Dynamic release of neuronal extracellular vesicles containing miR-21a-5p is induced by hypoxia

Biomarkers; Hypoxia; NeuronBiomarcadores; Hipoxia; NeuronaBiomarcadors; Hipòxia; NeuronaHypoxia induces changes in the secretion of extracellular vesicles (EVs) in several non-neuronal cells and pathological conditions. EVs are packed with biomolecules, such as microRNA(miR)-21-5p, which respond to hypoxia. However, the true EV association of miR-21-5p, and its functional or biomarker relevance, are inadequately characterised. Neurons are extremely sensitive cells, and it is not known whether the secretion of neuronal EVs and miR-21-5p are altered upon hypoxia. Here, we characterised the temporal EV secretion profile and cell viability of neurons under hypoxia. Hypoxia induced a rapid increase of miR-21a-5p secretion in the EVs, which preceded the elevation of hypoxia-induced tissue or cellular miR-21a-5p. Prolonged hypoxia induced cell death and the release of morphologically distinct EVs. The EVs protected miR-21a-5p from enzymatic degradation but a remarkable fraction of miR-21a-5p remained fragile and non-EV associated. The increase in miR-21a-5p secretion may have biomarker potential, as high blood levels of miR-21-5p in stroke patients were associated with significant disability at hospital discharge. Our data provides an understanding of the dynamic regulation of EV secretion from neurons under hypoxia and provides a candidate for the prediction of recovery from ischemic stroke.We thank Benita Löflund and Pasi Laurinmäki (University of Helsinki) for technical assistance in cryoEM. The facilities and expertise of the HiLIFE CryoEM unit at the University of Helsinki, a member of Instruct-ERIC Centre Finland, FINStruct, and Biocenter Finland are gratefully acknowledged. This work was carried out with the support of UEF Cell and Tissue Imaging Unit, University of Eastern Finland, Finland. Moreover, we express our great appreciation to Seppo Ylä-Herttuala and Petri Mäkinen for the access to the NTA facilities (University of Eastern Finland, A.I. Virtanen Institute, Finland). Finally, we would like to extend our thanks to Dora Brites for the facilitation of the N9 cell line (University of Lisbon, Faculty of Farmacy, Portugal) and to Mark Ansel and Eric Wigton (University of California San Francisco, US) for technical help with HITS-clip sequencing. This work was supported by the University of Eastern Finland, Emil Aaltonen Foundation, Paavo Nurmi Foundation, Saastamoinen Foundation, Instrumentarium Science Foundation and Business Finland (Grant number 4399/31/2019). Work with clinical samples was supported by the European Regional Development Fund - Project INBIO (No. CZ.02.1.01/0.0/0.0/16_026/0008451). Work with EVQuant was supported by the IMMPROVE Alpe d'HuZes grant of the Dutch Cancer Society (EMCR2015-8022) and the Daniel den Hoed Foundation grant for Erasmus MC Cancer Treatment Screening Facility. L.R. is supported by a predoctoral fellowship grant (IFI17/00012) and J.M. is the principal investigator of the grant PI18/804 ‘MULTI-BIO-TARGETS: a new strategy for stroke management combining outcome biomarkers and neuroprotection’, both from the Instituto de Salud Carlos III

Antibiotic Class and Outcome in Post-stroke Infections: An Individual Participant Data Pooled Analysis of VISTA-Acute

