MicroRNA Analysis of Human Stroke Brain Tissue Resected during Decompressive Craniectomy/Stroke-Ectomy Surgery

Background: Signaling pathways mediated by microRNAs (miRNAs) have been identified as one of the mechanisms that regulate stroke progression and recovery. Recent investigations using stroke patient blood and cerebrospinal fluid (CSF) demonstrated disease-specific alterations in miRNA expression. In this study, for the first time, we investigated miRNA expression signatures in freshly removed human stroke brain tissue. Methods: Human brain samples were obtained during craniectomy and brain tissue resection in severe stroke patients with life-threatening brain swelling. The tissue samples were subjected to histopathological and immunofluorescence microscopy evaluation, next generation miRNA sequencing (NGS), and bioinformatic analysis. Results: miRNA NGS analysis detected 34 miRNAs with significantly aberrant expression in stroke tissue, as compared to non-stroke samples. Of these miRNAs, 19 were previously identified in stroke patient blood and CSF, while dysregulation of 15 miRNAs was newly detected in this study. miRNA direct target gene analysis and bioinformatics approach demonstrated a strong association of the identified miRNAs with stroke-related biological processes and signaling pathways. Conclusions: Dysregulated miRNAs detected in our study could be regarded as potential candidates for biomarkers and/or targets for therapeutic intervention. The results described herein further our understanding of the molecular basis of stroke and provide valuable information for the future functional studies in the experimental models of stroke.

Neural network models for spinal implementation of muscle synergies

Muscle synergies have been proposed as functional modules to simplify the complexity of body motor control; however, their neural implementation is still unclear. Converging evidence suggests that output projections of the spinal premotor interneurons (PreM-INs) underlie the formation of muscle synergies, but they exhibit a substantial variation across neurons and exclude standard models assuming a small number of unitary "modules" in the spinal cord. Here we compared neural network models for muscle synergies to seek a biologically plausible model that reconciles previous clinical and electrophysiological findings. We examined three neural network models: one with random connections (non-synergy model), one with a small number of spinal synergies (simple synergy model), and one with a large number of spinal neurons representing muscle synergies with a certain variation (population synergy model). We found that the simple and population synergy models emulate the robustness of muscle synergies against cortical stroke observed in human stroke patients. Furthermore, the size of the spinal variation of the population synergy matched well with the variation in spinal PreM-INs recorded in monkeys. These results suggest that a spinal population with moderate variation is a biologically plausible model for the neural implementation of muscle synergies.

Platelet Activation and Chemokine Release Are Related to Local Neutrophil-Dominant Inflammation During Hyperacute Human Stroke

Experimental evidence has emerged that local platelet activation contributes to inflammation and infarct formation in acute ischemic stroke (AIS) which awaits confirmation in human studies. We conducted a prospective observational study on 258 consecutive patients undergoing mechanical thrombectomy (MT) due to large-vessel-occlusion stroke of the anterior circulation (08/2018–05/2020). Intraprocedural microcatheter aspiration of 1 ml of local (occlusion condition) and systemic arterial blood samples (self-control) was performed according to a prespecified protocol. The samples were analyzed for differential leukocyte counts, platelet counts, and plasma levels of the platelet-derived neutrophil-activating chemokine C-X-C-motif ligand (CXCL) 4 (PF-4), the neutrophil attractant CXCL7 (NAP-2), and myeloperoxidase (MPO). The clinical-biological relevance of these variables was corroborated by specific associations with molecular-cellular, structural-radiological, hemodynamic, and clinical-functional parameters. Seventy consecutive patients fulfilling all predefined criteria entered analysis. Mean local CXCL4 (+ 39%: 571 vs 410 ng/ml, P = .0095) and CXCL7 (+ 9%: 693 vs 636 ng/ml, P = .013) concentrations were higher compared with self-controls. Local platelet counts were lower (− 10%: 347,582 vs 383,284/µl, P = .0052), whereas neutrophil counts were elevated (+ 10%: 6022 vs 5485/µl, P = 0.0027). Correlation analyses revealed associations between local platelet and neutrophil counts (r = 0.27, P = .034), and between CXCL7 and MPO (r = 0.24, P = .048). Local CXCL4 was associated with the angiographic degree of reperfusion following recanalization (r = − 0.2523, P = .0479). Functional outcome at discharge correlated with local MPO concentrations (r = 0.3832, P = .0014) and platelet counts (r = 0.288, P = .0181). This study provides human evidence of cerebral platelet activation and platelet-neutrophil interactions during AIS and points to the relevance of per-ischemic thrombo-inflammatory mechanisms to impaired reperfusion and worse functional outcome following recanalization.

