Diesel exhaust particles exposure exacerbates pro-thrombogenic plasma features ex-vivo after cerebral ischemia and accelerates tPA-induced clot-lysis in hypertensive subjects.

The combustion of fossil fuels, mainly by diesel engines, generates Diesel Exhaust Particles (DEP) which are the main source of Particulate Matter (PM), a major air pollutant in urban areas. These particles are a risk factor for stroke with 5.6% of cases attributed to PM exposure. Our aim was to evaluate the effect of DEP exposure on clot formation and lysis in the context of stroke. An ex-vivo clot formation and lysis turbidimetric assay has been conducted in human and mouse plasma samples from ischemic stroke or control subjects exposed to DEP or control conditions. Experimental DEP exposure was achieved by nasal instillation in mice, or by ex-vivo exposure in human plasma. Results show consistent pro-thrombogenic features in plasma after human ischemic stroke and mouse cerebral ischemia (distal MCAo), boosted by the presence of DEP. Otherwise, thrombolysis times were increased after ischemia in chronically exposed mice but not in the DEP exposed group. Finally, subjects living in areas with high PM levels presented accelerated thrombolysis compared to those living in low polluted areas. Overall, our results point at a disbalance of the thrombogenic/lytic system in presence of DEP which could impact on ischemic stroke onset, clot size and thrombolytic treatment.

Distinct molecular profiles of skull bone marrow in health and neurological disorders.

The bone marrow in the skull is important for shaping immune responses in the brain and meninges, but its molecular makeup among bones and relevance in human diseases remain unclear. Here, we show that the mouse skull has the most distinct transcriptomic profile compared with other bones in states of health and injury, characterized by a late-stage neutrophil phenotype. In humans, proteome analysis reveals that the skull marrow is the most distinct, with differentially expressed neutrophil-related pathways and a unique synaptic protein signature. 3D imaging demonstrates the structural and cellular details of human skull-meninges connections (SMCs) compared with veins. Last, using translocator protein positron emission tomography (TSPO-PET) imaging, we show that the skull bone marrow reflects inflammatory brain responses with a disease-specific spatial distribution in patients with various neurological disorders. The unique molecular profile and anatomical and functional connections of the skull show its potential as a site for diagnosing, monitoring, and treating brain diseases.

Perspective Review of Myeloid Immune Cell Responses and Poststroke Immunosuppression.

Ischemic stroke profoundly influences the peripheral immune system, which responds quickly to brain ischemia and participates in the evolution of poststroke neuroinflammation, while a period of systemic immunosuppression ensues. Poststroke immunosuppression brings harmful consequences, including increased infection rates and escalated death. As the most abundant cell population in the fast-responding innate immune system, myeloid cells including neutrophils and monocytes play an indispensable role in systemic immunosuppression after stroke. The change in myeloid response after stroke can be regulated by circulating DAMPs (damage-associated molecular patterns) and neuromodulatory mechanisms, which contain sympathetic nervous system, hypothalamic-pituitary-adrenal, and parasympathetic nervous system. In this review, we summarize the emerging roles and newly identified mechanisms underlying myeloid cell response in poststroke immunosuppression. Deeper understanding of the above points may pave the way for future development of novel therapeutic strategies to treat poststroke immunosuppression.

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

Ischemic stroke is a major cause of death and disability worldwide. Translation into the clinical setting of neuroprotective agents showing promising results in pre-clinical studies has systematically failed. One possible explanation is that the animal models used to test neuroprotectants do not properly represent the population affected by stroke, as most of the pre-clinical studies are performed in healthy young male mice. Therefore, we aimed to determine if the response to cerebral ischemia differed depending on age, sex and the presence of comorbidities. Thus, we explored proteomic and transcriptomic changes triggered during the hyperacute phase of cerebral ischemia (by transient intraluminal middle cerebral artery occlusion) in the brain of: (1) young male mice, (2) young female mice, (3) aged male mice and (4) diabetic young male mice. Moreover, we compared each group's proteomic and transcriptomic changes using an integrative enrichment pathways analysis to disclose key common and exclusive altered proteins, genes and pathways in the first stages of the disease. We found 61 differentially expressed genes (DEG) in male mice, 77 in females, 699 in diabetics and 24 in aged mice. Of these, only 14 were commonly dysregulated in all groups. The enrichment pathways analysis revealed that the inflammatory response was the biological process with more DEG in all groups, followed by hemopoiesis. Our findings indicate that the response to cerebral ischemia regarding proteomic and transcriptomic changes differs depending on sex, age and comorbidities, highlighting the importance of incorporating animals with different phenotypes in future stroke research studies.

