PKM2 promotes neutrophil activation and cerebral thromboinflammation: therapeutic implications for ischemic stroke.

There is a critical need for cerebro-protective interventions to improve the suboptimal outcomes of patients with ischemic stroke who have been treated with reperfusion strategies. We found that nuclear pyruvate kinase muscle 2 (PKM2), a modulator of systemic inflammation, was upregulated in neutrophils after the onset of ischemic stroke in both humans and mice. Therefore, we determined the role of PKM2 in stroke pathogenesis by using murine models with preexisting comorbidities. We generated novel myeloid cell-specific PKM2-/- mice on wild-type (PKM2fl/flLysMCre+) and hyperlipidemic background (PKM2fl/flLysMCre+Apoe-/-). Controls were littermate PKM2fl/flLysMCre- or PKM2fl/flLysMCre-Apoe-/- mice. Genetic deletion of PKM2 in myeloid cells limited inflammatory response in peripheral neutrophils and reduced neutrophil extracellular traps after cerebral ischemia and reperfusion, suggesting that PKM2 promotes neutrophil hyperactivation in the setting of stroke. In the filament and autologous clot and recombinant tissue plasminogen activator stroke models, irrespective of sex, deletion of PKM2 in myeloid cells in either wild-type or hyperlipidemic mice reduced infarcts and enhanced long-term sensorimotor recovery. Laser speckle imaging revealed improved regional cerebral blood flow in myeloid cell-specific PKM2-deficient mice that was concomitant with reduced post-ischemic cerebral thrombo-inflammation (intracerebral fibrinogen, platelet [CD41+] deposition, neutrophil infiltration, and inflammatory cytokines). Mechanistically, PKM2 regulates post-ischemic inflammation in peripheral neutrophils by promoting STAT3 phosphorylation. To enhance the translational significance, we inhibited PKM2 nuclear translocation using a small molecule and found significantly reduced neutrophil hyperactivation and improved short-term and long-term functional outcomes after stroke. Collectively, these findings identify PKM2 as a novel therapeutic target to improve brain salvage and recovery after reperfusion.

Detection and Quantification of Symptomatic Atherosclerotic Plaques With High-Resolution Imaging in Cryptogenic Stroke.

BACKGROUND AND PURPOSE: High-resolution vessel wall imaging (HR-VWI) is a powerful tool in diagnosing intracranial vasculopathies not detected on routine imaging. We hypothesized that 7T HR-VWI may detect the presence of atherosclerotic plaques in patients with intracranial atherosclerosis disease initially misdiagnosed as cryptogenic strokes. METHODS: Patients diagnosed as cryptogenic stroke but suspected of having an intracranial arteriopathy by routine imaging were prospectively imaged with HR-VWI. If intracranial atherosclerotic plaques were identified, they were classified as culprit or nonculprit based on the likelihood of causing the index stroke. Plaque characteristics, such as contrast enhancement, degree of stenosis, and morphology, were analyzed. Contrast enhancement was determined objectively after normalization with the pituitary stalk. A cutoff value for plaque-to-pituitary stalk contrast enhancement ratio (CR) was determined for optimal prediction of the presence of a culprit plaque. A revised stroke cause was adjudicated based on clinical and HR-VWI findings. RESULTS: A total of 344 cryptogenic strokes were analyzed, and 38 eligible patients were imaged with 7T HR-VWI. Intracranial atherosclerosis disease was adjudicated as the final stroke cause in 25 patients. A total of 153 intracranial plaques in 374 arterial segments were identified. Culprit plaques (n=36) had higher CR and had concentric morphology when compared with nonculprit plaques (P≤0.001). CR ≥53 had 78% sensitivity for detecting culprit plaques and a 90% negative predictive value. CR ≥53 (P=0.008), stenosis ≥50% (P<0.001), and concentric morphology (P=0.030) were independent predictors of culprit plaques. CONCLUSIONS: 7T HR-VWI allows identification of underlying intracranial atherosclerosis disease in a subset of stroke patients with suspected underlying vasculopathy but otherwise classified as cryptogenic. Plaque analysis in this population demonstrated that culprit plaques had more contrast enhancement (CR ≥53), caused a higher degree of stenosis, and had a concentric morphology.

Cerebral Microbleeds are an Independent Predictor of Hemorrhagic Transformation Following Intravenous Alteplase Administration in Acute Ischemic Stroke.