Immune Cells Invade the Collateral Circulation during Human Stroke: Prospective Replication and Extension

It remains unclear if principal components of the local cerebral stroke immune response can be reliably and reproducibly observed in patients with acute large-vessel-occlusion (LVO) stroke. We prospectively studied a large independent cohort of n = 318 consecutive LVO stroke patients undergoing mechanical thrombectomy during which cerebral blood samples from within the occluded anterior circulation and systemic control samples from the ipsilateral cervical internal carotid artery were obtained. An extensive protocol was applied to homogenize the patient cohort and to standardize the procedural steps of endovascular sample collection, sample processing, and laboratory analyses. N = 58 patients met all inclusion criteria. (1) Mean total leukocyte counts were significantly higher within the occluded ischemic cerebral vasculature (I) vs. intraindividual systemic controls (S): +9.6%, I: 8114/µL ± 529 vs. S: 7406/µL ± 468, p = 0.0125. (2) This increase was driven by neutrophils: +12.1%, I: 7197/µL ± 510 vs. S: 6420/µL ± 438, p = 0.0022. Leukocyte influx was associated with (3) reduced retrograde collateral flow (R2 = 0.09696, p = 0.0373) and (4) greater infarct extent (R2 = 0.08382, p = 0.032). Despite LVO, leukocytes invade the occluded territory via retrograde collateral pathways early during ischemia, likely compromising cerebral hemodynamics and tissue integrity. This inflammatory response can be reliably observed in human stroke by harvesting immune cells from the occluded cerebral vascular compartment.

Isolation and identification of leukocyte populations in intracranial blood collected during mechanical thrombectomy

Using standard techniques during mechanical thrombectomy, the Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) protocol (NCT03153683) isolates intracranial arterial blood distal to the thrombus and proximal systemic blood in the carotid artery. We augmented the current protocol to study leukocyte subpopulations both distal and proximal to the thrombus during human stroke (n = 16 patients), and from patients with cerebrovascular disease (CVD) undergoing angiography for unrelated conditions (e.g. carotid artery stenosis; n = 12 patients). We isolated leukocytes for flow cytometry from small volume (<1 mL) intracranial blood and systemic blood (5–10 mL) to identify adaptive and innate leukocyte populations, in addition to platelets and endothelial cells (ECs). Intracranial blood exhibited significant increases in T cell representation and decreases in myeloid/macrophage representation compared to within-patient carotid artery samples. CD4+ T cells and classical dendritic cells were significantly lower than CVD controls and correlated to within-patient edema volume and last known normal. This novel protocol successfully isolates leukocytes from small volume intracranial blood samples of stroke patients at time of mechanical thrombectomy and can be used to confirm preclinical results, as well as identify novel targets for immunotherapies.

Mechanical Characterization of Thrombi Retrieved With Endovascular Thrombectomy in Patients With Acute Ischemic Stroke

Background and Purpose: Mechanical properties of thromboemboli play an important role in the efficacy of endovascular thrombectomy (EVT) for acute ischemic stroke. However, very limited data on mechanical properties of human stroke thrombi are available. We aimed to mechanically characterize thrombi retrieved with EVT, and to assess the relationship between thrombus composition and thrombus stiffness. Methods: Forty-one thrombi from 19 patients with acute stroke who underwent EVT between July and October 2019 were mechanically analyzed, directly after EVT. We performed unconfined compression experiments and determined tangent modulus at 75% strain (E t75 ) as a measure for thrombus stiffness. Thrombi were histologically analyzed for fibrin/platelets, erythrocytes, leukocytes, and platelets, and we assessed the relationship between histological components and E t75 with univariable and multivariable linear mixed regression. Results: Median E t75 was 560 (interquartile range, 393–1161) kPa. In the multivariable analysis, fibrin/platelets were associated with increased E t75 (aβ, 9 [95% CI, 5 to 13]) kPa, erythrocytes were associated with decreased E t75% (aβ, −9 [95% CI, −5 to −13]) kPa. We found no association between leukocytes and E t75 . High platelet values were strongly associated with increased E t75 (aβ, 56 [95% CI, 38–73]). Conclusions: Fibrin/platelet content of thrombi retrieved with EVT for acute ischemic stroke is strongly associated with increased thrombus stiffness. For thrombi with high platelet values, there was a very strong relationship with thrombus stiffness. Our data provide a basis for future research on the development of next-generation EVT devices tailored to thrombus composition.

