Evaluation and Characterization of Post-Stroke Lung Damage in a Murine Model of Cerebral Ischemia

After stroke and other brain injuries, there is a high incidence of respiratory complications such as pneumonia or acute lung injury. The molecular mechanisms that drive the brain-lung interaction post-stroke have not yet been elucidated. We performed transient middle cerebral artery occlusion (MCAO) and sham surgery on C57BL/6J mice and collected bronchoalveolar lavage fluid (BALF), serum, brain, and lung homogenate samples 24 h after surgery. A 92 proteins-panel developed by Olink Proteomics ® was used to analyze the content in BALF and lung homogenates. MCAO animals had higher protein concentration levels in BALF than sham -controls, but these levels did not correlate with the infarct volume. No alteration in alveolar-capillary barrier permeability was observed. A total of 12 and 14 proteins were differentially expressed between the groups (FDR < 0.1) in BALF and lung tissue homogenates, respectively. Of those, HGF, TGF-α, and CCL2 were identified as the most relevant to this study. Their protein expression patterns were verified by ELISA. This study confirmed that post-stroke lung damage was not associated with increased lung permeability or cerebral ischemia severity. Furthermore, the dysregulation of HGF, TGF-α, and CCL2 in BALF and lung tissue after ischemia could play an important role in the molecular mechanisms underlying stroke-induced lung damage

New candidate blood biomarkers potentially associated with white matter hyperintensities progression

We 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 studie

New candidate blood biomarkers potentially associated with white matter hyperintensities progression

We 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 studie

Blood Biomarker Panels for the Early Prediction of Stroke‐Associated Complications

Background Acute decompensated heart failure (ADHF) and respiratory tract infections (RTIs) are potentially life-threatening complications in patients experiencing stroke during hospitalization. We aimed to test whether blood biomarker panels might predict these complications early after admission. Methods and Results Nine hundred thirty-eight patients experiencing ischemic stroke were prospectively recruited in the Stroke-Chip study. Post-stroke complications during hospitalization were retrospectively evaluated. Blood samples were drawn within 6 hours after stroke onset, and 14 biomarkers were analyzed by immunoassays. Biomarker values were normalized using log-transformation and Z score. PanelomiX algorithm was used to select panels with the best accuracy for predicting ADHF and RTI. Logistic regression models were constructed with the clinical variables and the biomarker panels. The additional predictive value of the panels compared with the clinical model alone was evaluated by receiver operating characteristic curves. An internal validation through a 10-fold cross-validation with 3 repeats was performed. ADHF and RTI occurred in 19 (2%) and 86 (9.1%) cases, respectively. Three-biomarker panels were developed as predictors: vascular adhesion protein-1 >5.67, NT-proBNP (N-terminal pro-B-type natriuretic peptide) >4.98 and d-dimer >5.38 (sensitivity, 89.5%; specificity, 71.7%) for ADHF; and interleukin-6 >3.97, von Willebrand factor >3.67, and d-dimer >4.58 (sensitivity, 82.6%; specificity, 59.8%) for RTI. Both panels independently predicted stroke complications (panel for ADHF: odds ratio [OR] [95% CI], 10.1 [3-52.2]; panel for RTI: OR, 3.73 [1.95-7.14]) after adjustment by clinical confounders. The addition of the panel to clinical predictors significantly improved areas under the curve of the receiver operating characteristic curves in both cases. Conclusions Blood biomarkers could be useful for the early prediction of ADHF and RTI. Future studies should assess the usefulness of these panels in front of patients experiencing stroke with respiratory symptoms such as dyspnea

Circulating AQP4 Levels in Patients with Cerebral Amyloid Angiopathy-Associated Intracerebral Hemorrhage