Antibiotics; Post-stroke infections; Post-stroke pneumoniaAntibiòtics; Infeccions posteriors a un accident cerebrovascular; Pneumònia posterior a un accident cerebrovascularAntibióticos; Infecciones posteriores a un accidente cerebrovascular; Neumonía posterior a un accidente cerebrovascularIntroduction: Antibiotics used to treat post-stroke infections have differing antimicrobial and anti-inflammatory effects. Our aim was to investigate whether antibiotic class was associated with outcome after post-stroke infection. Methods: We analyzed pooled individual participant data from the Virtual International Stroke Trials Archive (VISTA)-Acute. Patients with ischemic stroke and with an infection treated with systemic antibiotic therapy during the first 2 weeks after stroke onset were eligible. Antibiotics were grouped into eight classes, according to antimicrobial mechanism and prevalence. The primary analysis investigated whether antibiotic class for any infection, or for pneumonia, was independently associated with a shift in 90 day modified Rankin Scale (mRS) using ordinal logistic regression. Results: 2,708 patients were eligible (median age [IQR] = 74 [65 to 80] y; 51% female; median [IQR] NIHSS score = 15 [11 to 19]). Pneumonia occurred in 35%. Treatment with macrolides (5% of any infections; 9% of pneumonias) was independently associated with more favorable mRS distribution for any infection [OR (95% CI) = 0.59 (0.42 to 0.83), p = 0.004] and for pneumonia [OR (95% CI) = 0.46 (0.29 to 0.73), p = 0.001]. Unfavorable mRS distribution was independently associated with treatment of any infection either with carbapenems, cephalosporins or monobactams [OR (95% CI) = 1.62 (1.33 to 1.97), p < 0.001], penicillin plus β-lactamase inhibitors [OR (95% CI) = 1.26 (1.03 to 1.54), p = 0.025] or with aminoglycosides [OR (95% CI) = 1.73 (1.22 to 2.46), p = 0.002]. Conclusion: This retrospective study has several limitations including effect modification and confounding by indication. Macrolides may have favorable immune-modulatory effects in stroke-associated infections. Prospective evaluation of the impact of antibiotic class on treatment of post-stroke infections is warranted.The Open Access Publication Fund of Charité—Universitätsmedizin Berlin and Professor Meisel as corresponding author will provide funding to cover the open access publication/article processing fe

Serum S-100B adds incremental value for the prediction of symptomatic intracranial hemorrhage and brain edema after acute ischemic stroke

Brain edema; Intracranial hemorrhage; Serum biomarkerEdema cerebral; Hemorragia intracraneal; Biomarcador séricoEdema cerebral; Hemorràgia intracranial; Biomarcador sèricBackground: Early identification of patients developing symptomatic intracranial hemorrhage and symptomatic brain edema after acute ischemic stroke is essential for clinical decision-making. Astroglial protein S-100B is a marker of blood-brain barrier disruption, which plays an important role in the formation of intracranial hemorrhage and brain edema. In this study, we assessed the prognostic value of serum S-100B for the development of these complications. Methods: Serum S-100B levels were measured within 24 h from symptom onset in 1749 consecutive acute ischemic stroke patients from the prospective, observational, multicenter BIOSIGNAL cohort study (mean age 72.0 years, 58.3% male). To determine symptomatic intracranial hemorrhage or symptomatic brain edema, follow-up neuroimaging was performed in all patients receiving reperfusion therapy or experiencing clinical worsening with an NIHSS increase of ⩾4. Results: Forty six patients (2.6%) developed symptomatic intracranial hemorrhage and 90 patients (5.2%) developed symptomatic brain edema. After adjustment for established risk factors, log10S-100B levels remained independently associated with both symptomatic intracranial hemorrhage (OR 3.41, 95% CI 1.7–6.9, p = 0.001) and symptomatic brain edema (OR 4.08, 95% CI 2.3–7.1, p < 0.001) in multivariable logistic regression models. Adding S-100B to the clinical prediction model increased the AUC from 0.72 to 0.75 (p = 0.001) for symptomatic intracranial hemorrhage and from 0.78 to 0.81 (p < 0.0001) for symptomatic brain edema. Conclusions: Serum S-100B levels measured within 24 h after symptom onset are independently associated with the development of symptomatic intracranial hemorrhage and symptomatic brain edema in acute ischemic stroke patients. Thus, S-100B may be useful for early risk-stratification regarding stroke complications.This study was supported with research grants from the Swiss National Science Foundation (142422), the Swiss Heart Foundation, the Göhner Foundation and the Swiss Seaside Foundation

