Central IRF4/5 Signaling Are Critical for Microglial Activation and Impact on Stroke Outcomes.

Microglia and monocytes play a critical role in immune responses to cerebral ischemia. Previous studies have demonstrated that interferon regulatory factor 4 (IRF4) and IRF5 direct microglial polarization after stroke and impact outcomes. However, IRF4/5 are expressed by both microglia and monocytes, and it is not clear if it is the microglial (central) or monocytic (peripheral) IRF4-IRF5 regulatory axis that functions in stroke. In this work, young (8-12 weeks) male pep boy (PB), IRF4 or IRF5 flox, and IRF4 or IRF5 conditional knockout (CKO) mice were used to generate 8 types of bone marrow chimeras, to differentiate the role of central (PB-to-IRF CKO) vs. peripheral (IRF CKO-to-PB) phagocytic IRF4-IRF5 axis in stroke. Chimeras generated from PB and flox mice were used as controls. All chimeras were subjected to 60-min middle cerebral artery occlusion (MCAO) model. Three days after the stroke, outcomes and inflammatory responses were analyzed. We found that PB-to-IRF4 CKO chimeras had more robust microglial pro-inflammatory responses than IRF4 CKO-to-PB chimeras, while ameliorated microglial response was seen in PB-to-IRF5 CKO vs. IRF5 CKO-to-PB chimeras. PB-to-IRF4 or IRF5 CKO chimeras had worse or better stroke outcomes respectively than their controls, whereas IRF4 or 5 CKO-to-PB chimeras had similar outcomes compared to controls. We conclude that the central IRF4/5 signaling is responsible for microglial activation and mediates stroke outcomes.

Heart Brain Clinic: An Integrated Approach to Stroke Care.

Background and Objectives: Multidisciplinary clinics have been shown to improve care. Patients with patent foramen ovale (PFO)-associated stroke need evaluation by cardiology and neurology specialists. We report our experience creating a multidisciplinary Structural Heart Brain Clinic (HBC) with a focus on patients with PFO-associated stroke. Methods: Demographic and clinical data were retrospectively collected for patients with PFO-associated ischemic stroke. Patients with PFO-associated stroke were divided into a standard care group and Heart Brain Clinic group for analysis. Outcome measures included time from stroke to PFO closure and number of clinic visits before decision regarding closure. Nonparametric analysis evaluated differences in median time to visit and clinical decision, while the chi square analysis compared differences in categorical variables between groups. Results: From February 2017 to December 2021, 120 patients were evaluated for PFO-associated stroke. The Structural HBC began in 12/2018 with coordination between Departments of Neurology and Cardiology. For this analysis, 41 patients were considered in the standard care group and 79 patients in the HBC group. During data analysis, 107 patients had received recommendations about PFO closure. HBC patients required fewer clinic visits (p = 0.001) before decision about closure; however, among patients who underwent PFO closure, there was no significant difference in weeks from stroke to PFO closure. Clinicians were more likely to recommend against PFO closure among patients seen in HBC compared with those seen in standard care (p = 0.021). Discussion: Our data demonstrate that a multidisciplinary, patient-centered approach to management of patients with PFO-associated ischemic stroke is feasible and may improve the quality of care in this younger patient population. The difference in recommendation to not pursue PFO closure between groups may reflect selection and referral bias. Additional work is needed to determine whether this approach improves other aspects of care and outcomes.

Strategies for Maintaining Brain Health: The Role of Stroke Risk Factors Unique to Elderly Women.

Stroke risk and prevalence increase with advanced age and women tend to be older than men at the time of their first stroke. Advanced age in women confers unique stroke risks that are beyond reproductive factors. Previous reviews and guidelines have largely focused on risk factors specific to women, with a predominant focus on reproductive factors and, therefore, younger to middle-aged women. This review aims to specifically describe stroke risk factors in elderly women, the population of women where the majority of strokes occur, with a focus on atrial fibrillation, hormone therapy, psychosocial risk factors, and cognitive impairment. Our review suggests that prevention and management of stroke risks that are unique or more prevalent in elderly women needs a coordinated system of care from general physicians, general neurologists, vascular and cognitive neurologists, psychologists, cardiologists, patients, and their caretakers. Early identification and management of the elderly woman-specific and traditional stroke risk factors is key for decreasing stroke burden in elderly women. Increased education among elderly women regarding stroke risk factors and their identification should be considered, and an update to the guidelines for prevention of stroke in women is strongly encouraged.

Sex differences in a murine model of Cerebral Amyloid Angiopathy.