Cerebral amyloid angiopathy (CAA) is a major cause of lobar intracerebral hemorrhage (ICH) in elderly patients. Growing evidence suggests a potential role of aquaporin 4 (AQP4) in amyloid-beta-associated diseases, including CAA pathology. Our aim was to investigate the circulating levels of AQP4 in a cohort of patients who had suffered a lobar ICH with a clinical diagnosis of CAA. AQP4 levels were analyzed in the serum of 60 CAA-related ICH patients and 19 non-stroke subjects by enzyme-linked immunosorbent assay (ELISA). The CAA-ICH cohort was divided according to the time point of the functional outcome evaluation: mid-term (12 +/- 18.6 months) and long-term (38.5 +/- 32.9 months) after the last ICH. Although no differences were found in AQP4 serum levels between cases and controls, lower levels were found in CAA patients presenting specific hemorrhagic features such as >= 2 lobar ICHs and >= 5 lobar microbleeds detected by magnetic resonance imaging (MRI). In addition, CAA-related ICH patients who presented a long-term good functional outcome had higher circulating AQP4 levels than subjects with a poor outcome or controls. Our data suggest that AQP4 could potentially predict a long-term functional outcome and may play a protective role after a lobar ICH

Noves estratègies per a la predicció i prevenció de la pneumònia associada a l'ictus