T cells modulate the microglial response to brain ischemia.

Neuroinflammation after stroke is characterized by the activation of resident microglia and the invasion of circulating leukocytes into the brain. Although lymphocytes infiltrate the brain in small number, they have been consistently demonstrated to be the most potent leukocyte subpopulation contributing to secondary inflammatory brain injury. However, the exact mechanism of how this minimal number of lymphocytes can profoundly affect stroke outcome is still largely elusive. Here, using a mouse model for ischemic stroke, we demonstrated that early activation of microglia in response to stroke is differentially regulated by distinct T cell subpopulations - with TH1 cells inducing a type I INF signaling in microglia and regulatory T cells (TREG) cells promoting microglial genes associated with chemotaxis. Acute treatment with engineered T cells overexpressing IL-10 administered into the cisterna magna after stroke induces a switch of microglial gene expression to a profile associated with pro-regenerative functions. Whereas microglia polarization by T cell subsets did not affect the acute development of the infarct volume, these findings substantiate the role of T cells in stroke by polarizing the microglial phenotype. Targeting T cell-microglia interactions can have direct translational relevance for further development of immune-targeted therapies for stroke and other neuroinflammatory conditions.

Systemic inflammation after stroke: implications for post-stroke comorbidities.

Immunological mechanisms have come into the focus of current translational stroke research, and the modulation of neuroinflammatory pathways has been identified as a promising therapeutic approach to protect the ischemic brain. However, stroke not only induces a local neuroinflammatory response but also has a profound impact on systemic immunity. In this review, we will summarize the consequences of ischemic stroke on systemic immunity at all stages of the disease, from onset to long-term outcome, and discuss underlying mechanisms of systemic brain-immune communication. Furthermore, since stroke commonly occurs in patients with multiple comorbidities, we will also overview the current understanding of the potential role of systemic immunity in common stroke-related comorbidities, such as cardiac dysfunction, atherosclerosis, diabetes, and infections. Finally, we will highlight how targeting systemic immunity after stroke could improve long-term outcomes and alleviate comorbidities of stroke patients.

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.

Ceruletide and Alpha-1 Antitrypsin as a Novel Combination Therapy for Ischemic Stroke.

Ischemic stroke is a primary cause of morbidity and mortality worldwide. Beyond the approved thrombolytic therapies, there is no effective treatment to mitigate its progression. Drug repositioning combinational therapies are becoming promising approaches to identify new uses of existing drugs to synergically target multiple disease-response mechanisms underlying complex pathologies. Here, we used a systems biology-based approach based on artificial intelligence and pattern recognition tools to generate in silico mathematical models mimicking the ischemic stroke pathology. Combinational treatments were acquired by screening these models with more than 5 million two-by-two combinations of drugs. A drug combination (CA) formed by ceruletide and alpha-1 antitrypsin showing a predicted value of neuroprotection of 92% was evaluated for their synergic neuroprotective effects in a mouse pre-clinical stroke model. The administration of both drugs in combination was safe and effective in reducing by 39.42% the infarct volume 24 h after cerebral ischemia. This neuroprotection was not observed when drugs were given individually. Importantly, potential incompatibilities of the drug combination with tPA thrombolysis were discarded in vitro and in vivo by using a mouse thromboembolic stroke model with t-PA-induced reperfusion, revealing an improvement in the forepaw strength 72 h after stroke in CA-treated mice. Finally, we identified the predicted mechanisms of action of ceruletide and alpha-1 antitrypsin and we demonstrated that CA modulates EGFR and ANGPT-1 levels in circulation within the acute phase after stroke. In conclusion, we have identified a promising combinational treatment with neuroprotective effects for the treatment of ischemic stroke.

Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery.

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options are limited to a small proportion of stroke patients within the first hours after stroke. Novel therapeutic strategies are being extensively investigated, especially to prolong the therapeutic time window. These current investigations include the study of important pathophysiological pathways after stroke, such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Over the last decade, there has been increasing concern about the poor reproducibility of experimental results and scientific findings among independent research groups. To overcome the so-called "replication crisis", detailed standardized models for all procedures are urgently needed. As an effort within the "ImmunoStroke" research consortium (https://immunostroke.de/), a standardized mouse model of transient middle cerebral artery occlusion (MCAo) is proposed. This model allows the complete restoration of the blood flow upon removal of the filament, simulating the therapeutic or spontaneous clot lysis that occurs in a large proportion of human strokes. The surgical procedure of this "filament" stroke model and tools for its functional analysis are demonstrated in the accompanying video.