Cerebral amyloid angiopathy (CAA) is one of the common causes of lobar intracerebral hemorrhage and vascular cognitive impairment (VCI) in the aging population. Increased amyloid plaque deposition within cerebral blood vessels, specifically the smooth muscle layer, is linked to increased cerebral microbleeds (CMBs) and impaired cognition in CAA. Studies in Alzheimer's disease (AD) have shown that amyloid plaque pathology is more prevalent in the brains of elderly women (2/3rd of the dementia population) compared with men, however, there is a paucity of studies on sex differences in CAA. The objective of this study was to discern the sexual dichotomies in CAA. We utilized male and female Tg-SwDI mice (mouse model of CAA) at 12-14 months of age for this study. We evaluated sex differences in CMBs, cognitive function and inflammation. Cognition was assessed using Y-maze (spatial working memory) and Fear Conditioning (contextual memory). CMBs were quantified by ex vivo brain MRI scans. Inflammatory cytokines in brain were quantified using ELISA. Our results demonstrated that aging Tg-SwDI female mice had a significantly higher burden of CMBs on MRI as compared to males. Interestingly, these aging Tg-SwDI female mice also had significantly impaired spatial and contextual memory on Y maze and Fear Conditioning respectively. Furthermore, female mice had significantly lower circulating inflammatory cytokines, IL-1α, IL-2, IL-9, and IFN-γ, as compared to males. Our results demonstrate that aging female Tg-SwDI mice are more cognitively impaired and have higher number of CMBs, as compared to males at 12-14 months of age. This may be secondary to reduced levels of neural repair cytokines (IL-1α, IL-2, IL-9 and IFN-γ) involved in sex specific inflammatory signaling in CAA.

Increased P450 aromatase levels in post-menopausal women after acute ischemic stroke.

BACKGROUND: Sex differences in stroke have been attributed to the neuroprotective effects of estrogen, yet most clinical trials of estrogen supplementation for stroke prevention have failed. The contribution of sex hormones to stroke outcome remains a subject of debate. Aromatization of testosterone to estradiol in neural tissue leads to sexual differentiation. Emerging data suggests aromatase activity increases in response to brain injury, and increased aromatase expression is seen in the ischemic penumbra in animal models. The objective of this study was to examine the levels of endogenous sex steroids after acute ischemic stroke and determine if levels of sex steroids were associated with acute stroke outcomes. METHODS: Peripheral blood from ischemic stroke patients and controls was collected under an approved IRB within 24 h of symptom onset. 17ß-estradiol, testosterone, and aromatase levels were measured in the serum of both men and women using ELISA. Hormone levels were compared in men vs. women in stroke and control groups and correlated with outcomes (NIHSS and change in the modified Rankin Scale (mRS), defined as the difference of premorbid and discharge mRS) using multivariate regression. RESULTS: We found no significant difference in estradiol levels 24 h after stroke in men (p = 0.86) or women (p = 0.10). In men, testosterone significantly decreased after stroke as compared with controls (1.83 ± 0.12 vs. 2.86 ± 0.65, p = 0.01). Aromatase levels were significantly increased in women after stroke as compared with controls (2.27 ± 0.22 vs. 0.97 ± 0.22, p = 0.002), but not in men (p = 0.84). Estradiol levels positively correlated with change in mRS in both women (r = 0.38, p = 0.02) and men (r = 0.3, p = 0.04). CONCLUSIONS: Estradiol levels correlated with functional outcomes (change in mRS) in both men and women, at least in the acute phase (24 h) of stroke. However, no significant difference in estradiol levels is seen 24 h post-stroke in men or women. Testosterone levels decrease at 24 h after stroke in men. As seen in animal models, aromatase levels increase after acute ischemic stroke, but this was only true for women. These indicate an active aromatization process in post-menopausal women after acute ischemic stroke.

Cerebral Amyloid Angiopathy, Alzheimer's Disease and MicroRNA: miRNA as Diagnostic Biomarkers and Potential Therapeutic Targets.

The protein molecules must fold into unique conformations to acquire functional activity. Misfolding, aggregation, and deposition of proteins in diverse organs, the so-called "protein misfolding disorders (PMDs)", represent the conformational diseases with highly ordered assemblies, including oligomers and fibrils that are linked to neurodegeneration in brain illnesses such as cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD). Recent studies have revealed several aspects of brain pathology in CAA and AD, but both the classification and underlying mechanisms need to be further refined. MicroRNAs (miRNAs) are critical regulators of gene expression at the post-transcriptional level. Increasing evidence with the advent of RNA sequencing technology suggests possible links between miRNAs and these neurodegenerative disorders. To provide insights on the small RNA-mediated regulatory circuitry and the translational significance of miRNAs in PMDs, this review will discuss the characteristics and mechanisms of the diseases and summarize circulating or tissue-resident miRNAs associated with AD and CAA.