La pneumònia associada a l'ictus (SAP, stroke-associated pneumonia) és una de les complicacions més freqüents després de l'ictus, amb una incidència del 12%, i augmenta el risc de mortalitat i discapacitat dels pacients amb ictus, així com la durada de la seva estada a l'hospital. Els factors que la causen són tant clínics (disfàgia, comorbiditats, alta exposició a microorganismes per hospitalització) com biològics, degut a la immunosupressió sistèmica que es dóna després de l'ictus i que fa als pacients més susceptibles a patir infeccions. Aquesta tesi pretén estudiar la SAP des de diferents punts de vista, buscant noves estratègies per a la seva predicció i prevenció. Consta de 4 objectius, cadascun dels quals s'ha intentat assolir amb un estudi independent. El primer objectiu és el d'avaluar biomarcadors descrits a la literatura prèvia en un estudi prospectiu multicèntric, i les proteïnes SAA, CRP i MR-proADM han estat els biomarcadors seleccionats que han format panells amb alta sensibilitat per a la predicció i diagnòstic de la SAP, i que han millorat el model de regressió logística amb les variables clíniques rellevants. Tot i així, el poder predictiu d'aquests panells no és òptim, i és per això que es necessita descobrir nous biomarcadors per a la SAP. En això s'han centrat el segon i tercer objectiu d'aquesta tesi. El segon objectiu consistia en el descobriment de nous biomarcadors predictors de la malaltia mitjançant l'ús de tècniques -òmiques per a l'estudi de les 3 subpoblacions leucocitàries majoritàries en pacients que desenvolupen SAP. S'han descrit els neutròfils com a la subpoblació amb més alteracions en la seva expressió gènica i proteica. A més, s'han seleccionat alguns candidats interessants, com S100A8 o S100A12, però és necessària la seva validació en nous pacients i per altres tècniques. El tercer objectiu s'ha basat en l'anàlisi retrospectiu de biomarcadors sanguinis per a la predicció de dues complicacions associades a l'ictus amb símptomes comuns com són la insuficiència cardíaca aguda descompensada (ADHF, acute decompensated heart failure) i les infeccions de tracte respiratori (que inclouen la SAP). S'han identificat dos panells diferents de 3 biomarcadors cadascun, que eren capaços de millorar de manera significativa el model de regressió logística corresponent amb les variables clíniques. Per últim, en el quart objectiu s'ha volgut avaluar l'afectació que pateix el pulmó després de la isquèmia cerebral. Per aquest motiu, s'ha realitzat un estudi experimental en un model d'isquèmia cerebral de ratolí, i s'ha observat un increment d'un marcador de dany pulmonar com és l'augment de concentració de proteïna al fluid del rentat broncoalveolar (BALF, bronchoalveolar lavage fluid), així com la identificació de 3 molècules alterades a nivell pulmonar per la isquèmia cerebral com són HGF, TGF-α i CCL2. La troballa de biomarcadors predictors de la SAP, així com el coneixement en profunditat dels processos moleculars que es donen a nivell sistèmic després de l'ictus isquèmic pot tenir implicacions clíniques molt importants com són el descobriment de noves dianes terapèutiques per a la prevenció de la SAP o la selecció d'un grup de pacients amb alt risc de desenvolupar-la, aconseguit així reduir les taxes de mortalitat i discapacitat dels pacients amb ictus.La neumonía asociada al ictus (SAP, stroke-associated pneumonia) es una de las complicaciones más frecuentes después del ictus, con una incidencia del 12%, y aumenta el riesgo de mortalidad y discapacidad de los pacientes con ictus, así como duración de su estancia en el hospital. Los factores que la causan son tanto clínicos (disfagia, comorbilidades, alta exposición a microorganismos por hospitalización) como biológicos, debido a la inmunosupresión sistémica que se da después del ictus y hace a los pacientes más susceptibles a sufrir infecciones. Esta tesis pretende estudiar la SAP desde diferentes puntos de vista, buscando nuevas estrategias para su predicción y prevención. Consta de 4 objetivos, cada uno de los cuales se ha intentado cumplir en un estudio independiente. El primer objetivo es evaluar biomarcadores descritos en la literatura previa en un estudi prospectivo multicentrico, y las proteínas SAA, CRP y MR-proADM han sido biomarcadores seleccionados que han formado paneles con alta sensibilidad para la predicción y diagnóstico de la SAP, y que han mejorado el modelo de regresión logística con las variables clínicas relevantes. Sin embargo, el poder predictivo de estos paneles no es óptimo, y es por eso que se necesita descubrir nuevos biomarcadores para la SAP. En eso se han centrado el segundo y tercer objetivo de esta tesis. El segundo objetivo consistía en el descubrimiento de nuevos biomarcadores predictores de la enfermedad mediante el uso de técnicas -ómicas para el estudio de las 3 subpoblaciones leucocitarias mayoritarias en pacientes que desarrollan SAP. Se han descrito los neutrófilos con más alteraciones en su expresión génica y proteica. Además, se han seleccionado algunos candidatos interesantes, como S100A8 o S100A12, pero es necesaria su validación en nuevos pacientes y por otras técnicas. El tercer objetivo se ha basado en el análisis retrospectivo de biomarcadores sanguíneos para la predicción de dos complicaciones asociadas al ictus con síntomas comunes como son la insuficiencia cardíaca aguda descompensada (ADHF, acute decompensated heart failure) y las infecciones de tracto respiratorio (que incluyen la SAP). Se han identificado dos paneles distintos de 3 biomarcadores cada uno, que eren capaces de mejorar de manera significativa el modelo de regresión logística correspondiente con las variables clínicas. Por último, en el cuarto objetivo se ha querido evaluar la afectación que sufre el pulmón después de la isquemia cerebral. Para eso, se ha realizado un estudio experimental en un modelo de isquemia cerebral de ratón, y se ha observado un incremento de un marcador de daño pulmonar como es el aumento de concentración de proteína en el fluido del lavado broncoalveolar (BALF, bronchoalveolar lavage fluid), así como la identificación de 3 moléculas alterades a nivel pulmonar para la isquèmia cerebral como son HGF, TGF-α y CCL2. El hallazgo de biomarcadores predictores de la SAP, así como el conocimiento en profundidad de los procesos moleculares que se dan a nivel sistémico después del ictus isquémico pueden tener implicaciones clínicas muy importantes como son el descubrimiento de nuevas dianas terapéuticas para la prevención de la SAP o la selección de un grupo de pacientes con alto riesgo de desarrollarla, consiguiendo así reducir las tasas de mortalidad y discapacidad de los pacientes con ictus.Stroke-associated pneumonia (SAP) is one of the most common complications after stroke, with a frequency of 12%. It increases the risk of mortality and disability of stroke patients, as well as the length of stay at the hospital. The factors that lead to SAP are both clinical (dysphagia, comorbidities, high exposure to microorganisms) and biological, due to the systemic immunosuppression that occurs after a stroke, making these patients more prone to suffer infections. This doctoral thesis intends to study SAP from different points of view, searching for novel strategies for its prediction and prevention. It comprises 4 objectives, and each of them has been explored in an independent study. The first objective aims to evaluate biomarkers previously described in the literature in a prospective multicentric study. The selected biomarkers have been SAA, MR-proADM, and CRP, which have formed panels with high sensitivity for the prediction and early diagnosis of SAP. They have improved the logistic regression models with the relevant clinical variables. However, the predictive value of these panels is not optimal, The second objective consisted in the discovery of new biomarkers for the prediction of SAP through the use of -omics for the study of 3 leukocyte subpopulations. Neutrophils are the subpopulation that suffers more alterations at gene and protein levels in SAP patients. Furthermore, some interesting candidates have been explored, as S100A8 or S100A12, but it is needed their validation in new patients and using different techniques. The third objective is based on a retrospective analysis of blood biomarkers for the prediction of two stroke-associated complications with common symptoms as acute decompensated heart failure (ADHF) and respiratory tract infections (which include SAP). Two different panels have been identified, with 3 biomarkers each, which were able to improve significantly the corresponding logistic regression models with clinical variables. Last but not least, the fourth objective has evaluated the alterations that cerebral ischemia causes to the lungs. For this reason, an experimental study has been performed, using a model of cerebral ischemia in mice. It has been observed an increase of a marker of lung damage (higher levels of protein concentration in bronchoalveolar lavage fluid). In addition, 3 molecules have been identified as altered at a pulmonary level after the cerebral ischemia: HGF, CCL2, and TGF-α The finding of predictive biomarkers for SAP, as well as the deeper knowledge of the molecular processes that occur systemically after an ischemic stroke, can have important clinical implications. These implications could be the discovery of new therapeutic targets for the prevention of SAP and the selection of those patients at higher risk of developing SAP. With this, it could be possible to reduce the mortality and disability rates of stroke patients