Blood biomarker changes following therapeutic hypothermia in ischemic stroke

Biomarkers; Hypothermia; IschemiaBiomarcadores; Hipotermia; IsquemiaBiomarcadors; Hipotèrmia; IsquèmiaIntroduction Therapeutic hypothermia is a promising candidate for stroke treatment although its efficacy has not yet been demonstrated in patients. Changes in blood molecules could act as surrogate markers to evaluate the efficacy and safety of therapeutic cooling. Methods Blood samples from 54 patients included in the EuroHYP-1 study (27 treated with hypothermia, and 27 controls) were obtained at baseline, 24 ± 2 h, and 72 ± 4 h. The levels of a panel of 27 biomarkers, including matrix metalloproteinases and cardiac and inflammatory markers, were measured. Results Metalloproteinase-3 (MMP-3), fatty-acid-binding protein (FABP), and interleukin-8 (IL-8) increased over time in relation to the hypothermia treatment. Statistically significant correlations between the minimum temperature achieved by each patient in the hypothermia group and the MMP-3 level measured at 72 h, FABP level measured at 24 h, and IL-8 levels measured at 24 and 72 h were found. No differential biomarker levels were observed in patients with poor or favorable outcomes according to modified Rankin Scale scores. Conclusion Although the exact roles of MMP3, FABP, and IL-8 in hypothermia-treated stroke patients are not known, further exploration is needed to confirm their roles in brain ischemia

Association of candidate genetic variants and circulating levels of ApoE/ApoJ with common neuroimaging features of cerebral amyloid angiopathy

MRI; LipoproteinsRessonància magnètica; LipoproteïnesResonancia magnética; LipoproteínasIntroduction: Cerebral amyloid angiopathy (CAA) is characterized by the accumulation of amyloid-β (Aβ) in brain vessels and is a main cause of lobar intracerebral hemorrhage (ICH) in the elderly. CAA is associated with magnetic resonance imaging (MRI) markers of small vessel disease (SVD). Since Aβ is also accumulated in Alzheimer’s disease (AD) in the brain parenchyma, we aimed to study if several single nucleotide polymorphisms (SNPs) previously associated with AD were also associated with CAA pathology. Furthermore, we also studied the influence of APOE and CLU genetic variants in apolipoprotein E (ApoE) and clusterin/apolipoprotein J (ApoJ) circulating levels and their distribution among lipoproteins. Methods: The study was carried out in a multicentric cohort of 126 patients with lobar ICH and clinical suspicion of CAA. Results: We observed several SNPs associated with CAA neuroimaging MRI markers [cortical superficial siderosis (cSS), enlarged perivascular spaces in the centrum semiovale (CSO-EPVS), lobar cerebral microbleeds (CMB), white matter hyperintensities (WMH), corticosubcortical atrophy and CAA-SVD burden score]. Concretely, ABCA7 (rs3764650), CLU (rs9331896 and rs933188), EPHA1 (rs11767557), and TREML2 (rs3747742) were significantly associated with a CAA-SVD burden score. Regarding circulating levels of apolipoproteins, protective AD SNPs of CLU [rs11136000 (T) and rs9331896 (C)] were significantly associated with higher HDL ApoJ content in the lobar ICH cohort. APOEε2 carriers presented higher plasma and LDL-associated ApoE levels whereas APOEε4 carriers presented lower plasma ApoE levels. Additionally, we observed that lower circulating ApoJ and ApoE levels were significantly associated with CAA-related MRI markers. More specifically, lower LDL-associated ApoJ and plasma and HDL-associated ApoE levels were significantly associated with CSO-EPVS, lower ApoJ content in HDL with brain atrophy and lower ApoE content in LDL with the extent of cSS. Discussion: This study reinforces the relevance of lipid metabolism in CAA and cerebrovascular functionality. We propose that ApoJ and ApoE distribution among lipoproteins may be associated with pathological features related to CAA with higher ApoE and ApoJ levels in HDL possibly enhancing atheroprotective, antioxidative, and anti-inflammatory responses in cerebral β-amyloidosis.This work was funded by the Instituto de Salud Carlos III (ISCIII), (PI17/00275, PI019/00421, PI20/00465, and PI20/00334) and co-financed by the European Regional Development Fund (FEDER). The Neurovascular Research Laboratory was part of the RICORS-ICTUS-Enfermedades Vasculares Cerebrales Network, ISCIII, Spain (RD21/0006/0007). CIBERDEM (CB07/08/0016) was an ISCIII Project

