Development and validation of a SCORing systEm for pre-thrombectomy diagnosis of IntraCranial Atherosclerotic Disease (Score-ICAD).

BACKGROUND: Early identification of intracranial atherosclerotic disease (ICAD) may impact the management of patients undergoing mechanical thrombectomy (MT). We sought to develop and validate a scoring system for pre-thrombectomy diagnosis of ICAD in anterior circulation large vessel/distal medium vessel occlusion strokes (LVOs/DMVOs). METHODS: Retrospective analysis of two prospectively maintained comprehensive stroke center databases including patients with anterior circulation occlusions spanning 2010-22 (development cohort) and 2018-22 (validation cohort). ICAD cases were matched for age and sex (1:1) to non-ICAD controls. RESULTS: Of 2870 MTs within the study period, 348 patients were included in the development cohort: 174 anterior circulation ICAD (6% of 2870 MTs) and 174 controls. Multivariable analysis ß coefficients led to a 20 point scale: absence of atrial fibrillation (5); vascular risk factor burden (1) for each of hypertension, diabetes, smoking, and hyperlipidemia; multifocal single artery stenoses on CT angiography (3); absence of territorial cortical infarct (3); presence of borderzone infarct (3); or ipsilateral carotid siphon calcification (2). The validation cohort comprised 56 ICAD patients (4.1% of 1359 MTs): 56 controls. Area under the receiver operating characteristic curve was 0.88 (0.84-0.91) and 0.82 (0.73-0.89) in the development and validation cohorts, respectively. Calibration slope and intercept showed a good fit for the development cohort although with overestimated risk for the validation cohort. After intercept adjustment, the overestimation was corrected (intercept 0, 95% CI -0.5 to -0.5; slope 0.8, 95% CI 0.5 to 1.1). In the full cohort (n=414), ≥11 points showed the best performance for distinguishing ICAD from non-ICAD, with 0.71 (95% CI 0.65 to 0.78) sensitivity and 0.82 (95% CI 0.77 to 0.87) specificity, and 3.92 (95% CI 2.92 to 5.28) positive and 0.35 (95% CI 0.28 to 0.44) negative likelihood ratio. Scores ≥12 showed 90% specificity and 63% sensitivity. CONCLUSION: The proposed scoring system for preprocedural diagnosis of ICAD LVOs and DMVOs presented satisfactory discrimination and calibration based on clinical and non-invasive radiological data.

Proteomic Signaling of Dual-Specificity Phosphatase 4 (DUSP4) in Alzheimer's Disease.

DUSP4 is a member of the DUSP (dual-specificity phosphatase) subfamily that is selective to the mitogen-activated protein kinases (MAPK) and has been implicated in a range of biological processes and functions in Alzheimer's disease (AD). In this study, we utilized the stereotactic delivery of adeno-associated virus (AAV)-DUSP4 to overexpress DUSP4 in the dorsal hippocampus of 5xFAD and wildtype (WT) mice, then used mass spectrometry (MS)-based proteomics along with the label-free quantification to profile the proteome and phosphoproteome in the hippocampus. We identified protein expression and phosphorylation patterns modulated in 5xFAD mice and examined the sex-specific impact of DUSP4 overexpression on the 5xFAD proteome/phosphoproteome. In 5xFAD mice, a substantial number of proteins were up- or down-regulated in both male and female mice in comparison to age and sex-matched WT mice, many of which are involved in AD-related biological processes, such as activated immune response or suppressed synaptic activities. Many proteins in pathways, such as immune response were found to be suppressed in response to DUSP4 overexpression in male 5xFAD mice. In contrast, such a shift was absent in female mice. For the phosphoproteome, we detected an array of phosphorylation sites regulated in 5xFAD compared to WT and modulated via DUSP4 overexpression in each sex. Interestingly, 5xFAD- and DUSP4-associated phosphorylation changes occurred in opposite directions. Strikingly, both the 5xFAD- and DUSP4-associated phosphorylation changes were found to be mostly in neurons and play key roles in neuronal processes and synaptic functions. Site-centric pathway analysis revealed that both the 5xFAD- and DUSP4-associated phosphorylation sites were enriched for a number of kinase sets in females but only a limited number of sets of kinases in male mice. Taken together, our results suggest that male and female 5xFAD mice responded to DUSP4 overexpression via shared and sex-specific molecular mechanisms, which might underly similar reductions in amyloid pathology in both sexes while learning deficits were reduced in only females with DUSP4 overexpression. Finally, we validated our findings with the sex-specific AD-associated proteomes in human cohorts and further developed DUSP4-centric proteomic network models and signaling maps for each sex.

Cellular Proteomic Profiling Using Proximity Labeling by TurboID-NES in Microglial and Neuronal Cell Lines.