Integrative Multi-omics Analysis to Characterize Human Brain Ischemia.

Stroke is a major cause of death and disability. A better comprehension of stroke pathophysiology is fundamental to reduce its dramatic outcome. The use of high-throughput unbiased omics approaches and the integration of these data might deepen the knowledge of stroke at the molecular level, depicting the interaction between different molecular units. We aimed to identify protein and gene expression changes in the human brain after ischemia through an integrative approach to join the information of both omics analyses. The translational potential of our results was explored in a pilot study with blood samples from ischemic stroke patients. Proteomics and transcriptomics discovery studies were performed in human brain samples from six deceased stroke patients, comparing the infarct core with the corresponding contralateral brain region, unveiling 128 proteins and 2716 genes significantly dysregulated after stroke. Integrative bioinformatics analyses joining both datasets exposed canonical pathways altered in the ischemic area, highlighting the most influential molecules. Among the molecules with the highest fold-change, 28 genes and 9 proteins were selected to be validated in five independent human brain samples using orthogonal techniques. Our results were confirmed for NCDN, RAB3C, ST4A1, DNM1L, A1AG1, A1AT, JAM3, VTDB, ANXA1, ANXA2, and IL8. Finally, circulating levels of the validated proteins were explored in ischemic stroke patients. Fluctuations of A1AG1 and A1AT, both up-regulated in the ischemic brain, were detected in blood along the first week after onset. In summary, our results expand the knowledge of ischemic stroke pathology, revealing key molecules to be further explored as biomarkers and/or therapeutic targets.

Blood Biomarkers to Differentiate Ischemic and Hemorrhagic Strokes.

OBJECTIVE: To validate a panel of blood biomarkers to differentiate between ischemic stroke (IS) and intracerebral hemorrhage (ICH) in patients with suspected stroke. METHODS: Patients with suspected stroke admitted within 4.5 hours after onset were enrolled. Blood samples were collected at hospital admission. Glial fibrillary acid protein (GFAP), retinol binding protein 4 (RBP-4), N-terminal proB-type natriuretic peptide (NT-proBNP), and endostatin were measured by immunoassays. Cutoff points were obtained for 100% specificity for IS. A high-sensitivity assay to measure GFAP and rapid point-of-care tests (POCTs) to measure RBP-4 and NT-proBNP were used in subsets of patients. Biomarker panels were evaluated in another cohort of 62 stroke mimics. RESULTS: A total of 189 patients (154 IS and 35 ICH) were enrolled. Patients with IS had higher RBP-4, NT-proBNP, and endostatin and lower GFAP levels than patients with ICH. The best biomarker combination for the identification of IS was RBP-4+NT-proBNP, which was able to identify 29.7% of patients with IS with 100% specificity. In the subset of patients for whom GFAP was measured with the high-sensitivity assay, RBP-4, NT-proBNP, and GFAP identified 51.5% of patients with IS with 100% specificity. When stroke mimics were included, specificities were reduced to 98.4 and 96.8%, respectively. POCTs of RBP-4 and NT-proBNP showed results similar results to those of conventional ELISAs. CONCLUSIONS: A biomarker panel including RBP-4, NT-proBNP, and GFAP provided moderate but potentially useful sensitivity rates at 100% specificity for IS diagnosis. If confirmed in future studies, this strategy might allow prehospital treatment in selected patients. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that a biomarker panel including RBP-4, NT-proBNP, and GFAP distinguishes IS from ICH with moderate accuracy.

A Mouse Brain-based Multi-omics Integrative Approach Reveals Potential Blood Biomarkers for Ischemic Stroke.

Stroke remains a leading cause of death and disability worldwide. Despite continuous advances, the identification of key molecular signatures in the hyper-acute phase of ischemic stroke is still a primary interest for translational research on stroke diagnosis, prognosis, and treatment. Data integration from high-throughput -omics techniques has become crucial to unraveling key interactions among different molecular elements in complex biological contexts, such as ischemic stroke. Thus, we used advanced data integration methods for a multi-level joint analysis of transcriptomics and proteomics data sets obtained from mouse brains at 2 h after cerebral ischemia. By modeling net-like correlation structures, we identified an integrated network of genes and proteins that are differentially expressed at a very early stage after stroke. We validated 10 of these deregulated elements in acute stroke, and changes in their expression pattern over time after cerebral ischemia were described. Of these, CLDN20, GADD45G, RGS2, BAG5, and CTNND2 were next evaluated as blood biomarkers of cerebral ischemia in mice and human blood samples, which were obtained from stroke patients and patients presenting stroke-mimicking conditions. Our findings indicate that CTNND2 levels in blood might potentially be useful for distinguishing ischemic strokes from stroke-mimicking conditions in the hyper-acute phase of the disease. Furthermore, circulating GADD45G content within the first 6 h after stroke could also play a key role in predicting poor outcomes in stroke patients. For the first time, we have used an integrative biostatistical approach to elucidate key molecules in the initial stages of stroke pathophysiology and highlight new notable molecules that might be further considered as blood biomarkers of ischemic stroke.