Predictive model for atrial fibrillation in hypertensive diabetic patients

Diabetes; Hpertension; Prediction modelsDiabetes; Hipertensión; Modelos de predicciónDiabetis; Hipertensió; Models de prediccióBackground Several scores to identify patients at high risk of suffering atrial fibrillation have been developed. Their applicability in hypertensive diabetic patients, however, remains uncertain. Our aim is to develop and validate a diagnostic predictive model to calculate the risk of developing atrial fibrillation at five years in a hypertensive diabetic population. Methods The derivation cohort consisted of patients with both hypertension and diabetes attended in any of the 52 primary healthcare centres of Barcelona; the validation cohort came from the 11 primary healthcare centres of Terres de l’Ebre (Catalonia South) from January 2013 to December 2017. Multivariable Cox regression identified clinical risk factors associated with the development of atrial fibrillation. The overall performance, discrimination and calibration of the model were carried out. Results The derivation data set comprised 54 575 patients. The atrial fibrillation rate incidence was 15.3 per 1000 person/year. A 5-year predictive model included age, male gender, overweight, heart failure, valvular heart disease, peripheral vascular disease, chronic kidney disease, number of antihypertensive drugs, systolic and diastolic blood pressure, heart rate, thromboembolism, stroke and previous history of myocardial infarction. The discrimination of the model was good (c-index = 0.692; 95% confidence interval, 0.684-0.700), and calibration was adequate. In the validation cohort, the discrimination was lower (c-index = 0.670). Conclusions The model accurately predicts future atrial fibrillation in a population with both diabetes and hypertension. Early detection allows the prevention of possible complications arising from this disease

Fine-tuning a brain spheroid model as a screening platform for neuroprotective therapies against ischemic stroke.

Motivation: Stroke is the second cause of death and a leading cause of disability worldwide. Ischemic stroke represents more than 80% of all events and it is due to the presence of a clot blocking the blood flow. European legislation has already assumed the goal of no longer needing to use animals for scientific research in the future. To improve current in vitro models, a two-stage 3D brain spheroid model was previously described to recreate the blood-brain barrier and serve as a tool to assess ischemic damage and neuroinflammation. Our objective is to fine tune this organoid model, so we can use it as a screening platform of different neuroprotective therapies against ischemic stroke. Methods: Six different human brain cells were cultured following manufacturer´s recommendations. In order to be able to visualize each cell type in the 3D model, immunocytochemistry protocols were stablished. Cells were fixed and immunolabeled using a suitable primary antibody for each type of cell followed by the appropriate Alexa 568 secondary antibody. Negative controls were also prepared following the same protocol without the addition of the primary antibody. As a fist approach for spheroid formation, astrocytes, microglia, oligodendrocytes and cortical neurons were co-cultured in round bottom 96 well plates building the spheroid core. After 48h microvascular cells and pericytes were added to the culture creating the surface area. Finally, spheroids images were taken employing bright field microscopy and diameter was measured. Results: Fluorescent images confirmed that the immunolabeling protocols for each cell type were set up appropriately. Red fluorescence staining indicated cell´s detection by their specific markers. Fluorescent signal was absent in negative controls, confirming the specific binding. Finally, two-stage spheroids (4.000 cells) were formed. After 48, one round-shaped spheroid per well was observed. Spheroid diameter was 313.9± 22 μm after 48 h and 690.9±64 μm after 4 days. Conclusions: We have fine-tuned the immunocytochemical technique to identify each cell line that will be used to analyse brain spheroids. The first trial for spheroid formation showed homogenous shape and size. Next step would be testing smaller spheroids (2.000 cells) and verifying that each cell type is correctly located in the 3D structure. If results are satisfactory these spheroids could be used as a tool to test potential neuroprotective therapies

Prenatal Stroke: Detection and Neuroprotection

Neonatal stroke is the most common cause of death and disability in newborns and is often associated with persistent motor, sensory and cognitive impairments. Recently, great efforts have been made towards neuroprotection with bioactive compounds and because of to the promising results found in adult stroke patients; we consider evaluating it in the pediatric population. In this line of research, our group has developed a neonatal hypoxic-ischemic (HI) mouse model in which the supplementation of pregnant females with olive leaf extract (OLE) is being tested as a neuroprotection tool against the potencial ischemic damage. Through studies of histology, immunohistochemistry and behavior test, we are evaluating the degree of damage between the control group and the one subjected to HI in the presence and absence of supplementation with OLE. On the other hand, the discovery of biomarkers is key for the early diagnosis of neonatal stroke, which would favor the immediate application of neuroprotective therapy with the aim of minimizing the possible sequelae. Based on this, the second part of the project is the collection and analysis of plasma and dried blood samples (as a minimally invasive sample) from each of the different groups under study. Neonatal stroke is a disease with a high emotional burden for patients as well as their families, aside from severe repercussions that have a significant impact on the quality of life of both patients and their families. The design of a neuroprotective strategy and an early diagnosis protocol for perinatal stroke will represent a great advance in the prevention of brain damage and help to increase the life expectancy and the standard of living of this population