BACKGROUND AND PURPOSE: Intravenous alteplase (rt-PA) increases the risk of hemorrhagic transformation of acute ischemic stroke. The objective of our study was to evaluate clinical, laboratory, and imaging predictors on forecasting the risk of hemorrhagic transformation following treatment with rt-PA. We also evaluated the factors associated with cerebral microbleeds that increase the risk of hemorrhagic transformation. METHODS: Consecutive patients with acute ischemic stroke admitted between January 1, 2009 and December 31, 2013 were included in the study if they received IV rt-PA, had magnetic resonance imaging (MRI) of the brain on admission, and computed tomography or MRI of the brain at 24 (18-36) hours later to evaluate for the presence of hemorrhagic transformation. The clinical data, lipid levels, platelet count, MRI, and computed tomography images were retrospectively reviewed. RESULTS: The study included 366 patients, with mean age 67 ± 15 years; 46% were women and 88% were white. The median National Institutes of Health Stroke Scale (NIHSS) score was 6 (interquartile range 3-15). Hemorrhagic transformation was observed in 87 (23.8%) patients and cerebral microbleeds were noted in 95 (25.9%). Patients with hemorrhagic transformation tended to be older, nonwhite, have atrial fibrillation, higher baseline NIHSS score, lower cholesterol and triglyceride levels, and cerebral microbleeds and nonlacunar infarcts. Patients with cerebral microbleeds were more likely to be older, have hypertension, hyperlipidemia, previous history of stroke, and prior use of antithrombotics. On multivariate analysis race, NIHSS score, nonlacunar infarct, and presence of cerebral microbleeds were independently associated with hemorrhagic transformation following treatment with rt-PA. CONCLUSIONS: Presence of cerebral microbleeds is an independent predictor of hemorrhagic transformation of acute ischemic stroke following treatment with rt-PA.

Lack of international consensus on ethical aspects of acute stroke trials.

Acute stroke trials are becoming increasingly multinational. Working toward a shared ethical standard for acute stroke research necessitates evaluating the degree of consensus among international researchers. We surveyed all 275 coinvestigators and coordinators who participated in the AbESTT II study (evaluating abciximab vs placebo) about their experience with their local institutional review board (IRB) or equivalent, as well as, about their personal beliefs regarding the ethical aspects of acute stroke trials. A total of 90 coinvestigators from 15 different countries responded to our survey. Among the IRBs represented by the responding coinvestigators, only 18% allowed surrogate consent to be obtained over the phone. Although 52% allowed the participation of subjects with aphasia, only 5% allowed the participation of subjects with neglect/hemi-inattention. The National Institutes of Health Stroke Scale score was deemed adequate to establish decisional capacity based on language by 62% of the coinvestigators and 36% of the IRBs. A belief that IRB regulations cause unnecessary delays and fear in relatives/patients was reported by 67% of coinvestigators, and the belief that granting an exemption from informed consent under specific circumstances is appropriate was reported by 41%. There appears to be considerable international diversity in the ethical priorities and informed consent standards among different IRBs and investigators in stroke research. The stroke community should make an attempt to standardize the consent process used in research. Given the critical nature of the time to treatment in stroke care, these standards should be integrated into current frameworks of clinical care and research. The absence of an ethical consensus can become a barrier to advancing stroke treatment internationally.

Multiple sclerosis patients have an altered gut mycobiome and increased fungal to bacterial richness.

Trillions of microbes such as bacteria, fungi, and viruses exist in the healthy human gut microbiome. Although gut bacterial dysbiosis has been extensively studied in multiple sclerosis (MS), the significance of the fungal microbiome (mycobiome) is an understudied and neglected part of the intestinal microbiome in MS. The aim of this study was to characterize the gut mycobiome of patients with relapsing-remitting multiple sclerosis (RRMS), compare it to healthy controls, and examine its association with changes in the bacterial microbiome. We characterized and compared the mycobiome of 20 RRMS patients and 33 healthy controls (HC) using Internal Transcribed Spacer 2 (ITS2) and compared mycobiome interactions with the bacterial microbiome using 16S rRNA sequencing. Our results demonstrate an altered mycobiome in RRMS patients compared with HC. RRMS patients showed an increased abundance of Basidiomycota and decreased Ascomycota at the phylum level with an increased abundance of Candida and Epicoccum genera along with a decreased abundance of Saccharomyces compared to HC. We also observed an increased ITS2/16S ratio, altered fungal and bacterial associations, and altered fungal functional profiles in MS patients compared to HC. This study demonstrates that RRMS patients had a distinct mycobiome with associated changes in the bacterial microbiome compared to HC. There is an increased fungal to bacterial ratio as well as more diverse fungal-bacterial interactions in RRMS patients compared to HC. Our study is the first step towards future studies in delineating the mechanisms through which the fungal microbiome can influence MS disease.

Previous statin use is not associated with an increased prevalence or degree of gradient-echo lesions in patients with acute ischemic stroke or transient ischemic attack.

BACKGROUND AND PURPOSE: Microhemorrhages on gradient-echo T2*-weighted MRI sequences are often found in patients with cerebrovascular disease and are related to intracerebral hemorrhage. Because statin therapy is associated with increased risk of intracerebral hemorrhage, we investigated whether statin use was also associated with microhemorrhages in patients with acute ischemic stroke or transient ischemic attack. METHODS: We performed a retrospective analysis on prospectively collected data from a stroke registry containing patients with acute ischemic stroke or transient ischemic attack. The primary and secondary outcome variables were the prevalence and degree of microhemorrhages as detected on gradient-echo MRI sequences and categorized as mild (1-2), moderate (3-10), or severe (>10). The location of the microhemorrhages was noted and rated by 2 neuroradiologists. Previous use of statins and other covariates were assessed as potential predictors. RESULTS: Three hundred forty-nine patients were admitted from June 2008 to July 2009, and 300 of which were analyzed. Microhemorrhages were detected in 70 subjects (23%); 35 had only lobar lesions, 16 had only deep lesions, and 19 had both lobar and deep lesions. On univariate and multivariate analysis, statin therapy was not associated with the prevalence (OR, 0.73; 95% CI, 0.36-1.51; P=0.40) or degree of microhemorrhages modeled for lesser severity (OR, 2.31; 95% CI, 0.61-8.75; P=0.22). CONCLUSIONS: Previous statin therapy was not associated with the prevalence or degree of microhemorrhages in patients with acute ischemic stroke or transient ischemic attack. The association between statins and intracerebral hemorrhage does not appear to be mediated through microhemorrhages.

