Utilidad del estudio Doppler transcraneal en la detección de microembolias cerebrales

Introducción Las intervenciones terapéuticas, tanto en la fase aguda del ictus como en la prevención secundaria del infarto cerebral, requieren un diagnóstico etiológico preciso. La incorporación de técnicas diagnósticas más sofisticadas como la ultrasonografía dúplex-color, el Doppler transcraneal (DTC) y la ecocardiografía transesofágica (ETE), han demostrado la importancia clínica del mecanismo embólico que ha sido aceptado como la causa más frecuente de infarto cerebral en los bancos de datos de ictus más modernos. Sin embargo la fuente potencial de embolia cerebral es desconocida en un porcentaje importante de pacientes. En la actualidad el interés se centra en considerar al embolismo cerebral como un fenómeno ocasionado por la oclusión de la arteria receptora del émbolo, independientemente de que la fuente del émbolo sea conocida (corazón, aorta o arterias proximales) o no haya sido determinada. La reciente introducción de técnicas Doppler transcraneal capaces de detectar la existencia de microembolias cerebrales (MEs) resultan muy esperanzadores y, aunque el significado de las MEs continúa siendo parcialmente desconocido, cada vez existen más evidencias que sugieren que la detección de este fenómeno puede tener implicaciones tanto pronósticas como terapéuticas. Objetivos del estudio Objetivo principal Determinar la utilidad clínica de la detección de microembolias cerebrales mediante Doppler transcraneal en pacientes con ictus isquémico agudo. Para ello se plantearon los objetivos concretos que detallamos a continuación y que desglosamos para cada etapa de estudio. Etapa I. Investigación I. Utilidad del DTC con contraste (DTC-c) en el diagnóstico del shunt derecha-izquierda (ShDI) en el adulto joven. 1. Analizar la prevalencia de ShDI en el ictus isquémico en adultos jóvenes. 2. Comparar la eficacia del ecocardiograma-transtorácico con contraste (ETT-c) respecto al DTC-c. 3. Evaluar la importancia del ShDI como factor de riesgo potencial de ictus isquémico criptogénico. Etapa II. Investigación II. Utilidad del DTC-c en el diagnóstico del ShDI: la necesidad de cuantificar la magnitud del ShDI. 1. Determinar la prevalencia de ShDI en la población sana. 2. Determinar la prevalencia de ShDI en el ictus isquémico agudo, sin sesgos de edad ni subtipo de ictus. 3. Determinar la importancia de la presencia del ShDI como factor de riesgo vascular. 4. Determinar la sensibilidad y especificidad del DTC-c respecto al ecocardiograma-transesofágico con contraste (ETE-c) en pacientes con ictus criptogénico. 5. Determinar la capacidad del DTC-c en la cuantificación de la magnitud del ShDI cardiaco. Etapa II. Investigación III. Detección de microembolias sólidas mediante DTC en el ictus isquémico agudo. 1. Estudiar la prevalencia de MEs en una población no sesgada de pacientes con ictus isquémico en fase aguda. 2. Detectar y confirmar la actividad embolígena de fuentes sospechadas o conocidas de ictus embólico. 3. Determinar el valor pronóstico de la detección de MEs en el ictus isquémico agudo: gravedad del ictus, existencia de recurrencias precoces y grado de dependencia al alta. Material y método El trabajo que presentamos en esta tesis se desarrolla en 2 etapas, que se diferencian fundamentalmente en el planteamiento de un objetivo más inmediato, con disponibilidad de una tecnología más elemental en la primera de ellas. Los resultados del primer estudio no llevaron a diseñar y desarrollar una investigación más amplia y ambiciosa tanto en los objetivos propuestos como en los medios utilizados. El desarrollo del proyecto y las publicaciones resultado de cada fase se resumen en la siguiente tabla:Introduction Therapeutic intervention, both in the acute phase of stroke and in the secondary prevention of ischaemic stroke, requires a precise aetiological diagnosis. The introduction of more sophisticated diagnostic techniques such as duplex colour ultrasonography, transcranial Doppler (TCD) and transoesophageal echocardiography (TEE) has shown the clinical importance of the embolic mechanism, which most modern stroke data banks show to be the most frequent cause of ischaemic stroke. However, the potential source of cerebral emboli is unknown in a high percentage of patients. Currently cerebral embolism is considered to be a phenomenon caused by an occlusion of the artery in which the embolus occurs, independently to whether or not the source of the embolus is known (heart, aorta, or proximal arteries). New transcranial Doppler techniques which are capable of detecting the existence of cerebral microemboli (MES) are giving encouraging results and although the full significance of MES is still not fully understood, it is becoming ever more apparent that the detection of this phenomenon may have both prognostic and therapeutic implications. Objectives of the study Primary objective To determine the clinical utility of the detection of cerebral microemboli through transcranial Doppler in patients with acute ischaemic stroke. In order to achieve this the following specific objectives, outlined for each stage of the study, were set. Stage I. Investigation I. The usefulness of TCD with contrast (TCD-c) in the diagnosis of Right-to-Left shunt (RLSh) in young adults. 4. To analyse the prevalence of RLSh in ischaemic stroke in young adults. 5. To compare the efficacy of transthoracic echocardiogram with contrast (TEE-c) with TCD-c. 6. To evaluate the importance of RLSh as a potential risk factor in cryptogenic ischaemic stroke. Stage II. Investigation II. The usefulness of TCD-c in the diagnosis of RLSh: the need to quantify RLSh. 6. To determine the prevalence of RLSh in a healthy population. 7. To determine the prevalence of RLSh in acute ischaemic stroke in all age groups and stroke subtypes. 8. To determine the importance of RLSh as a vascular risk factor. 9. To determine the sensibility and specificity of TCD-c with respect to the transoesophageal echocardiogram with contrast (TEE-c) in patients with cryptogenic stroke. 10. To determine the capacity of TCD-c in the quantification of the magnitude of cardiac RLSh. Stage II. Investigation III. The detection of solid microemboli through TCD in acute ischaemic stroke. 4. To study the prevalence of MES in a non-selected patient group with acute ischaemic stroke. 5. To detect and confirm the embolic origin of suspected or known sources of embolic strokes. 6. To determine the prognostic value of the detection of MES in acute ischaemic stroke: severity of stroke, early recurrence and dependency. Material and method The study which we present in this thesis is undertaken in 2 stages, which differ from one another essentially in that the first has a more immediate objective and uses more elementary and widely available technology. The results of the first study led us to design and develop a wider and more ambitious study both with respect to the aims which were proposed and the resources which were used. The development of the project and the resulting publications are summarised in the following table