Management of Acute Ischemic Stroke.

The treatment of acute ischemic stroke includes both intravenous (IV) thrombolysis and mechanical thrombectomy. Important advances regarding both treatment modalities have occurred recently that all physicians who see patients at risk for or who have had a stroke should be aware of. This review will focus on recent clinical trials of IV thrombolysis both positive and negative. Additionally, the results of a large number of early and late time window thrombectomy trials will be presented that demonstrate the remarkable efficacy of this treatment for appropriately selected patients.

Early Magnetic Resonance Imaging Decreases Hospital Length of Stay in Patients with Ischemic Stroke.

BACKGROUND: Imaging modalities are important part of stroke evaluation. Noncontrast head computed tomography (CT) is the initial imaging modality in acute stroke and although important to rule out acute hemorrhage and making a decision on thrombolytic treatment, ischemic changes may not be visible on CT for up to 24 hours. Magnetic resonance imaging (MRI) brain is an invaluable tool to confirm an ischemic stroke and facilitates stroke evaluation. Objective of this study was to investigate the correlation between time to MRI and length of hospital stay. METHODS: A total of 432 patients admitted to Hartford Hospital (Comprehensive Stroke Center) with a focal neurological deficit in the year 2014 and got a CT head and MRI brain were enrolled in the study. Data collection was done via stroke database and retrospective chart review. Patients with any hemorrhage or age <18 years were excluded from the study. Patients were categorized as having had an early (within 12 hours) or a late (more than 12 hours) MRI. We used chi-square and Wilcoxon ranked sum test to compare time from arrival to MRI and length of stay in the hospital. RESULTS: There was a statistically significant difference in hospital length of stay between patients who obtained MRI within 12 hours, as compared with patients who had MRI greater than 12 hours after admission, early MRI group 3 days (1.8, 4.9) versus 4 days (2.6, 7.0), P < .001. CONCLUSIONS: Our study suggests that brain MRI performed within 12 hours of admission facilitates stroke evaluation and decreases hospital length of stay. It provides evidence for cost effectiveness of MRI in ischemic stroke.

Sex differences in ischemic stroke sensitivity are influenced by gonadal hormones, not by sex chromosome complement.

Epidemiologic studies have shown sex differences in ischemic stroke. The four core genotype (FCG) mouse model, in which the testes determining gene, Sry, has been moved from Y chromosome to an autosome, was used to dissociate the effects of sex hormones from sex chromosome in ischemic stroke outcome. Middle cerebral artery occlusion (MCAO) in gonad intact FCG mice revealed that gonadal males (XXM and XYM) had significantly higher infarct volumes as compared with gonadal females (XXF and XYF). Serum testosterone levels were equivalent in adult XXM and XYM, as was serum estrogen in XXF and XYF mice. To remove the effects of gonadal hormones, gonadectomized FCG mice were subjected to MCAO. Gonadectomy significantly increased infarct volumes in females, while no change was seen in gonadectomized males, indicating that estrogen loss increases ischemic sensitivity. Estradiol supplementation in gonadectomized FCG mice rescued this phenotype. Interestingly, FCG male mice were less sensitive to effects of hormones. This may be due to enhanced expression of the transgene Sry in brains of FCG male mice. Sex differences in ischemic stroke sensitivity appear to be shaped by organizational and activational effects of sex hormones, rather than sex chromosomal complement.

Inhibition of mitogen-activated protein kinase phosphatase-1 (MKP-1) increases experimental stroke injury.