Extending acute trials to remote populations: a pilot study during interhospital helicopter transfer.

BACKGROUND AND PURPOSE: Methods to increase recruitment into acute stroke trials are needed. The purposes of this study were to evaluate the safety and acceptability of initiating acute stroke trials during early helicopter evacuation and to test an intervention to facilitate informed consent. METHODS: A randomized, controlled trial was done with patients with acute stroke who were transferred by helicopter to the University of Iowa Hospitals and Clinics from February 2007 to January 2008. The intervention to be evaluated was the use of fax and a telephone call to the patient/surrogate ahead of helicopter arrival at the outside emergency department. The aim was to improve the rate of subsequent consent (primary outcome) for a pilot trial of a potentially beneficial, low-risk medical intervention (ranitidine) to prevent aspiration pneumonitis. Consenting eligible patients received the infusion during the flight to University of Iowa Hospitals and Clinics. RESULTS: One hundred patients were enrolled. Consent rate was 54% in the intervention group and 50% in the control group (P=0.69). However, the consent rate was higher (69%) when prearrival communications between the coinvestigator and potential subjects were successful (P=0.04). This approach resulted in an average gain of 59 minutes as compared with initiating recruitment on arrival to University of Iowa Hospitals and Clinics. CONCLUSIONS: Enrollment into stroke intervention trials is feasible during helicopter transportation from a community hospital emergency department to a tertiary stroke center. This underused resource may improve trial efficiency by enabling and expediting participation of remote populations currently excluded from research. Consent rates might be further improved by communication strategies that are more successful in reaching patients at outside emergency departments.

An autoimmune disease risk SNP, rs2281808, in SIRPG is associated with reduced expression of SIRPγ and heightened effector state in human CD8 T-cells.

Multiple GWAS studies have shown that the SNP rs2281808 TT variant, present within the SIRPG gene, is associated with autoimmune diseases, such as type 1 diabetes. However, the role of SIRPγ in human T-cells is not known, neither is the functional significance of TT variant. Here we investigated SIRPG genotypes and their effects on the fate and function of human T-cells. We found that the presence of T variant resulted in reduction of SIRPγ expression on T-cells. Functionally, SIRPγlow CD8 T-cells in CT and TT individuals existed in a heightened effector state with lower activation threshold and had greater expression of genes and molecules associated with migratory and cytotoxic potential. Further, SIRPγlow CD8 T-cells were deficient in transcription factors associated with long-term functional memory formation. Our study reveals biological consequences of the SNP rs2281808 and provides novel insights into the potential mechanisms by which SIRPγ might regulate human immune responses.

Effect of helicopter transport on neurological outcomes in a mouse model of embolic stroke with reperfusion: AIR-MICE pilot study.

BACKGROUND: Patients often suffer a stroke at a significant distance from a center capable of delivering endovascular therapy, thus requiring rapid transport by helicopter emergency medical services while receiving a recombinant tissue plasminogen activator infusion that was initiated locally. But little is known about how a helicopter flight may impact the safety and efficacy of recombinant tissue plasminogen activator-induced reperfusion and patient outcomes. AIM: To establish a new animal method to address with fidelity the safety and overall effect of helicopter emergency medical services during thrombolysis. METHODS: Prospective randomized open blinded end-point study of an actual helicopter flight exposure. Adult C57BL/6 male mice were treated with a 10 mg/kg recombinant tissue plasminogen activator infusion two-hours after an embolic middle cerebral artery occlusion. Mice were randomized in pairs to simultaneously receive the infusion during a local helicopter flight or in a ground hangar. RESULTS: Eighteen mice (nine pairs) were analyzed. The paired t-test analysis showed nonsignificant smaller infarction volumes in the helicopter-assigned animals (mean pair difference 33 mm(3) , P = 0·33). The amount of hemorrhagic transformation between the helicopter and ground groups was 4·08 vs. 4·56 µl, respectively (paired t-test, P = 0·45). CONCLUSIONS: This study shows that helicopter emergency medical services do not have an inherent adverse effect on outcome in a mouse model of ischemic stroke with reperfusion. These results endorse the safety of the practice of using helicopter emergency medical services in stroke patients. The observed potential synergistic effect of helicopter-induced factors, such as vibration and changes in altitude, with reperfusion merits further exploration in animal experimental models and in stroke patients.