Stroke-induced immunosuppression: implications for the prevention and prediction of post-stroke infections

Abstract Stroke produces a powerful inflammatory cascade in the brain, but also a suppression of the peripheral immune system, which is also called stroke-induced immunosuppression (SIIS). The main processes that lead to SIIS are a shift from a lymphocyte phenotype T-helper (Th) 1 to a Th2 phenotype, a decrease of the lymphocyte counts and NK cells in the blood and spleen, and an impairment of the defense mechanisms of neutrophils and monocytes. The direct clinical consequence of SIIS in stroke patients is an increased susceptibility to stroke-associated infections, which is enhanced by clinical factors like dysphagia. Among these infections, stroke-associated pneumonia (SAP) is the one that accounts for the highest impact on stroke outcome, so research is focused on its early diagnosis and prevention. Biomarkers indicating modifications in SIIS pathways could have an important role in the early prediction of SAP, but currently, there are no individual biomarkers or panels of biomarkers that are accurate enough to be translated to clinical practice. Similarly, there is still no efficient therapy to prevent the onset of SAP, and clinical trials testing prophylactic antibiotic treatment and β-blockers have failed. However, local immunomodulation could open up a new research opportunity to find a preventive therapy for SAP. Recent studies have focused on the pulmonary immune changes that could be caused by stroke similarly to other acquired brain injuries. Some of the traits observed in animal models of stroke include lung edema and inflammation, as well as inflammation of the bronchoalveolar lavage fluid

SAA (Serum Amyloid A): A Novel Predictor of Stroke-Associated Infections

BACKGROUND AND PURPOSE The aim of this study was to evaluate and independently validate SAA (serum amyloid A)-a recently discovered blood biomarker-to predict poststroke infections. METHODS The derivation cohort (A) was composed of 283 acute ischemic stroke patients and the independent validation cohort (B), of 367 patients. The primary outcome measure was any stroke-associated infection, defined by the criteria of the US Centers for Disease Control and Prevention, occurring during hospitalization. To determine the association of SAA levels on admission with the development of infections, logistic regression models were calculated. The discriminatory ability of SAA was assessed, by calculating the area under the receiver operating characteristic curve. RESULTS After adjusting for all predictors that were significantly associated with any infection in the univariate analysis, SAA remained an independent predictor in study A (adjusted odds ratio, 1.44 [95% CI, 1.16-1.79]; P=0.001) and in study B (adjusted odds ratio, 1.52 [1.05-2.22]; P=0.028). Adding SAA to the best regression model without the biomarker, the discriminatory accuracy improved from 0.76 (0.69-0.83) to 0.79 (0.72-0.86; P<0.001; likelihood ratio test) in study A. These results were externally validated in study B with an improvement in the area under the receiver operating characteristic curve, from 0.75 (0.70-0.81) to 0.76 (0.71-0.82; P<0.038). CONCLUSIONS Among patients with ischemic stroke, blood SAA measured on admission is a novel independent predictor of infection after stroke. SAA improved the discrimination between patients who developed an infection compared with those who did not in both derivation and validation cohorts. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT00390962

Influence of sex, age and diabetes on brain transcriptome and proteome modifications following cerebral ischemia

The online version contains supplementary material available at 10.1186/s12868-023-00775-