BACKGROUND AND PURPOSE: Activation of mitogen-activated protein kinases (MAPKs), particularly c-jun-N-terminal kinases (JNK) and p38 exacerbates stroke injury by provoking pro-apoptotic and pro-inflammatory cellular signaling. MAPK phosphatase-1 (MKP-1) restrains the over-activation of MAPKs via rapid de-phosphorylation of the MAPKs. We therefore examined the role of MKP-1 in stroke and studied its inhibitory effects on MAPKs after experimental stroke. METHODS: Male mice were subjected to transient middle cerebral artery occlusion (MCAO). MKP-1 knockout (KO) mice and a MKP-1 pharmacological inhibitor were utilized. We utilized flow cytometry, immunohistochemistry (IHC), and Western blots analysis to explore MKP-1 signaling and its effects on apoptosis/inflammation in the brain and specifically in microglia after stroke. RESULTS: MKP-1 was highly expressed in the nuclei of both neurons and microglia after stroke. MKP-1 genetic deletion exacerbated stroke outcome by increasing infarct, neurological deficits and hemorrhagic transformation. Additionally, delayed treatment of the MKP-1 pharmacological inhibitor worsened stroke outcome in wild type (WT) mice but had no effect in MKP-1 KO mice. Furthermore, MKP-1 deletion led to increased c-jun-N-terminal kinase (JNK) activation and microglial p38 activation after stroke. Finally, MKP-1 deletion or inhibition increased inflammatory and apoptotic response as evidenced by the increased levels of interleukin-6 (IL-6), tumor necrosis factor α (TNFα), ratio of p-c-jun/c-jun and cleaved caspase-3 following ischemia. CONCLUSIONS: We have demonstrated that MKP-1 signaling is an endogenous protective mechanism in stroke. Our data imply that MKP-1 possesses anti-apoptotic and anti-inflammatory properties by simultaneously controlling the activities of JNK and microglial p38.

Perfusion of ischemic brain in young and aged animals: a laser speckle flowmetry study.

BACKGROUND AND PURPOSE: Aging is an important determinant of ischemic stroke outcomes. Both clinical and experimental stroke studies have shown that aging negatively correlates with infarct volumes but is associated with worsened functional recovery after stroke. This may correspond to a differing cellular and molecular response to stroke in the aged versus young brain. It was hypothesized in this study that the smaller injury seen in the aged ischemic brain is because of structural differences in microvasculature with aging or differences in intraischemic tissue perfusion. METHODS: Both young and aged C57BL6 mice were subject to middle cerebral artery occlusion modeling. Laser speckle flowmetry was used to study the functional dynamics of cerebral perfusion, and fluorescein isothiocyanate (FITC)-dextran staining was performed to examine the structural change in microvasculature. In separate cohorts, cresyl violet staining and immunohistochemistry with CD31 and IgG antibodies were applied to further assess the microvascular density and blood-brain barrier breakdown after stroke. RESULTS: No difference in cerebral blood flow was seen at the baseline, intraischemically, and postreperfusion in young versus aged mice. FITC-dextran and CD31 staining did not show significant differences in the microvascular density between young and aged ischemic brains. More extravasation of IgG through the blood-brain barrier was found in the young versus aged cohort at both 24 and 72 hours after stroke. CONCLUSIONS: Cerebrovascular dynamics and perfusion are not responsible for the different stroke phenotypes seen in the young versus aged animals, which may be more related to different levels of blood-brain barrier breakdown.

Differential effects of aging and sex on stroke induced inflammation across the lifespan.

Aging and biological sex are critical determinants of stroke outcome. Post-ischemic inflammatory response strongly contributes to the extent of ischemic brain injury, but how this response changes with age and sex is unknown. We subjected young (5-6 months), middle aged (14-15 months) and aged (20-22 months), C57BL/6 male and female mice to transient middle cerebral artery occlusion (MCAO) and found that a significant age by sex interaction influenced histological stroke outcomes. Acute functional outcomes were worse with aging. Neutrophils, inflammatory macrophages, macrophages, dendritic cells (DCs) and microglia significantly increased in the brain post MCAO. Cycling females had higher Gr1(-) non-inflammatory macrophages and lower T cells in the brain after stroke and these correlated with serum estradiol levels. Estrogen loss in acyclic aged female mice exacerbated stroke induced splenic contraction. Advanced age increased T cells, DCs and microglia at the site of injury, which may be responsible for the exacerbated behavioral deficits in the aged. We conclude that aging and sex have differential effects on the post stroke inflammatory milieu. Putative immunomodulatory therapies need to account for this heterogeneity.

Function of the master energy regulator adenosine monophosphate-activated protein kinase in stroke.