Multilevel omics for the discovery of biomarkers and therapeutic targets for stroke.

Despite many years of research, no biomarkers for stroke are available to use in clinical practice. Progress in high-throughput technologies has provided new opportunities to understand the pathophysiology of this complex disease, and these studies have generated large amounts of data and information at different molecular levels. The integration of these multi-omics data means that thousands of proteins (proteomics), genes (genomics), RNAs (transcriptomics) and metabolites (metabolomics) can be studied simultaneously, revealing interaction networks between the molecular levels. Integrated analysis of multi-omics data will provide useful insight into stroke pathogenesis, identification of therapeutic targets and biomarker discovery. In this Review, we detail current knowledge on the pathology of stroke and the current status of biomarker research in stroke. We summarize how proteomics, metabolomics, transcriptomics and genomics are all contributing to the identification of new candidate biomarkers that could be developed and used in clinical stroke management.

CCL23: A Chemokine Associated with Progression from Mild Cognitive Impairment to Alzheimer's Disease.

CCL23 is a chemokine implicated in inflammation and host defense responses. It has been recently associated with acquired brain damage and stroke outcomes. In this study, we reported the role of CCL23 in Alzheimer's disease (AD). We evaluated the levels of CCL23 in 659 individuals: cognitively normal, mild cognitive impaired (MCI), and AD patients. Two cross-sectional (study 1, n = 53; study 2, n = 200) and two longitudinal (study 3, n = 74; study 4, n = 332) studies were analyzed separately. CCL23 levels in the blood and/or cerebrospinal fluid (CSF) of each study were measured by immunoassays. Globally, our results suggest a predictive role of CCL23 protein levels both in the plasma in study 3 (hazard ratio (HR) = 2.5 (confidence interval (CI) 95% : 1.2-5.3), p = 0.02) and in the CSF in study 4 (HR = 3.05 (CI 95% : 1.02-5), p = 0.04) in cases of MCI that progress to AD. Moreover, we observed that the APOEɛ4 allele was associated with higher levels of CCL23 in study 2 (470.33 pg/mL (interquartile range (IQR): 303.33-597.76) versus 377.94 pg/mL (IQR: 267.16-529.19), p = 0.01) (APOE genotypes were available in studies 2 and 4). Together, these findings support the role of CCL23 in neuroinflammation in the early stages of AD, suggesting that CCL23 might be a candidate blood biomarker for MCI to AD progression.

Circulating Aquaporin-4 as A biomarker of early neurological improvement in stroke patients: A pilot study.

Patients' outcome prediction after ischemic stroke is still challenging. Aquaporin-4 (AQP4) is a water channel that is up-regulated in the brain after the ischemic event, but its presence in bloodstream of stroke patients has not been previously studied. The aim of this pilot study was to investigate circulating AQP4 levels after stroke and its correlation with infarct growth and neurological outcome. AQP4 level was determined by ELISA in serum from 42 t-PA-treated ischemic stroke patients at admission (before t-PA) and 13 healthy subjects. To assess infarct growth, serial brain diffusion-weighted magnetic resonance images were performed at hospital admission and 1-3 days after. Neurological improvement was defined as a ≥4-point decrease in NIHSS score compared to baseline score. Despite stroke patients and healthy controls had similar baseline circulating AQP4 levels, among strokes AQP4 level negatively correlated with NIHSS score at admission (R= -0.34, p = 0.029) and with infarct growth after 1-3 days of stroke onset (R=-0.36; p = 0.018). Furthermore, baseline AQP4 level was higher in those stroke patients showing a neurological improvement 48 h after stroke onset (p = 0.030) and at hospital discharge (p = 0.037). Baseline AQP4 levels also resulted to be an independent predictor of good neurological outcome at both studied time points (ORadj: 14.33[1.82-112.92], p = 0.012 at 48 h; ORadj: 4.86[0.98-24.12], p = 0.053 at discharge) in logistic regression analysis, adjusted by age, sex, baseline NIHSS and significant variables in the univariate analysis. Overall, we have explored circulating AQP4 levels, and our data suggest that AQP4 could be used as a biomarker of neurological recovery in the acute-subacute phase of ischemic stroke.