Different brain cell types play distinct roles in brain development and disease. Molecular characterization of cell-specific mechanisms using cell type-specific approaches at the protein (proteomic) level can provide biological and therapeutic insights. To overcome the barriers of conventional isolation-based methods for cell type-specific proteomics, in vivo proteomic labeling with proximity-dependent biotinylation of cytosolic proteins using biotin ligase TurboID, coupled with mass spectrometry (MS) of labeled proteins, emerged as a powerful strategy for cell type-specific proteomics in the native state of cells without the need for cellular isolation. To complement in vivo proximity labeling approaches, in vitro studies are needed to ensure that cellular proteomes using the TurboID approach are representative of the whole-cell proteome and capture cellular responses to stimuli without disruption of cellular processes. To address this, we generated murine neuroblastoma (N2A) and microglial (BV2) lines stably expressing cytosolic TurboID to biotinylate the cellular proteome for downstream purification and analysis using MS. TurboID-mediated biotinylation captured 59% of BV2 and 65% of N2A proteomes under homeostatic conditions. TurboID labeled endolysosome, translation, vesicle, and signaling proteins in BV2 microglia and synaptic, neuron projection, and microtubule proteins in N2A neurons. TurboID expression and biotinylation minimally impacted homeostatic cellular proteomes of BV2 and N2A cells and did not affect lipopolysaccharide-mediated cytokine production or resting cellular respiration in BV2 cells. MS analysis of the microglial biotin-labeled proteins captured the impact of lipopolysaccharide treatment (>500 differentially abundant proteins) including increased canonical proinflammatory proteins (Il1a, Irg1, and Oasl1) and decreased anti-inflammatory proteins (Arg1 and Mgl2).

BIN1 is a key regulator of proinflammatory and neurodegeneration-related activation in microglia.

BACKGROUND: The BIN1 locus contains the second-most significant genetic risk factor for late-onset Alzheimer's disease. BIN1 undergoes alternate splicing to generate tissue- and cell-type-specific BIN1 isoforms, which regulate membrane dynamics in a range of crucial cellular processes. Whilst the expression of BIN1 in the brain has been characterized in neurons and oligodendrocytes in detail, information regarding microglial BIN1 expression is mainly limited to large-scale transcriptomic and proteomic data. Notably, BIN1 protein expression and its functional roles in microglia, a cell type most relevant to Alzheimer's disease, have not been examined in depth. METHODS: Microglial BIN1 expression was analyzed by immunostaining mouse and human brain, as well as by immunoblot and RT-PCR assays of isolated microglia or human iPSC-derived microglial cells. Bin1 expression was ablated by siRNA knockdown in primary microglial cultures in vitro and Cre-lox mediated conditional deletion in adult mouse brain microglia in vivo. Regulation of neuroinflammatory microglial signatures by BIN1 in vitro and in vivo was characterized using NanoString gene panels and flow cytometry methods. The transcriptome data was explored by in silico pathway analysis and validated by complementary molecular approaches. RESULTS: Here, we characterized microglial BIN1 expression in vitro and in vivo and ascertained microglia expressed BIN1 isoforms. By silencing Bin1 expression in primary microglial cultures, we demonstrate that BIN1 regulates the activation of proinflammatory and disease-associated responses in microglia as measured by gene expression and cytokine production. Our transcriptomic profiling revealed key homeostatic and lipopolysaccharide (LPS)-induced inflammatory response pathways, as well as transcription factors PU.1 and IRF1 that are regulated by BIN1. Microglia-specific Bin1 conditional knockout in vivo revealed novel roles of BIN1 in regulating the expression of disease-associated genes while counteracting CX3CR1 signaling. The consensus from in vitro and in vivo findings showed that loss of Bin1 impaired the ability of microglia to mount type 1 interferon responses to proinflammatory challenge, particularly the upregulation of a critical type 1 immune response gene, Ifitm3. CONCLUSIONS: Our convergent findings provide novel insights into microglial BIN1 function and demonstrate an essential role of microglial BIN1 in regulating brain inflammatory response and microglial phenotypic changes. Moreover, for the first time, our study shows a regulatory relationship between Bin1 and Ifitm3, two Alzheimer's disease-related genes in microglia. The requirement for BIN1 to regulate Ifitm3 upregulation during inflammation has important implications for inflammatory responses during the pathogenesis and progression of many neurodegenerative diseases.

Cell type-specific biotin labeling in vivo resolves regional neuronal and astrocyte proteomic differences in mouse brain.

Proteomic profiling of brain cell types using isolation-based strategies pose limitations in resolving cellular phenotypes representative of their native state. We describe a mouse line for cell type-specific expression of biotin ligase TurboID, for in vivo biotinylation of proteins. Using adenoviral and transgenic approaches to label neurons, we show robust protein biotinylation in neuronal soma and axons throughout the brain, allowing quantitation of over 2000 neuron-derived proteins spanning synaptic proteins, transporters, ion channels and disease-relevant druggable targets. Next, we contrast Camk2a-neuron and Aldh1l1-astrocyte proteomes and identify brain region-specific proteomic differences within both cell types, some of which might potentially underlie the selective vulnerability to neurological diseases. Leveraging the cellular specificity of proteomic labeling, we apply an antibody-based approach to uncover differences in neuron and astrocyte-derived signaling phospho-proteins and cytokines. This approach will facilitate the characterization of cell-type specific proteomes in a diverse number of tissues under both physiological and pathological states.