New candidate blood biomarkers potentially associated with white matter hyperintensities progression

Barrera hematoencefàlica; Interaccions neurovasculars; NeurociènciaBarrera hematoencefálica; Interacciones neurovasculares; NeurocienciaBlood-brain barrier; Neuro-vascular interactions; NeuroscienceWe aimed to discover blood biomarkers associated with longitudinal changes in white matter hyperintensities (WMH). This study was divided into a discovery phase and a replication phase. Subjects in both studies were patients with hypertension, aged 50-70, who underwent two magnetic resonance imaging (MRI) sessions and blood extractions over a 4-year follow-up period. In the discovery phase, we screened 1305 proteins in 12 subjects with WMH progression and in 12 matched control subjects. We found that 41 proteins were differentially expressed: 13 were upregulated and 28 were downregulated. We subsequently selected three biomarkers for replication in baseline and follow-up samples in 80 subjects with WMH progression and in 80 control subjects. The selected protein candidates for the replication were MMP9 (matrix metalloproteinase-9), which was higher in cases, MET (hepatocyte growth factor receptor) and ASAH2 (neutral ceramidase), which were both lower in cases of WMH progression. Baseline biomarker concentrations did not predict WMH progression. In contrast, patients with WMH progression presented a steeper decline in MET over time. Furthermore, cases showed higher MMP9 and lower ASAH2 levels than controls at the follow-up. These results indicate that MMP9, MET, and ASAH2 are potentially associated with the progression of WMH, and could therefore be interesting candidates to validate in future studies.Funds were obtained from the Instituto de Salud Carlos III (Grant Numbers: PI14/01535, PI17/02222, ICI14/307, PI19/00217, CP15/00010, and JR15/00032), incorporation of scientists and technicians to research groups (PERIS, SLT006/17/00266) and the AGAUR (FI_DGR 2017, Grant Number 2017_FI_B 00064), with the support of the Secretary of Universities and Research (Department of Economy and Knowledge, Generalitat de Catalunya), and was cofinanced by the European Regional Development Fund. The neurovascular research laboratory receives funds from the Spanish research stroke network (RD/16/0019/0021)

Peripheral inflammation preceeding ischemia impairs neuronal survival through mechanisms involving miR‐127 in aged animals

Envelliment; Inflamació; MicroARNEnvejecimiento; Inflamación; MicroARNAging; Inflammation; MicroRNAIschemic stroke, the third leading cause of death in the Western world, affects mainly the elderly and is strongly associated with comorbid conditions such as atherosclerosis or diabetes, which are pathologically characterized by increased inflammation and are known to influence the outcome of stroke. Stroke incidence peaks during influenza seasons, and patients suffering from infections such as pneumonia prior to stroke exhibit a worse stroke outcome. Earlier studies have shown that comorbidities aggravate the outcome of stroke, yet the mediators of this phenomenon remain obscure. Here, we show that acute peripheral inflammation aggravates stroke‐induced neuronal damage and motor deficits specifically in aged mice. This is associated with increased levels of plasma proinflammatory cytokines, rather than with an increase of inflammatory mediators in the affected brain parenchyma. Nascent transcriptomics data with mature microRNA sequencing were used to identify the neuron‐specific miRNome, in order to decipher dysregulated miRNAs in the brains of aged animals with stroke and co‐existing inflammation. We pinpoint a previously uninvestigated miRNA in the brain, miR‐127, that is highly neuronal, to be associated with increased cell death in the aged, LPS‐injected ischemic mice. Target prediction tools indicate that miR‐127 interacts with several basally expressed neuronal genes, and of these we verify miR‐127 binding to Psmd3. Finally, we report reduced expression of miR‐127 in human stroke brains. Our results underline the impact of peripheral inflammation on the outcome of stroke in aged subjects and pinpoint molecular targets for restoring endogenous neuronal capacity to combat ischemic stroke.This study was supported by Emil Aaltonen Foundation, Academy of Finland and Finnish Cultural Foundation