Application of an Aptamer-Based Proteomics Assay (SOMAscan™) in Rat Cerebrospinal Fluid.

The exploration of the cerebrospinal fluid (CSF) through proteomics techniques might help in the search of molecular biomarkers relevant to neurological pathologies. Aiming this, we describe here a commercially available multiplexed proteomics technology based on the use of modified aptamers (SOMAscan™ assay). From our experience in exploring the rat CSF proteome, we detail the basic principles of this oligonucleotide-based proteomics approach, as well as the main data-processing steps to obtain relative quantitative values for proteins that could discriminate among different brain conditions, as an attempt in the search of neurological biomarkers. Finally, we give some tips on performing the SOMAscan assay and key recommendations on the verification analyses of the resulting candidate biomarkers.

Matrix metalloproteinases and ADAMs in stroke.

Stroke is a leading cause of death and disability worldwide. However, after years of in-depth research, the pathophysiology of stroke is still not fully understood. Increasing evidence shows that matrix metalloproteinases (MMPs) and "a disintegrin and metalloproteinase" (ADAMs) participate in the neuro-inflammatory cascade that is triggered during stroke but also in recovery phases of the disease. This review covers the involvement of these proteins in brain injury following cerebral ischemia which has been widely studied in recent years, with efforts to modulate this group of proteins in neuroprotective therapies, together with their implication in neurorepair mechanisms. Moreover, the review also discusses the role of these proteins in specific forms of neurovascular disease, such as small vessel diseases and intracerebral hemorrhage. Finally, the potential use of MMPs and ADAMs as guiding biomarkers of brain injury and repair for decision-making in cases of stroke is also discussed.

Inflammatory molecules might become both biomarkers and therapeutic targets for stroke management.

Stroke is the fifth leading cause of death and the most frequent cause of disability worldwide. Currently, stroke diagnosis is based on neuroimaging; therefore, the lack of a rapid tool to diagnose stroke is still a major concern. In addition, therapeutic approaches to combat ischemic stroke are still scarce, since the only approved therapies are directed toward restoring blood flow to the affected brain area. However, due to the reduced time window during which these therapies are effective, few patients benefit from them; therefore, alternative treatments are urgently needed to reduce stroke brain damage in order to improve patients' outcome. The inflammatory response triggered after the ischemic event plays an important role in the progression of stroke; consequently, the study of inflammatory molecules in the acute phase of stroke has attracted increasing interest in recent decades. Here, we provide an overview of the inflammatory processes occurring during ischemic stroke, as well as the potential for these inflammatory molecules to become stroke biomarkers and the possibility that these candidates will become interesting neuroprotective therapeutic targets to be blocked or stimulated in order to modulate inflammation after stroke.

Characterization of the rat cerebrospinal fluid proteome following acute cerebral ischemia using an aptamer-based proteomic technology.

The limited accessibility to the brain has turned the cerebrospinal fluid (CSF) into a valuable source that may contribute to the complete understanding of the stroke pathophysiology. Here we have described the CSF proteome in the hyper-acute phase of cerebral ischemia by performing an aptamer-based proteomic assay (SOMAscan) in CSF samples collected before and 30 min after male Wistar rats had undergone a 90 min Middle Cerebral Artery Occlusion (MCAO) or sham-surgery. Proteomic results indicated that cerebral ischemia acutely increased the CSF levels of 716 proteins, mostly overrepresented in leukocyte chemotaxis and neuronal death processes. Seven promising candidates were further evaluated in rat plasma and brain (CKB, CaMK2A, CaMK2B, CaMK2D, PDXP, AREG, CMPK). The 3 CaMK2 family-members and CMPK early decreased in the infarcted brain area and, together with AREG, co-localized with neurons. Conversely, CKB levels remained consistent after the insult and specifically matched with astrocytes. Further exploration of these candidates in human plasma revealed the potential of CKB and CMPK to diagnose stroke, while CaMK2B and CMPK resulted feasible biomarkers of functional stroke outcome. Our findings provided insights into the CSF proteome following cerebral ischemia and identified new outstanding proteins that might be further considered as potential biomarkers of stroke.