Anticoagulation Therapy Reduces Recurrent Stroke in Embolic Stroke of Undetermined Source Patients With Elevated Coagulation Markers or Severe Left Atrial Enlargement.

Background: The objective of this study was to evaluate if anticoagulation therapy reduces recurrent stroke in embolic stroke of undetermined source (ESUS) patients with left atrial enlargement (LAE) or abnormal markers of coagulation and hemostatic activity (MOCHA) compared to antiplatelet therapy. Methods: ESUS patients from January 1, 2017, to June 30, 2019, underwent outpatient cardiac monitoring and the MOCHA profile (serum d-dimer, prothrombin fragment 1.2, thrombin-antithrombin complex, and fibrin monomer). Anticoagulation was offered to patients with abnormal MOCHA (≥2 elevated markers) or left atrial volume index 40 mL/m2. Patients were evaluated for recurrent stroke or major hemorrhage at routine clinical follow-up. We compared this patient cohort (cohort 2) to a historical cohort (cohort 1) who underwent the same protocol but remained on antiplatelet therapy. Results: Baseline characteristics in cohort 2 (n = 196; mean age = 63 ± 16 years, 59% female, 49% non-White) were similar to cohort 1 (n = 42) except that cohort 2 had less diabetes (43 vs. 24%, p = 0.01) and more tobacco use (26 vs. 43%, p = 0.04). Overall, 45 patients (23%) in cohort 2 initiated anticoagulation based on abnormal MOCHA or LAE. During mean follow-up of 13 ± 10 months, cohort 2 had significantly lower recurrent stroke rates than cohort 1 (14 vs. 3%, p = 0.009) with no major hemorrhages. Conclusions: Anticoagulation therapy in a subgroup of ESUS patients with abnormal MOCHA or severe LAE may be associated with a reduced rate of recurrent stroke compared to antiplatelet therapy. A prospective, randomized study is warranted to validate these results.

The Utility of the Markers of Coagulation and Hemostatic Activation Profile in the Management of Embolic Strokes of Undetermined Source.

BACKGROUND: Potential causes of embolic stroke of undetermined source (ESUS) include occult malignancy, venous thrombosis (VTE) with paradoxical embolism, and hypercoagulable disorders. Given the association of markers of coagulation and hemostatic activation (MOCHA) with these causes, the objective of this study was to validate the utility of the MOCHA profile in identifying the underlying cause of stroke. METHODS: We prospectively identified ESUS patients from January 1, 2017 to December 1, 2019 who underwent MOCHA profile (plasma d-dimer, prothrombin fragment 1.2, thrombin-antithrombin complex, fibrin monomer) testing. Abnormal MOCHA profile was defined as ≥ 2 abnormal markers. New diagnoses of malignancy, VTE, hypercoagulable disorders and recurrent stroke were identified during routine clinical follow-up. RESULTS: Of 236 ESUS patients, 104 (44%) patients had an abnormal MOCHA profile. In multivariable analyses the number of MOCHA abnormalities was significantly associated with malignancy, VTE, and hypercoagulable disorders (OR 2.59, CI 95% 1.78-3.76, p<0.001). Sensitivity, specificity, positive predictive value, and negative predictive value of an abnormal MOCHA profile for the combined outcome of malignancy, VTE, and hypercoagulability was 96%, 62%, 23%, and 99% respectively. DISCUSSION: The MOCHA profile was able to identify ESUS patients more likely to have malignancy, VTE, and hypercoagulable disorders during follow-up. Our results show that a normal MOCHA profile in ESUS patients can effectively rule out these potential causes of ESUS.

Cerebrovascular events and outcomes in hospitalized patients with COVID-19: The SVIN COVID-19 Multinational Registry.

BACKGROUND: Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has been associated with a significant risk of thrombotic events in critically ill patients. AIM: To summarize the findings of a multinational observational cohort of patients with SARS-CoV-2 and cerebrovascular disease. METHODS: Retrospective observational cohort of consecutive adults evaluated in the emergency department and/or admitted with coronavirus disease 2019 (COVID-19) across 31 hospitals in four countries (1 February 2020-16 June 2020). The primary outcome was the incidence rate of cerebrovascular events, inclusive of acute ischemic stroke, intracranial hemorrhages (ICH), and cortical vein and/or sinus thrombosis (CVST). RESULTS: Of the 14,483 patients with laboratory-confirmed SARS-CoV-2, 172 were diagnosed with an acute cerebrovascular event (1.13% of cohort; 1130/100,000 patients, 95%CI 970-1320/100,000), 68/171 (40.5%) were female and 96/172 (55.8%) were between the ages 60 and 79 years. Of these, 156 had acute ischemic stroke (1.08%; 1080/100,000 95%CI 920-1260/100,000), 28 ICH (0.19%; 190/100,000 95%CI 130-280/100,000), and 3 with CVST (0.02%; 20/100,000, 95%CI 4-60/100,000). The in-hospital mortality rate for SARS-CoV-2-associated stroke was 38.1% and for ICH 58.3%. After adjusting for clustering by site and age, baseline stroke severity, and all predictors of in-hospital mortality found in univariate regression (p < 0.1: male sex, tobacco use, arrival by emergency medical services, lower platelet and lymphocyte counts, and intracranial occlusion), cryptogenic stroke mechanism (aOR 5.01, 95%CI 1.63-15.44, p < 0.01), older age (aOR 1.78, 95%CI 1.07-2.94, p = 0.03), and lower lymphocyte count on admission (aOR 0.58, 95%CI 0.34-0.98, p = 0.04) were the only independent predictors of mortality among patients with stroke and COVID-19. CONCLUSIONS: COVID-19 is associated with a small but significant risk of clinically relevant cerebrovascular events, particularly ischemic stroke. The mortality rate is high for COVID-19-associated cerebrovascular complications; therefore, aggressive monitoring and early intervention should be pursued to mitigate poor outcomes.