Adenosine monophosphate-activated protein kinase (AMPK) is an evolutionarily conserved signaling molecule that is emerging as one of the most important energy sensors in the body. AMPK monitors cellular energy status and is activated via phosphorylation when energy stores are low. This allows for maintenance of energy homeostasis by promoting catabolic pathways for ATP production and limiting processes that consume ATP. Growing number of stimuli have been shown to activate AMPK, and AMPK has been implicated in many diverse biological processes, including cell polarity, autophagy, and senescence. The effect of AMPK activation and its biological functions are extremely diverse and depend on both the overall energy "milieu" and the location and duration of activation. AMPK has tissue- and isoform-specific functions in the brain vs. periphery. These functions and the pathways activated also appear to differ by cell location (hypothalamus vs. cortex), cell type (astrocyte vs. neuron), and duration of exposure. Short bursts of AMPK activation have been found to be involved in ischemic preconditioning and neuronal survival; however, prolonged AMPK activity during ischemia leads to neuronal cell death. AMPK may also underlie some of the beneficial effects of hypothermia, a potential therapy for ischemic brain injury. This review discusses the role of AMPK in ischemic stroke, a condition of severe energy depletion.

Protection from cerebral ischemia by inhibition of TGFß-activated kinase.

OBJECTIVE: Transforming growth factor-ß-activated kinase (TAK1) is a member of the mitogen-activated protein kinase family that plays important roles in apoptosis and inflammatory signaling, both of which are critical components of stroke pathology. TAK1 has recently been identified as a major upstream kinase that phosphorylates and activates adenosine monophosphate-activated protein kinase (AMPK), a major mediator of neuronal injury after experimental cerebral ischemia. We studied the functional role of TAK1 and its mechanistic link with AMPK after stroke. METHODS: Male mice were subjected to transient middle cerebral artery occlusion (MCAO). The TAK1 inhibitor 5Z-7-oxozeaenol was injected either intracerebroventricularly or intraperitoneally at various doses and infarct size and functional outcome after long term survival was assessed. Mice with deletion of the AMPK α2 isoform were utilized to assess the contribution of downstream AMPK signaling to stroke outcomes. Levels of pTAK1, pAMPK, and other TAK1 targets including the pro-apoptotic molecule c-Jun-N-terminal kinase (JNK)/c-Jun and the pro-inflammatory protein cyclooxygenase-2 were also examined. RESULTS: TAK1 is critical in stroke pathology. Delayed treatment with a TAK1 inhibitor reduced infarct size and improved behavioral outcome even when given several hours after stroke onset. This protective effect may be independent of AMPK activation but was associated with a reduction in JNK and c-Jun signaling. CONCLUSIONS: Enhanced TAK1 signaling, via activation of JNK, contributes to cell death in ischemic stroke. TAK1 inhibition is a novel therapeutic approach for stroke as it is neuroprotective with systemic administration, has a delayed therapeutic window, and demonstrates sustained neuroprotective effects.

Functional recovery in aging mice after experimental stroke.

Aging is a non-modifiable risk factor for stroke. Since not all strokes can be prevented, a major emerging area of research is the development of effective strategies to enhance functional recovery after stroke. However, in the vast majority of pre-clinical stroke studies, the behavioral tests used to assess functional recovery have only been validated for use in young animals, or are designed for rats. Mice are increasingly utilized in stroke models but well validated behavioral tests designed for rats are not necessarily reproducible in mice. We examined a battery of behavioral tests to evaluate functional recovery in an aging murine model of stroke. We found that the vertical pole, hanging wire and open field can accurately assess acute behavioral impairments after stroke in both young and aging male mice, but animals recover rapidly on these tasks. The corner test can accurately and repeatedly differentiate stroke from sham animals up to 30 days post stroke and can be performed reliably in aging mice. Aging male mice had significantly worse behavioral impairment compared to young male mice in the first two weeks after stroke but eventually recovered to the same degree as young mice. In contrast, chronic infarct size, as measured by ipsilateral cerebral atrophy, was significantly lower in aging male mice compared to young male mice. Reactive gliosis, formation of glial scar, and an enhanced innate immune response was seen in the aging brain and may contribute to the delayed behavioral recovery seen in the aging animals.

Sexual dimorphism in ischemic stroke: lessons from the laboratory.

Ischemic stroke is emerging as a major health problem for elderly women. Women have lower stroke incidence than men until an advanced age, when the epidemiology of ischemic stroke shifts and incidence rises dramatically in women. Experimental models of rodent stroke have replicated this clinical epidemiology, with exacerbated injury in older compared with young female rodents. Many of the detrimental effects of aging on ischemic stroke outcome in females can be replicated by ovariectomy, suggesting that hormones such as estrogen play a neuroprotective role. However, emerging data suggest that the molecular mechanisms leading to ischemic cell death differ in the two sexes, and these effects may be independent of circulating hormone levels. This article highlights recent clinical and experimental literature on sex differences in stroke outcomes and mechanisms.