Prevalence of the Frank's sign by aetiopathogenic stroke subtype: A prospective analysis

Enfermedades cerebrovasculares; Pabellón auricularCerebrovascular disorders; Ear auricleMalalties cerebrovasculars; Pavelló auricularBackground and purpose: The Frank's sign is a diagonal earlobe crease running from the tragus to the edge of the auricle at an angle of 45°. Many studies have associated this sign with coronary artery disease and some with cerebrovascular disease. The objective of this study was to analyse the prevalence of the Frank's sign in patients suffering from acute stroke with a particular focus on its prevalence in each of the five aetiopathogenic stroke subtypes. Special interest is given to embolic stroke of undetermined source (ESUS), correlating the sign with clinical and radiological markers that support an underlying causal profile in this subgroup. Methods: Cross-sectional descriptive study including 124 patients admitted consecutively to a stroke unit after suffering an acute stroke. The Frank's sign was evaluated by the same blinded member of the research team from photographs taken of the patients. The stroke subtype was classified following SSS-TOAST criteria and the aetiological study was performed following the ESO guidelines. Results: The Frank's sign was present in 75 patients and was more prevalent in patients with an ischaemic stroke in comparison with haemorrhagic stroke (63.9 vs. 37.5, p<0.05). A similar prevalence was found in the different ischaemic stroke subtypes. The Frank's sign was significantly associated with age, particularly in patients older than 70 who had vascular risk factors. Atherosclerotic plaques found in carotid ultrasonography were significantly more frequent in patients with the Frank's sign (63.6%, p<0.05). Analysing the ESUS, we also found an association with age and a higher prevalence of the Frank's sign in patients with vascular risk factors and a tendency to a high prevalence of atherosclerosis markers. Conclusion: The Frank's sign is prevalent in all aetiopathogenic ischaemic stroke subtypes, including ESUS, where it could be helpful in suspecting the underlying cardioembolic or atherothrombotic origin and guiding the investigation of atherosclerosis in patients with ESUS and the Frank's sign.JS: 3 -Spanish Ministry of Economy and Competitiveness for grants RETICS-INVICTUSPLUS (RD0016/0019/0003) funded by Instituto de Salud Carlos III and cofunded by the European Regional Development Fund [ERDF]. -Instituto de Salud Carlos III with a Grant for Health Research (PI16/01540) -Government of Catalonia-Agència de Gestio´ d’Ajuts Universitaris i de Recerca (2017 SGR 1730). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Validation of housekeeping genes for quantitative real-time PCR in in-vivo and in-vitro models of cerebral ischaemia

