 (4.2 to 6.0) pg/ml, p = 0.032) and control group (1.8 (1.5 to 2.3) pg/ml, p<0.0001). Increased values of MMP-9 were found in KC (59.4 (50.6 to 66.1) ng/ml) vs subclinical KC eye (7.0 (4.8 to 8.6) ng/ml) (p<0.0001). MMP-9 levels in the control group (6.1 (3.9 to 8.3) ng/ml) and subclinical KC were similar (p = 0.203). Conclusions: IL-6 and TNF-α are overexpressed in the tears of subclinical and KC eyes. Increased MMP-9 levels were found only in the KC eye. These results indicate that the pathogenesis of KC may involve chronic inflammatory events.

Commensal microbiota affects ischemic stroke outcome by regulating intestinal γδ T cells

Commensal gut bacteria impact the host immune system and can influence disease processes in several organs, including the brain. However, it remains unclear whether the microbiota has an impact on the outcome of acute brain injury. Here we show that antibiotic-induced alterations in the intestinal flora reduce ischemic brain injury in mice, an effect transmissible by fecal transplants. Intestinal dysbiosis alters immune homeostasis in the small intestine, leading to an increase in regulatory T cells and a reduction in interleukin (IL)-17–positive γδ T cells through altered dendritic cell activity. Dysbiosis suppresses trafficking of effector T cells from the gut to the leptomeninges after stroke. Additionally, IL-10 and IL-17 are required for the neuroprotection afforded by intestinal dysbiosis. The findings reveal a previously unrecognized gut-brain axis and an impact of the intestinal flora and meningeal IL-17+ γδ T cells on ischemic injury.

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.

Toll-like receptors 2 and 4 in ischemic stroke: Outcome and therapeutic values

Stroke triggers an intense inflammatory response that could be a consequence of Toll-like receptors (TLRs) activation. However, the clinical significance and the therapeutic possibilities of TLR in stroke is not completely clear. In this study, we analyze the association between the expression of TLR2 and TLR4, inflammatory molecules and endogenous ligands, and clinical outcome of ischemic stroke patients, and we test the potential of TLR2/TLR4 and their endogenous ligands as therapeutic targets. For this purpose, we included 110 patients with ischemic stroke finding that TLR2 and TLR4 are independently associated to poor outcome and correlated with higher serum levels of interleukin (IL)1β, IL6, tumor necrosis factor α, and VCAM1, and that TLR4 was independently associated to lesion volume. In addition, we have developed an in vitro model to test the potential therapeutic value of blocking TLR2/TLR4 or their endogenous ligands. Cultured cells (monocytes and human umbilical vein endothelial cells) were treated with serum from ischemic stroke patients, showing a strong inflammatory response that was blocked when TLR2/4 or cellular fibronectin (cFN) or HSP60 were blocked. In conclusion, TLR2 and TLR4 are associated to outcome in stroke patients and TLR2/4 or their endogenous ligands, cFN/HSP60 could be new therapeutic targets for ischemic stroke.

Neuroprotection by glutamate oxaloacetate transaminase in ischemic stroke: an experimental study

As ischemic stroke is associated with an excessive release of glutamate into the neuronal extracellular space, a decrease in blood glutamate levels could provide a mechanism to remove it from the brain tissue, by increasing the brain-blood gradient. In this regard, the ability of glutamate oxaloacetate transaminase (GOT) to metabolize glutamate in blood could represent a potential neuroprotective tool for ischemic stroke. This study aimed to determine the neuroprotective effects of GOT in an animal model of cerebral ischemia by means of a middle cerebral arterial occlusion (MCAO) following the Stroke Therapy Academic Industry Roundtable (STAIR) group guidelines. In this animal model, oxaloacetate-mediated GOT activation inhibited the increase of blood and cerebral glutamate after MCAO. This effect is reflected in a reduction of infarct size, smaller edema volume, and lower sensorimotor deficits with respect to controls. Magnetic resonance spectroscopy confirmed that the increase of glutamate levels in the brain parenchyma after MCAO is inhibited after oxaloacetate-mediated GOT activation. These findings show the capacity of the GOT to remove glutamate from the brain by means of blood glutamate degradation, and suggest the applicability of this enzyme as an efficient and novel neuroprotective tool against ischemic stroke.

Inflammatory and Neuroimmunomodulatory Changes in Acute Cerebral Ischemia

Neuronal death produced by cerebral ischemia activates innate immunity by Toll-like receptors and triggers inflammatory response. This response is necessary to remove cell debris and to start regenerative process. However, inflammatory response could exacerbate cerebral damage and it is involved in secondary brain damage. Therefore, organisms have developed different mechanisms to regulate inflammatory response. An accurate balance between inflammation and anti-inflammation is necessary to assure the removal of cell debris and to avoid secondary cell damage. New therapeutic targets could be designed to obtain a correct modulation of the immune system and to reduce cerebral brain damage after cerebral ischemia. In this paper, we review the function of the immune system in cerebral ischemia, particularly inflammation and immunomodulation.