Circadian rhythm of ischaemic core progression in human stroke

IntroductionExperimental stroke studies suggest an influence of the time of day of stroke onset on infarct progression. Whether this holds true after human stroke is unknown, but would have implications for the design of randomised controlled trials, especially those on neuroprotection.MethodsWe pooled data from 583 patients with anterior large-vessel occlusion stroke from three prospectively recruited cohorts. Ischaemic core and penumbra volumes were determined with CT perfusion using automated thresholds. Core growth was calculated as the ratio of core volume and onset-to-imaging time. To determine circadian rhythmicity, we applied multivariable linear and sinusoidal regression analysis adjusting for potential baseline confounders.ResultsPatients with symptom onset at night showed larger ischaemic core volumes on admission compared with patients with onset during the day (median, 40.2 mL vs 33.8 mL), also in adjusted analyses (p=0.008). Sinusoidal analysis indicated a peak of core volumes with onset at 11pm. Core growth was faster at night compared with day onset (adjusted p=0.01), especially for shorter onset-to-imaging times. In contrast, penumbra volumes did not change across the 24-hour cycle.DiscussionThese results suggest that human infarct progression varies across the 24-hour cycle with potential implications for the design and interpretation of neuroprotection trials.

X chromosome escapee genes are involved in ischemic sexual dimorphism through epigenetic modification of inflammatory signals

Background Stroke is a sexually dimorphic disease. Previous studies have found that young females are protected against ischemia compared to males, partially due to the protective effect of ovarian hormones, particularly estrogen (E2). However, there are also genetic and epigenetic effects of X chromosome dosage that contribute to stroke sensitivity and neuroinflammation after injury, especially in the aged. Genes that escape from X chromosome inactivation (XCI) contribute to sex-specific phenotypes in many disorders. Kdm5c and kdm6a are X escapee genes that demethylate H3K4me3 and H3K27me3, respectively. We hypothesized that the two demethylases play critical roles in mediating the stroke sensitivity. Methods To identify the X escapee genes involved in stroke, we performed RNA-seq in flow-sorted microglia from aged male and female wild type (WT) mice subjected to middle cerebral artery occlusion (MCAO). The expression of these genes (kdm5c/kdm6a) were confirmed in four core genotypes (FCG) mice and in post-mortem human stroke brains by immunohistochemistry (IHC), Western blot, and RT-PCR. Chromatin immunoprecipitation (ChIP) assays were conducted to detect DNA levels of inflammatory interferon regulatory factor (IRF) 4/5 precipitated by histone H3K4 and H3K27 antibodies. Manipulation of kdm5c/kdm6a expression with siRNA or lentivirus was performed in microglial culture, to determine downstream pathways and examine the regulatory roles in inflammatory cytokine production. Results Kdm5c and kdm6a mRNA levels were significantly higher in aged WT female vs. male microglia, and the sex difference also existed in ischemic brains from FCG mice and human stroke patients. The ChIP assay showed the IRF 4/5 had higher binding levels to demethylated H3K4 or H3K27, respectively, in female vs. male ischemic microglia. Knockdown or over expression of kdm5c/kdm6a with siRNA or lentivirus altered the methylation of H3K4 or H3K27 at the IRF4/5 genes, which in turn, impacted the production of inflammatory cytokines. Conclusions The KDM-Histone-IRF pathways are suggested to mediate sex differences in cerebral ischemia. Epigenetic modification of stroke-related genes constitutes an important mechanism underlying the ischemic sexual dimorphism.