<p>Abstract</p> <p>Background</p> <p>Studies of gene expression in experimental cerebral ischaemia models can contribute to understanding the pathophysiology of brain ischaemia and to identifying prognostic markers and potential therapeutic targets. The normalization of relative qRT-PCR data using a suitable reference gene is a crucial prerequisite for obtaining reliable conclusions. No validated housekeeping genes have been reported for the relative quantification of the mRNA expression profile activated in in-vitro ischaemic conditions, whereas for the in-vivo model different reference genes have been used.</p> <p>The present study aims to determine the expression stability of ten housekeeping genes (Gapdh, β2m, Hprt, Ppia, Rpl13a, Oaz1, 18S rRNA, Gusb, Ywhaz and Sdha) to establish their suitability as control genes for in-vitro and in-vivo cerebral ischaemia models.</p> <p>Results</p> <p>The expression stability of the candidate reference genes was evaluated using the 2^-ΔC'Tmethod and ANOVA followed by Dunnett's test. For the in-vitro model using primary cultures of rat astrocytes, all genes analysed except for Rpl13a and Sdha were found to have significantly different levels of mRNA expression. These different levels were also found in the case of the in-vivo model of pMCAO in rats except for Hprt, Sdha and Ywhaz mRNA, where the expression did not vary. Sdha and Ywhaz were identified by geNorm and NormFinder as the two most stable genes.</p> <p>Conclusion</p> <p>We have validated endogenous control genes for qRT-PCR analysis of gene expression in in-vitro and in-vivo cerebral ischaemia models. For normalization purposes, Rpl13a and Sdha are found to be the most suitable genes for the in-vitro model and Sdha and Ywhaz for the in-vivo model. Genes previously used as housekeeping genes for the in-vivo model in the literature were not validated as good control genes in the present study, showing the need for careful evaluation for each new experimental setup.</p

Regional subsidence modelling in Murcia city (SE Spain) using 1-D vertical finite element analysis and 2-D interpolation of ground surface displacements

Subsidence is a hazard that may have natural or anthropogenic origin causing important economic losses. The area of Murcia city (SE Spain) has been affected by subsidence due to groundwater overexploitation since the year 1992. The main observed historical piezometric level declines occurred in the periods 1982–1984, 1992–1995 and 2004–2008 and showed a close correlation with the temporal evolution of ground displacements. Since 2008, the pressure recovery in the aquifer has led to an uplift of the ground surface that has been detected by the extensometers. In the present work an elastic hydro-mechanical finite element code has been used to compute the subsidence time series for 24 geotechnical boreholes, prescribing the measured groundwater table evolution. The achieved results have been compared with the displacements estimated through an advanced DInSAR technique and measured by the extensometers. These spatio-temporal comparisons have showed that, in spite of the limited geomechanical data available, the model has turned out to satisfactorily reproduce the subsidence phenomenon affecting Murcia City. The model will allow the prediction of future induced deformations and the consequences of any piezometric level variation in the study area.This work has been supported by the Spanish Ministry of Economy and Competitiveness and EU FEDER funds under projects ESP2013-47780-C2-2-R and TEC2011-28201-C02-02 and by the project 15224/PI/10 from the Regional Agency of Science and Technology in Murcia. The European Space Agency (ESA) Terrafirma project funded all the SAR data processing with the SPN technique

Modeling anti-IL-6 therapy using breast cancer patient-derived xenografts

The pleiotropic cytokine IL-6 accelerates the progression of breast cancer in a variety of preclinical models through the activation of the STAT3 (signal transducer and activator of transcription 3) signaling pathway. However, the proportion of breast cancers sensitive to anti-IL-6 therapies is not known. This study evaluates the efficacy of anti-IL-6 therapies using breast cancer patient derived xenografts (PDXs). During the generation of our collection of PDXs, we showed that the successful engraftment of tumor tissue in immunodeficient mice correlates with bad prognosis. Four PDXs out of six were resistant to anti-IL-6 therapies and the expression of IL-6, its receptor or the levels of phospho-STAT3 (the active form of the signal transducer) did not correlate with sensitivity. Using cell cultures established from the PDXs as well as samples from in vivo treatments, we showed that only tumors in which the activation of STAT3 depends on IL-6 respond to the blocking antibodies. Our results indicate that only a fraction of breast tumors are responsive to anti-IL-6 therapies. In order to identify responsive tumors, a functional assay to determine the dependence of STAT3 activation on IL-6 should be performed

Cav-1 Protein Levels in Serum and Infarcted Brain Correlate with Hemorrhagic Volume in a Mouse Model of Thromboembolic Stroke, Independently of rt-PA Administration.