Hyperdense vessel sign as a potential guide for the choice of stent retriever versus contact aspiration as first-line thrombectomy strategy.

BACKGROUND: The first-pass effect (FPE) has emerged as a key metric for efficacy in mechanical thrombectomy (MT). The hyperdense vessel sign (HDVS) on non-contrast head CT (NCCT) indicates a higher clot content of red blood cells. OBJECTIVE: To assess whether the HDVS could serve as an imaging biomarker for guiding first-line device selection in MT. METHODS: A prospective MT database was reviewed for consecutive patients with anterior circulation large vessel occlusion stroke who underwent thrombectomy with stent retriever (SR) or contact aspiration (CA) as first-line therapy between January 2012 and November 2018. Pretreatment NCCT scans were evaluated for the presence of HDVS. The primary outcome was FPE (modified Thrombolysis in Cerebral Infarction score 2c/3). The primary analysis was the interaction between HDVS and thrombectomy modality on FPE. Secondary analyses aimed to evaluate the predictors of FPE. RESULTS: A total of 779 patients qualified for the analysis. HDVS and FPE were reported in 473 (60.7%) and 286 (36.7%) patients, respectively. The presence of HDVS significantly modified the effect of thrombectomy modality on FPE (p=0.01), with patients with HDVS having a significantly higher rate of FPE with a SR (41.3% vs 22.2%, p=0.001; adjusted OR 2.11 (95% CI 1.20 to 3.70), p=0.009) and non-HDVS patients having a numerically better response to CA (41.4% vs 33.9%, p=0.28; adjusted OR 0.58 (95% CI 0.311 to 1.084), p=0.088). Age (OR 1.01 (95% CI 1.00 to 1.02), p=0.04) and balloon guide catheter (OR 2.08 (95% CI 1.24 to 3.47), p=0.005) were independent predictors of FPE in the overall population. CONCLUSION: Our data suggest that patients with HDVS may have a better response to SRs than CA for the FPE. Larger confirmatory prospective studies are warranted.

Flow Cytometry Approach to Characterize Phagocytic Properties of Acutely-Isolated Adult Microglia and Brain Macrophages In Vitro.

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in neurological diseases including neurodegeneration and stroke. CNS-MPs are involved in phagocytic clearance of pathological proteins, debris and neuronal synapses, each with distinct underlying molecular pathways. Characterizing these phagocytic properties can provide a functional readout that compliments molecular profiling of microglia using traditional flow cytometry, transcriptomics and proteomics approaches. Phagocytic profiling of microglia has relied on microscopic visualization and in vitro cultures of mouse neonatal microglia. The former approach suffers from limited sampling while the latter approach is inherently poorly reflective of the true in vivo state of adult CNS-MPs. This paper describes optimized protocols to phenotype phagocytic properties of acutely-isolated mouse CNS-MPs by flow cytometry. CNS-MPs are acutely isolated from adult mouse brain using mechanical dissociation followed by density gradient centrifugation, incubated with fluorescent microspheres or fluorescent Aß fibrils, washed, and then labeled with panels of antibodies against surface markers (CD11b, CD45). Using this approach, it is possible to compare phagocytic properties of brain-resident microglia with CNS-infiltrating macrophages and then assess the effect of aging and disease pathology on these phagocytic phenotypes. This rapid method also holds potential to functionally phenotype acutely-isolated human CNS-MPs from post-mortem or surgical brain specimens. Additionally, specific mechanisms of phagocytosis by CNS-MP subsets can be investigated by inhibiting select phagocytic pathways.

Kv1.3 modulates neuroinflammation and neurodegeneration in Parkinson's disease.