Inflammation as Therapeutic Objective in Stroke

Ischemic stroke is the most frequent cause of persistent neurologic disability in modern Western societies. Albeit it is still not clear whether inflammation is merely an epiphenomenon or rather has a disease-promoting function, accumulating evidence implicates inflammation in many forms of acute neurodegenerative disorders including ischemia. The immune cell influx during a neuropathological event is thought to be elicited by glial cells, especially microglia. This article reviews the cellular and molecular pathways involved in stroke-induced inflammatory response in the CNS. We focused on how CNS innate immune cells including microglia and macrophages play integral roles in receiving and propagating inflammatory signals, and how activated microglia secrete a wide range of factors. We present the relevance of the expression of adhesion molecules after ischemia including selectin, immunoglobulin superfamily, integrins, and the role of inflammatory mediators such as cytokines, chemokines and matrix metalloproteinases. Further, we explore the role of transcription factors in inflammation, and the function of immunomodulation and innate and adaptive immunity in brain ischemia, focusing on immunosupression therapies for acute stroke. Although several approaches for anti-inflammatory treatment have proven effective in animal models, clinical trials of immune system modulation therapy after stroke have not yet proved successful. There is still much to be done in order to translate interesting findings into therapies, but undoubtedly studying the cellular and molecular pathways may not only improve our understanding of inflammatory mechanism but also serve as a basis for designing effective therapies.

Endothelial progenitor cells A new key for endothelial dysfunction in migraine

Objective: We aimed to study endothelial function with biochemical and ultrasonographic markers and its relation with endothelial progenitor cells (EPCs) in patients with migraine. Methods: We performed a case-control study including 47 patients with episodic migraine (International Headache Society 2004 criteria) and 23 control subjects. We analyzed flow-mediated dilation (FMD) in the dominant brachial artery, calcitonin gene−related peptide (CGRP), and vascular endothelial growth factor (VEGF) levels by ELISA, nitric oxide stable metabolites (NOx) by high-performance liquid chromatography, and EPCs in peripheral blood samples, obtained during interictal periods (n = 47) and migraine attacks (n = 19). Frequency, severity, duration of attacks, and time of evolution of migraine were also recorded. Results: Patients with migraine showed lower numbers of EPCs than control subjects (9.4 ± 5.0 vs 17.9 ± 6.0 colony forming unit−endothelial cells [CFU-ECs]; p < 0.0001) and higher levels of CGRP (164.2 ± 139.1 vs 37.1 ± 38.5 pg/mL), VEGF (473.4 ± 398.7 vs 72.6 ± 56.6 pg/mL), and NOx (1225.2 ± 466.1 vs 671.9 ± 358.6 μM) (all p < 0.05). During attacks, higher levels for CGRP (298.2 ± 100.3 pg/mL) and NOx (1,656.8 ± 259.5 μM) and lower numbers of EPC (7.2 ± 3.2 CFU-ECs) were observed (all p < 0.05). No changes were found for FMD in interictal periods or during headache. In relation to clinical parameters, EPCs decreased with the time of evolution of migraine (r = −0.592; p < 0.0001). Conclusions: Patients with migraine show reduced numbers of EPCs and increased levels of CGRP, NOx, and VEGF than control subjects. Furthermore, EPC counts decrease as migraine progresses in time. These findings suggest altered endothelial function in patients with migraine.

Toll-like receptors 7 and 8 expression is associated with poor outcome and greater inflammatory response in acute ischemic stroke

Toll-like receptors are innate immunity receptors that activate inflammation and adaptive immunity. Our objectives were to analyze the association between TLR3, 7, 8 and 9 expressions and clinical outcome in patients with ischemic stroke and the expression of inflammatory molecules. One hundred-ten patients with ischemic stroke were included within 12h of symptoms onset. Stroke severity was evaluated by the NIHSS, and functional outcome was assessed at 3 months by the modified Rankin Scale. Infarct volume at 4-7 days was measured on Computed Tomography imaging. TLR7 and TLR8 at different time points were independently associated with poor outcome. TLR8 was also correlated with infarct volumes. Furthermore, TLR7 and TLR8 on admission were correlated with levels of IL6 and IL1β at 24h, 72 h and 7 days. In conclusion, TLR 7 and TLR8 expressions are associated with poor outcome and greater inflammatory response in acute ischemic stroke.