Thrombolytic therapy with recombinant tissue plasminogen activator (rt-PA) is currently the only FDA-approved drug for acute ischemic stroke. However, its administration is still limited due to the associated increased risk of hemorrhagic transformation (HT). rt-PA may exacerbate blood-brain barrier (BBB) injury by several mechanisms that have not been fully elucidated. Caveolin-1 (Cav-1), a major structural protein of caveolae, has been linked to the endothelial barrier function. The effects of rt-PA on Cav-1 expression remain largely unknown. Here, Cav-1 protein expression after ischemic conditions, with or without rt-PA administration, was analyzed in a murine thromboembolic middle cerebral artery occlusion (MCAO) and in brain microvascular endothelial bEnd.3 cells subjected to oxygen/glucose deprivation (OGD). Our results show that Cav-1 is overexpressed in endothelial cells of infarcted area and in bEnd.3 cell line after ischemia but there is disagreement regarding rt-PA effects on Cav-1 expression between both experimental models. Delayed rt-PA administration significantly reduced Cav-1 total levels from 24 to 72 h after reoxygenation and increased pCav-1/Cav-1 at 72 h in the bEnd.3 cells while it did not modify Cav-1 immunoreactivity in the infarcted area at 24 h post-MCAO. Importantly, tissue Cav-1 positively correlated with Cav-1 serum levels at 24 h post-MCAO and negatively correlated with the volume of hemorrhage after infarction, the latter supporting a protective role of Cav-1 in cerebral ischemia. In addition, the negative association between baseline serum Cav-1 levels and hemorrhagic volume points to a potential usefulness of baseline serum Cav-1 levels to predict hemorrhagic volume, independently of rt-PA administration.This work was supported by grants from Instituto de Salud Carlos III and co-fnanced by the European Development Regional Fund “A Way to Achieve Europe” Health Strategic Action Program PI13/02258 and PI17/02123 (MC), PI20/00535 (IL), and Spanish Stroke Research Network RETICS RD12/0014/0010 (MC), and RD16/0019/0003 (JS), RD16/0019/0004 (MC), and RD16/0019/0009 (IL); from Regional Madrid Government B2017/BMD- 3688 (IL); from Spanish Ministry of Science and Innovation PID2019-106581RBI00 (MAM); from Leducq Foundation for Cardiovascular Research TNE-19CVD01 (MAM); and from Fundación La Caixa HR17_00527 (MAM). P. Comajoan was a recipient of a predoctoral fellowship from the University of Girona (IF-UdG 2015).S

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

Interleukin-10 facilitates the selection of patients for systemic thrombolysis

Background Clinical-Diffusion mismatch (CDM; NIHSS score ≥8 & DWI lesion volume ≤25 mL) and Perfusion-Diffusion mismatch (PDM; difference >20% between initial DWI and MTT lesion volumes) have been proposed as surrogates for ischemic brains that are at risk of infarction. However, their utility to improve the selection of patients for thrombolytic treatment remains controversial. Our aim was to identify molecular biomarkers that can be used with neuroimaging to facilitate the selection of ischemic stroke patients for systemic thrombolysis. Methods We prospectively studied 595 patients with ischemic stroke within 12 h of the stroke onset. A total of 184 patients received thrombolytic treatment according to the SITS-MOST criteria. DWI and MTT volumes were measured at admission. The main outcome variable was good functional outcome at 3 months (modified Rankin scale <3). Serum levels of glutamate (Glu), IL-10, TNF-α, IL-6, NSE, and active MMP-9 also were determined at admission. Results Patients treated with t-PA who presented with PDM had higher IL-10 levels at admission (p < 0.0001). In contrast, patients with CDM had higher levels of IL-10 (p < 0.0001) as well as Glu and TNF-α (all p < 0.05) and lower levels of NSE and active MMP-9 (all p < 0.0001). IL-10 ≥ 30 pg/mL predicts good functional outcome at 3 months with a specificity of 88% and a sensitibity of 86%. IL-10 levels ≥30 pg/mL independently in both patients with PDM (OR, 18.9) and CDM (OR, 7.5), after an adjustment for covariates. Conclusions Serum levels of IL-10 facilitate the selection of ischemic stroke patients with CDM and PDM for systemic thrombolysis.This project has been partially supported by grants from the Spanish Ministry of Science and Innovation (Fondo de Investigaciones Sanitarias, Instituto Salud Carlos III, RETICS-RD06/0026 and PI081472) and Xunta de Galicia (Consellería de Economía e Industria: 09CSA057918PR, Consellería de Sanidade: PS09/32)S