Characterization of the key cellular targets contributing to sustained microglial activation in neurodegenerative diseases, including Parkinson's disease (PD), and optimal modulation of these targets can provide potential treatments to halt disease progression. Here, we demonstrated that microglial Kv1.3, a voltage-gated potassium channel, was transcriptionally upregulated in response to aggregated α-synuclein (αSynAgg) stimulation in primary microglial cultures and animal models of PD, as well as in postmortem human PD brains. Patch-clamp electrophysiological studies confirmed that the observed Kv1.3 upregulation translated to increased Kv1.3 channel activity. The kinase Fyn, a risk factor for PD, modulated transcriptional upregulation and posttranslational modification of microglial Kv1.3. Multiple state-of-the-art analyses, including Duolink proximity ligation assay imaging, revealed that Fyn directly bound to Kv1.3 and posttranslationally modified its channel activity. Furthermore, we demonstrated the functional relevance of Kv1.3 in augmenting the neuroinflammatory response by using Kv1.3-KO primary microglia and the Kv1.3-specific small-molecule inhibitor PAP-1, thus highlighting the importance of Kv1.3 in neuroinflammation. Administration of PAP-1 significantly inhibited neurodegeneration and neuroinflammation in multiple animal models of PD. Collectively, our results imply that Fyn-dependent regulation of Kv1.3 channels plays an obligatory role in accentuating the neuroinflammatory response in PD and identify Kv1.3 as a potential therapeutic target for PD.

Flow-cytometric microglial sorting coupled with quantitative proteomics identifies moesin as a highly-abundant microglial protein with relevance to Alzheimer's disease.

BACKGROUND: Proteomic characterization of microglia provides the most proximate assessment of functionally relevant molecular mechanisms of neuroinflammation. However, microglial proteomics studies have been limited by low cellular yield and contamination by non-microglial proteins using existing enrichment strategies. METHODS: We coupled magnetic-activated cell sorting (MACS) and fluorescence activated cell sorting (FACS) of microglia with tandem mass tag-mass spectrometry (TMT-MS) to obtain a highly-pure microglial proteome and identified a core set of highly-abundant microglial proteins in adult mouse brain. We interrogated existing human proteomic data for Alzheimer's disease (AD) relevance of highly-abundant microglial proteins and performed immuno-histochemical and in-vitro validation studies. RESULTS: Quantitative multiplexed proteomics by TMT-MS of CD11b + MACS-enriched (N = 5 mice) and FACS-isolated (N = 5 mice), from adult wild-type mice, identified 1791 proteins. A total of 203 proteins were highly abundant in both datasets, representing a core-set of highly abundant microglial proteins. In addition, we found 953 differentially enriched proteins comparing MACS and FACS-based approaches, indicating significant differences between both strategies. The FACS-isolated microglia proteome was enriched with cytosolic, endoplasmic reticulum, and ribosomal proteins involved in protein metabolism and immune system functions, as well as an abundance of canonical microglial proteins. Conversely, the MACS-enriched microglia proteome was enriched with mitochondrial and synaptic proteins and higher abundance of neuronal, oligodendrocytic and astrocytic proteins. From the 203 consensus microglial proteins with high abundance in both datasets, we confirmed microglial expression of moesin (Msn) in wild-type and 5xFAD mouse brains as well as in human AD brains. Msn expression is nearly exclusively found in microglia that surround Aß plaques in 5xFAD brains. In in-vitro primary microglial studies, Msn silencing by siRNA decreased Aß phagocytosis and increased lipopolysaccharide-induced production of the pro-inflammatory cytokine, tumor necrosis factor (TNF). In network analysis of human brain proteomic data, Msn was a hub protein of an inflammatory co-expression module positively associated with AD neuropathological features and cognitive dysfunction. CONCLUSIONS: Using FACS coupled with TMT-MS as the method of choice for microglial proteomics, we define a core set of highly-abundant adult microglial proteins. Among these, we validate Msn as highly-abundant in plaque-associated microglia with relevance to human AD.

Markers of coagulation and hemostatic activation aid in identifying causes of cryptogenic stroke.

OBJECTIVE: To test the hypothesis that markers of coagulation and hemostatic activation (MOCHA) help identify causes of cryptogenic stroke, we obtained serum measurements on 132 patients and followed them up to identify causes of stroke. METHODS: Consecutive patients with cryptogenic stroke who met embolic stroke of undetermined source (ESUS) criteria from January 1, 2017, to October 31, 2018, underwent outpatient cardiac monitoring and the MOCHA profile (serum D-dimer, prothrombin fragment 1.2, thrombin-antithrombin complex, and fibrin monomer) obtained ≥2 weeks after the index stroke; abnormal MOCHA profile was defined as ≥2 elevated markers. Prespecified endpoints monitored during routine clinical visits included new atrial fibrillation (AF), malignancy, venous thromboembolism (VTE), or other defined hypercoagulable states (HS). RESULTS: Overall, 132 patients with ESUS (mean age 64 ± 15 years, 61% female, 51% nonwhite) met study criteria. During a median follow-up of 10 (interquartile range 7-14) months, AF, malignancy, VTE, or HS was identified in 31 (23%) patients; the 53 (40%) patients with ESUS with abnormal MOCHA were significantly more likely than patients with normal levels to have subsequent new diagnoses of malignancy (21% vs 0%, p < 0.001), VTE (9% vs 0%, p = 0.009), or HS (11% vs 0%, p = 0.004) but not AF (8% vs 9%, p = 0.79). The combination of 4 normal MOCHA and normal left atrial size (n = 30) had 100% sensitivity for ruling out the prespecified endpoints. CONCLUSION: The MOCHA profile identified patients with cryptogenic stroke more likely to have new malignancy, VTE, or HS during short-term follow-up and may be useful in direct evaluation for underlying causes of cryptogenic stroke.

