Bharti Manwani

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.

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.

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.

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 TGFβ-activated kinase

OBJECTIVEnTransforming growth factor-β-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.nnnMETHODSnMale 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.nnnRESULTSnTAK1 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.nnnCONCLUSIONSnEnhanced 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.

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

BACKGROUND AND PURPOSEnActivation 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.nnnMETHODSnMale 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.nnnRESULTSnMKP-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.nnnCONCLUSIONSnWe 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.

Estrogen in ischaemic stroke: the debate continues

Sex differences abound in the epidemiological literature on ischemic stroke. Females enjoy a lower stroke incidence throughout most of the lifespan; however this benefit dissipates with aging [1]. Women over 80 have higher mortality and poorer functional outcomes compared to age-matched men after stroke. Over the next twenty years this disparity is expected to increase, and women will bear the brunt of stroke related death and disability. Much of the “female-protected” phenotype has been attributed to life-long exposure to estrogen, and the rise in stroke incidence occurs concomitantly with loss of ovarian function. Since a low estrogenic state (menopause) is associated with this transition into the higher stroke risk state, the link between estrogen and stroke has been an area of interest for many stroke investigators (reviewed recently in [2]). It is now well recognized that estradiol plays a vital role in the ischemic brain. Animal models of experimental stroke have shown that estrogen (17-β estradiol) is a robust neuroprotective agent in both males and females [3]. n nDespite the strong experimental evidence of estrogens beneficial effects in pre-clinical stroke models, estrogen supplementation in clinical trials involving post-menopausal women have not been successful. In the Womens Health Initiative (WHI), administration of conjugated equine estrogen (0.625 mg/day) to post menopausal women without a history of stroke increased stroke incidence [4]. In the Womens Estrogen For Stroke Trial (WEST) (1mg/day) of 17 beta-estradiol surprisingly increased mortality and morbidity in post-menopausal women with a recent TIA or non-disabling stroke [5]. Although as noted by many, numerous issues make these trials somewhat difficult to interpret such as the initiation of therapy well after menopause, use in patients with established vascular disease, and administration of supraphysiological doses of estrogen, this has led to a reassessment of the efficacy of exogenous estrogen for the prevention of stroke in post-menopausal women. However, much less is known regarding estrogen effects in the acute injury period. In this issue of The European Journal of Neurology, Pappa and collegues evaluated serum levels of endogenous estradiol in postmenopausal women with acute ischemic or hemorrhagic stroke [6]. n nIn this study, serum levels of estradiol were evaluated in 302 postmenopausal women (age 73.6+/-12.9 years; approximately 26.7+/-10.6 years post menopause) admitted with acute ischemic stroke. Patients on steroid or hormone replacement therapy or women with transient ischemic attack or subarachnoid hemorrhage were excluded. This is a novel approach as these women were evaluated in the acute period, in which data is sorely lacking. Historically, most clinical studies have focused on the effects of chronic exogenous administration of estrogen on stroke incidence. The main question addressed was whether the levels of endogenous estradiol in elderly women after stroke were associated with changes in stroke severity or outcome. The authors utilized the National Insititute of Health Stroke Scale (NIHSS) and modified Rankin Scale (mRS) at both admission and one month post stroke. Estradiol levels, measured acutely post stroke, were significantly higher in non-survivors as compared to survivors one month after stroke. They also found that endogenous estradiol levels were an independent predictor of stroke severity as quantified by NIHSS at the time of admission. In a multivariate analysis, estradiol levels continued to be a significant independent predictor of adverse functional outcomes and handicap (mRS≥4) one-month post stroke even after removing confounders such as age and initial NIHSS. As hemorrhagic and ischemic stroke pathophysiology may differ, the authors also removed the 13% of patients with hemorrhage, and repeated the analysis (261 patients) and found similar results. Importantly, as testosterone can be peripherally converted to estrogen via the actions of aromatase in adipose tissue, the authors also measured BMIs and found no association between BMI and outcomes. These findings are consistent with recent reports showing increased mortality [7] and stroke risk (also associated with greater central adiposity) [8] in post menopausal women with higher estrogen levels. n nThe findings of this study suggest that high levels of endogenous estradiol are deleterious in acute stroke. The authors suggest that the concept of estrogen as a neuroprotective agent “should be challenged”. However, these results should be interpreted with caution, and certainly these results do not definitively answer the question of whether acute estrogen is detrimental or beneficial, as well noted by the authors. There is a clear association between estradiol levels and stroke outcomes, but this does not imply causation. The poorer outcomes seen in women with the highest levels estradiol levels may not be a direct biological effect but simply reflective of larger lesion sizes (infarct volumes) which were not assessed in this study. Indeed, raising endogenous estradiol levels may even reflect an endogenous protective mechanism, as has been seen in animal models in which up-regulation of aromatase occurs in the infarct area, and infarct damage worsens with loss or inhibition of aromatase in the acutely injured brain [9]. Another important limitation of this paper is that the authors do not mention the exact time points at which blood samples were collected from the patients, although the authors previous studies (using the same cohort) state that these were collected in the morning 2-3 days after onset [10]. However, biomarkers are known to vary widely, and can be especially sensitive to stroke acuity, post-stroke infection rates, medication use etc. many which were not controlled for in this initial study. Additionally, levels of other endogenous estrogens (i.e., estrone) were not assessed. Future studies examining serial samples and the utilization of an age-matched control group (for example, post menopausal women admitted to the hospital for reasons other than stroke) may provide some clarity on levels of estradiol in stress and sickness. Overall, these results although intriguing, and a large step forward, yet again demonstrate the many uncertainties related to the role of estrogen in postmenopausal women with ischemic stroke.

Stroke as the Initial Manifestation of the Human Immunodeficiency Virus

A 51-year-old man presented to the emergency department with acute onset of left-sided sensory loss and hemiparesis leading to a fall. The hemisensory loss had resolved at the time of presentation. He denied associated dysarthria, ataxia, visual field deficit, or aphasia. Furthermore, he had no current or history of headaches or seizures. The patient’s past medical history was significant for hypertension, anxiety, posttraumatic stress disorder and unilateral hearing loss after an explosion. Home medications included quetiapine, desvenlafaxine, amlodipine, benazepril/hydrochlorothiazide, and nebivolol, for which he was compliant. He did not smoke, drink alcohol, or use recreational drugs and was independent in activities of daily living. He denied unprotected sexual intercourse, intravenous drug abuse, and blood transfusions.nnHe presented 1 hour 29 minutes after last seen normal with a blood pressure of 163/97 mm Hg and was in normal sinus rhythm. Neurological examination revealed left hemiparesis with motor strength of 0/5 in left upper and 4/5 in left lower extremity; sensory deficits and cortical findings were absent. National Institutes of Health Stroke Scale score was 5. Given the acuity of focal symptoms, stroke was the leading diagnosis. An emergent head computed tomography did not show any acute intracranial pathology. Intravenous tissue-type plasminogen activator was administered and the patient subsequently admitted to the neurointensive care unit. Magnetic resonance imaging brain demonstrated acute right frontoparietal infarcts, with a distribution suggestive of a watershed infarct between the anterior and middle cerebral artery territories, and a second acute infarct in the left pons (Figure [A]).nnnnFigure. nNeuroimaging confirms multifocal acute infarctions and intracranial vasculopathy. A , Magnetic resonance imaging brain demonstrates acute infarcts in right frontoparietal lobe with distribution suggestive of a watershed infarct involving the right middle and anterior cerebral arteries; and left pontine paramedian perforator infarct …