Anticoagulant vs. antiplatelet therapy in patients with cryptogenic stroke and patent foramen ovale: an individual participant data meta-analysis

Aims The preferred antithrombotic strategy for secondary prevention in patients with cryptogenic stroke (CS) and patent foramen ovale (PFO) is unknown. We pooled multiple observational studies and used propensity score-based methods to estimate the comparative effectiveness of oral anticoagulation (OAC) compared with antiplatelet therapy (APT). Methods and results Individual participant data from 12 databases of medically treated patients with CS and PFO were analysed with Cox regression models, to estimate database-specific hazard ratios (HRs) comparing OAC with APT, for both the primary composite outcome [recurrent stroke, transient ischaemic attack (TIA), or death] and stroke alone. Propensity scores were applied via inverse probability of treatment weighting to control for confounding. We synthesized database-specific HRs using random-effects meta-analysis models. This analysis included 2385 (OAC = 804and APT = 1581) patients with 227 composite endpoints (stroke/TIA/death). The difference between OAC and APT was not statistically significant for the primary composite outcome [adjusted HR = 0.76, 95% confidence interval (CI) 0.52-1.12] or for the secondary outcome of stroke alone (adjusted HR = 0.75, 95% CI 0.44-1.27). Results were consistent in analyses applying alternative weighting schemes, with the exception that OAC had a statistically significant beneficial effect on the composite outcome in analyses standardized to the patient population who actually received APT (adjusted HR = 0.64, 95% CI 0.42-0.99). Subgroup analyses did not detect statistically significant heterogeneity of treatment effects across clinically important patient groups. Conclusion We did not find a statistically significant difference comparing OAC with APT; our results justify randomized trials comparing different antithrombotic approaches in these patient

Association of High Serum Levels of Growth Factors with Good Outcome in Ischemic Stroke : a Multicenter Study

Altres ajuts: This project was partially supported by grants from Xunta de Galicia (Consellería Educación: GRC2014/027 and IN607A2018/3), Spanish Research Network on Cerebrovascular Diseases RETICS-INVICTUS PLUS (RD16/0019), and by the European Union FEDER program.The main objective of this research work was to study the association of serum levels of growth factors (GF) and SDF-1α with the functional outcome and reduction of lesion volume in ischemic stroke patients. In this multicenter study, 552 patients with non-lacunar stroke (male, 62.1%; mean age, 68.2 ± 11.4) were included within 24 h from symptom onset. The main outcome variable was good functional outcome (modified Rankin Scale [mRS] ≤ 2) at 12 months. Secondary outcome variable was infarct volume (in mL) after 6 ± 3 months. Serum levels of VEGF, Ang-1, G-CSF, BDNF, and SDF-1α were measured by ELISA at admission, 7 ± 1 days, at 3 ± 1 months, and 12 ± 3 months. Except for BDNF, all GF and SDF-1α serum levels showed a peak value at day 7 and remained elevated during the first 3 months (all p < 0.01). High serum levels at day 7 of VEGF (OR, 19.3), Ang-1 (OR, 14.7), G-CSF (OR, 9.6), and SDF-1α (OR, 28.5) were independently associated with good outcome at 12 months (all p < 0.0001). On the other hand, serum levels of VEGF (B, − 21.4), G-CSF (B, − 14.0), Ang-1 (B, − 13.3), and SDF-1α (B, − 44.6) measured at day 7 were independently associated with lesion volume at 6 months (p < 0.01). In summary, high serum levels of VEGF, Ang-1, G-CSF, and SDF-1α at day 7 and 3 months after ischemic stroke are associated with good functional outcome and smaller residual lesion at 1 year of follow-up