Recurrent Hemispheric Stroke Syndromes in Symptomatic Atherosclerotic Internal Carotid Artery Occlusions: The Carotid Occlusion Surgery Study Randomized Trial.

BACKGROUND: There are limited data on outcomes of extracranial-intracranial (EC-IC) bypass in patients with recurrent hemispheric syndromes due to atherosclerotic internal carotid artery occlusion (AICAO). OBJECTIVE: To compare clinical outcomes and efficacy of EC-IC bypass surgery in patients with and without recurrent hemispheric syndromes associated with AICAO in the Carotid Occlusion Surgery Study (COSS). METHODS: In patients enrolled in the COSS trial, we compared baseline characteristics and clinical outcomes for participants with (rHEMI+) and without recurrent hemispheric ischemia (rHEMI-) prior to randomization into surgical vs medical groups. The primary outcome was all stroke and death from randomization through 30 d and ipsilateral ischemic stroke within 2 yr. RESULTS: Of 195 randomized participants, 100 were rHEMI+ (50 in each group). Baseline characteristics between rHEMI+ and rHEMI- participants were similar except rHEMI+ were more likely to have had previous stroke prior to randomization (61% vs 20%, P < .01) and to have TIA as the entry event (59% vs 21%, P < .01). All primary endpoints were ipsilateral ischemic strokes. There were no significant differences in occurrence of the primary endpoint between nonsurgical and surgical participants in rHEMI+ (26.3% vs 22.4%, P = .660) and rHEMI- (18.9% vs 19.5%, P = .943). For nonsurgical participants, there was no significant difference in the primary endpoint for rHEMI+ vs rHEMI- patients (P = .410). CONCLUSION: Patients with recurrent hemispheric stroke syndromes enrolled in the COSS trial did not show benefit from EC-IC bypass compared to medical treatment. Early aggressive risk factor measures should be prioritized to reduce recurrent strokes in these patients.

Estimating the social value of mechanical thrombectomy randomized trials on an established stroke network.

BACKGROUND: Given the relative strength of prior mechanical thrombectomy (MT) data in face of the perceived poor natural history of emergent large vessel occlusion strokes, randomization to medical therapy during the recent clinical MT trials raised ethical challenges at individual and institutional levels despite overall clinical equipoise. METHODS: In a thrombectomy stroke registry, we compared treatment rates preceding and following the SWIFT-PRIME and DAWN trials. Based on effect sizes of treatment in both trials, we estimated missed opportunities due to randomization to medical therapy and estimated the societal benefit resulting from additional patients who benefited as a result of these studies. RESULTS: The average monthly thrombectomy rate in the SWIFT-PRIME time window increased from 14.1±4 patients-per-month (ppm) to 23.8±6 ppm (p<0·001). Twelve subjects were enrolled in SWIFT-PRIME and we estimated a missed opportunity of benefiting 2.3 of six subjects randomized to medical therapy. This was offset by providing treatment to an additional 9.7 ppm, resulting in an additional functional benefit to 3.7 ppm. Similarly, the thrombectomy rate in the DAWN window increased from 8.6±4 ppm pre-DAWN to 11.9±3.6 ppm post-DAWN (p<0.01). 38 subjects were enrolled in the DAWN trial with a missed opportunity to benefit eight of 16 subjects randomized to medical therapy. This was offset by the ability to offer MT to an additional 3.3 ppm, bringing definite benefit to an additional 1.65 ppm. CONCLUSION: Completion of recent trials resulted in observable increases in rates of thrombectomy, translating to functional benefits that rapidly offset any missed opportunities due to randomization to medical therapy arms.

Deep Hypothermia With Retrograde Cerebral Perfusion Versus Moderate Hypothermia With Antegrade Cerebral Perfusion for Arch Surgery.

BACKGROUND: Patients undergoing aortic arch replacement are at high risk for neurologic injury. This study compared two different established neuroprotective strategies in patients undergoing elective transverse hemiarch replacement. METHODS: Twenty patients undergoing hemiarch replacement were prospectively randomized to receive deep hypothermic circulatory arrest with retrograde cerebral perfusion (DHCA+RCP) or moderate hypothermic circulatory arrest with antegrade cerebral perfusion (MHCA+ACP). All patients received neurologist-adjudicated examinations and magnetic resonance imaging before discharge. The primary end point was a composite of stroke, transient ischemic attack, and magnetic resonance imaging-adjudicated injury. Secondary end points were transient neurologic dysfunction, and the National Institutes of Health Stroke Scale, and neurocognitive scores. RESULTS: Randomization resulted in 11 DHCA+RCP patients and 9 MHCA+ACP patients. There was no difference in cardiopulmonary bypass, cross-clamp, or circulatory arrest times. MHCA+ACP patients underwent circulatory arrest at significantly warmer temperatures (26.3° ± 1.8°C) than DHCA+RCP patients (19.9° ± 0.1°C, p < 0.0001). There were no deaths or renal failure in either group. There was 1 stroke in each group. National Institute of Health stroke scale scores and neurocognitive test results were equivalent. Diffusion-weighted magnetic resonance imaging demonstrated lesions in 100% (9 of 9) of MHCA+ACP patients compared with 45% (5 of 11) of DHCA+RCP patients (p < 0.01). MHCA+ACP patients had a significantly higher number of lesions than DHCA+RCP patients (p < 0.01). The primary end point was achieved in 100% of MHCA+ACP patients compared with 45% of DHCA+RCP patients (p < 0.01). CONCLUSIONS: Although there was no significant difference in clinically evident neurologic injury, this pilot study suggests that MHCA+ACP may be associated with a higher incidence of radiographic neurologic injury than DHCA+RCP in patients undergoing elective hemiarch replacement.

Prognostic importance of CT ASPECTS and CT perfusion measures of infarction in anterior emergent large vessel occlusions.

BACKGROUND: Age, neurologic deficits, core volume (CV), and clinical core or radiographic mismatch are considered in selection for endovascular therapy (ET) in anterior circulation emergent large vessel occlusion (aELVO). Semiquantitative CV estimation by Alberta Stroke Programme Early CT Score (CT ASPECTS) and quantitative CV estimation by CT perfusion (CTP) are both used in selection paradigms. OBJECTIVE: To compare the prognostic value of CTP CV with CT ASPECTS in aELVO. METHODS: Patients in an institutional endovascular registry who had aELVO, pre-ET National Institutes of Health Stroke Scale (NIHSS) score, non-contrast CT head and CTP imaging, and prospectively collected 3-month modified Rankin Scale (mRS) score were included. Age- and NIHSS-adjusted models, including either CT ASPECTS or CTP volumes (relative cerebral blood flow <30% of normal tissue, total hypoperfusion, and radiographic mismatch), were compared using receiver operator characteristic analyses. RESULTS: We included 508 patients with aELVO (60.8% M1 middle cerebral artery, 34% internal carotid artery, mean age 64.1±15.3 years, median baseline NIHSS score 16 (12-20), median baseline CT ASPECTS 8 (7-9), mean CV 16.7±24.8 mL). Age, pre-ET NIHSS, CT ASPECTS, CV, hypoperfusion, and perfusion imaging mismatch volumes were predictors of good outcome (mRS score 0-2). There were no differences in prognostic accuracies between reference (age, baseline NIHSS, CT ASPECTS; area under the curve (AUC)=0.76) and additional models incorporating combinations of age, NIHSS, and CTP metrics including CV, total hypoperfusion or mismatch volume (AUCs 0.72-0.75). Predicted outcomes from CT ASPECTS or CTP CV-based models had excellent agreement (R2=0.84, p<0.001). CONCLUSIONS: Incorporating CTP measures of core or penumbral volume, instead of CT ASPECTS, did not improve prognostication of 3-month outcomes, suggesting prognostic equivalence of CT ASPECTS and CTP CV.

Coagulation markers and echocardiography predict atrial fibrillation, malignancy or recurrent stroke after cryptogenic stroke.

We evaluated the utility of left atrial volume index (LAVI) and markers of coagulation and hemostatic activation (MOCHA) in cryptogenic stroke (CS) patients to identify those more likely to have subsequent diagnosis of atrial fibrillation (AF), malignancy or recurrent stroke during follow-up.Consecutive CS patients who met embolic stroke of undetermined source (ESUS) who underwent transthoracic echocardiography and outpatient cardiac monitoring following stroke were identified from the Emory cardiac registry. In a subset of consecutive patients, d-dimer, prothrombin fragment 1.2, thrombin-antithrombin complex and fibrin monomer (MOCHA panel) were obtained ≥2 weeks post-stroke and repeated ≥4 weeks later if abnormal; abnormal MOCHA panel was defined as ≥2 elevated markers which did not normalize when repeated. We assessed the predictive abilities of LAVI and the MOCHA panel to identify patients with subsequent diagnosis of AF, malignancy, recurrent stroke or the composite outcome during follow-up.Of 94 CS patients (mean age 64 ± 15 years, 54% female, 63% non-white, mean follow-up 1.4 ± 0.8 years) who underwent prolonged cardiac monitoring, 15 (16%) had new AF. Severe LA enlargement (vs normal) was associated with AF (P < .06). In 42 CS patients with MOCHA panel testing (mean follow-up 1.1 ± 0.6 years), 14 (33%) had the composite outcome and all had abnormal MOCHA. ROC analysis showed LAVI and abnormal MOCHA together outperformed either test alone with good predictive ability for the composite outcome (AUC 0.84).We report the novel use of the MOCHA panel in CS patients to identify a subgroup of patients more likely to have occult AF, occult malignancy or recurrent stroke during follow-up. A normal MOCHA panel identified a subgroup of CS patients at low risk for recurrent stroke on antiplatelet therapy. Further study is warranted to evaluate whether the combination of an elevated LAVI and abnormal MOCHA panel identifies a subgroup of CS patients who may benefit from early anticoagulation for secondary stroke prevention.

Predictors of Good Outcome After Endovascular Therapy for Vertebrobasilar Occlusion Stroke.

BACKGROUND AND PURPOSE: Endovascular therapy is increasingly used in acute ischemic stroke treatment and is now considered the gold standard approach for selected patient populations. Prior studies have demonstrated that eventual patient outcomes depend on both patient-specific factors and procedural considerations. However, these factors remain unclear for acute basilar artery occlusion stroke. We sought to determine prognostic factors of good outcome in acute posterior circulation large vessel occlusion strokes treated with endovascular therapy. METHODS: We reviewed our prospectively collected endovascular databases at 2 US tertiary care academic institutions for patients with acute posterior circulation strokes from September 2005 to September 2015 who had 3-month modified Rankin Scale documented. Baseline characteristics, procedural data, and outcomes were evaluated. A good outcome was defined as a 90-day modified Rankin Scale score of 0 to 2. The association between clinical and procedural parameters and functional outcome was assessed. RESULTS: A total of 214 patients qualified for the study. Smoking status, creatinine levels, baseline National Institutes of Health Stroke Scale score, anesthesia modality (conscious sedation versus general anesthesia), procedural length, and reperfusion status were significantly associated with good outcomes in the univariate analysis. Multivariate logistic regression indicated that only smoking (odds ratio=2.61; 95% confidence interval, 1.23-5.56; P=0.013), low baseline National Institutes of Health Stroke Scale score (odds ratio=1.09; 95% confidence interval, 1.04-1.13; P<0.0001), and successful reperfusion status (odds ratio=10.80; 95% confidence interval, 1.36-85.96; P=0.025) were associated with good outcome. CONCLUSIONS: In our retrospective case series, only smoking, low baseline National Institutes of Health Stroke Scale score, and successful reperfusion status were associated with good outcome in patients with posterior circulation stroke treated with endovascular therapy.

A systems pharmacology-based approach to identify novel Kv1.3 channel-dependent mechanisms in microglial activation.

BACKGROUND: Kv1.3 potassium channels regulate microglial functions and are overexpressed in neuroinflammatory diseases. Kv1.3 blockade may selectively inhibit pro-inflammatory microglia in neurological diseases but the molecular and cellular mechanisms regulated by Kv1.3 channels are poorly defined. METHODS: We performed immunoblotting and flow cytometry to confirm Kv1.3 channel upregulation in lipopolysaccharide (LPS)-activated BV2 microglia and in brain mononuclear phagocytes freshly isolated from LPS-treated mice. Quantitative proteomics was performed on BV2 microglia treated with control, LPS, ShK-223 (highly selective Kv1.3 blocker), and LPS+ShK-223. Gene ontology (GO) analyses of Kv1.3-dependent LPS-regulated proteins were performed, and the most representative proteins and GO terms were validated. Effects of Kv1.3-blockade on LPS-activated BV2 microglia were studied in migration, focal adhesion formation, reactive oxygen species production, and phagocytosis assays. In vivo validation of protein changes and predicted molecular pathways were performed in a model of systemic LPS-induced neuroinflammation, employing antigen presentation and T cell proliferation assays. Informed by pathway analyses of proteomic data, additional mechanistic experiments were performed to identify early Kv1.3-dependent signaling and transcriptional events. RESULTS: LPS-upregulated cell surface Kv1.3 channels in BV2 microglia and in microglia and CNS-infiltrating macrophages isolated from LPS-treated mice. Of 144 proteins differentially regulated by LPS (of 3141 proteins), 21 proteins showed rectification by ShK-223. Enriched cellular processes included MHCI-mediated antigen presentation (TAP1, EHD1), cell motility, and focal adhesion formation. In vitro, ShK-223 decreased LPS-induced focal adhesion formation, reversed LPS-induced inhibition of migration, and inhibited LPS-induced upregulation of EHD1, a protein involved in MHCI trafficking. In vivo, intra-peritoneal ShK-223 inhibited LPS-induced MHCI expression by CD11b+CD45low microglia without affecting MHCI expression or trafficking of CD11b+CD45high macrophages. ShK-223 inhibited LPS-induced MHCI-restricted antigen presentation to ovalbumin-specific CD8+ T cells both in vitro and in vivo. Kv1.3 co-localized with the LPS receptor complex and regulated LPS-induced early serine (S727) STAT1 phosphorylation. CONCLUSIONS: We have unraveled novel molecular and functional roles for Kv1.3 channels in pro-inflammatory microglial activation, including a Kv1.3 channel-regulated pathway that facilitates MHCI expression and MHCI-dependent antigen presentation by microglia to CD8+ T cells. We also provide evidence for neuro-immunomodulation by systemically administered ShK peptides. Our results further strengthen the therapeutic candidacy of microglial Kv1.3 channels in neurologic diseases.