Stroke in the Time of Circadian Medicine.

Time-of-day significantly influences the severity and incidence of stroke. Evidence has emerged not only for circadian governance over stroke risk factors, but also for important determinants of clinical outcome. In this review, we provide a comprehensive overview of the interplay between chronobiology and cerebrovascular disease. We discuss circadian regulation of pathophysiological mechanisms underlying stroke onset or tolerance as well as in vascular dementia. This includes cell death mechanisms, metabolism, mitochondrial function, and inflammation/immunity. Furthermore, we present clinical evidence supporting the link between disrupted circadian rhythms and increased susceptibility to stroke and dementia. We propose that circadian regulation of biochemical and physiological pathways in the brain increase susceptibility to damage after stroke in sleep and attenuate treatment effectiveness during the active phase. This review underscores the importance of considering circadian biology for understanding the pathology and treatment choice for stroke and vascular dementia and speculates that considering a patient's chronotype may be an important factor in developing precision treatment following stroke.

Posterior circulation ischaemic stroke diagnosis and management.

This narrative review provides an overview of the posterior circulation and the clinical features of common posterior circulation stroke (PCS) syndromes in the posterior arterial territories and how to distinguish them from mimics. We outline the hyperacute management of patients with suspected PCS with emphasis on how to identify those who are likely to benefit from intervention based on imaging findings. Finally, we review advances in treatment options, including developments in endovascular thrombectomy (EVT) and intravenous thrombolysis (IVT), and the principles of medical management and indications for neurosurgery. Observational and randomised clinical trial data have been equivocal regarding EVT in PCS, but more recent studies strongly support its efficacy. There have been concomitant advances in imaging of posterior stroke to guide optimal patient selection for thrombectomy. Recent evidence suggests that clinicians should have a heightened suspicion of posterior circulation events with the resultant implementation of timely, evidence-based management.

Placental capillary pericytes release excess extracellular vesicles under hypoxic conditions inducing a pro-angiogenic profile in term pregnancy.

Pericytes are multifunctional cells wrapped around capillary endothelia, essential for vascular health, development, and blood flow regulation, although their role in human placental chorionic villi has not been fully explored. The second half of normal pregnancy is characterized by a progressive decline in placental and fetal oxygen levels which, by term, comprises a substantial degree of hypoxia. We hypothesized this hypoxia would stimulate pericyte regulation of chorionic villous capillary function. This study's objective was to investigate the role of hypoxia on normal term placental pericytes (PLVP) and their signaling to endothelial cells. First, we confirmed fetoplacental hypoxia at term by a new analysis of umbilical arterial blood oxygen tension of 3,010 healthy singleton neonates sampled at caesarean section and before labor. We then measured the release of cytokines, chemokines, and small extracellular vesicles (PLVPsv), from PLVP cultured at 20%, 8% and 1% O2. As O2 levels decreased, secreted cytokines and chemokines [interleukin-6 (IL-6), interleukin-1α (IL-1α) and vascular endothelial growth factor (VEGF)], and small extracellular vesicle markers, (Alix, Syntenin and CD9) increased significantly in the culture supernatants. When primary human umbilical vein endothelial cells (HUVEC) were cultured with PLVPsv, polygon formation, number, and tube formation length was significantly increased compared to cells not treated with PLVPsv, indicating PLVPsv stimulated angiogenesis. We conclude that adding PLVPsv stimulates angiogenesis and vessel stabilization on neighboring endothelial cells in response to hypoxia in term pregnancy compared to no addition of PLVPsv. Our finding that PLVP can release angiogenic molecules via extracellular vesicles in response to hypoxia may apply to other organ systems.

Automated quantification of atrophy and acute ischemic volume for outcome prediction in endovascular thrombectomy.

Background: Short- and long-term outcomes from endovascular thrombectomy (EVT) for large vessel occlusion stroke remain variable. Numerous relevant predictors have been identified, including severity of neurological deficits, age, and imaging features. The latter is typically defined as acute changes (most commonly Alberta Stroke Programme Early CT Score, ASPECTS, at presentation), but there is little information on the impact of imaging assessment of premorbid brain health as a determinant of outcome. Aims: To examine the impact of automated measures of stroke severity and underlying brain frailty on short- and long-term outcomes in acute stroke treated with EVT. Methods: In 215 patients with anterior circulation stroke, who subsequently underwent EVT, automated analysis of presenting non-contrast CT scans was used to determine acute ischemic volume (AIV) and e-ASPECTS as markers of stroke severity, and cerebral atrophy as a marker of brain frailty. Univariate and multivariate logistic regression were used to identify significant predictors of NIHSS improvement, modified Rankin scale (mRS) at 90 and 30 days, mortality at 90 days and symptomatic intracranial hemorrhage (sICH) following successful EVT. Results: For long-term outcome, atrophy and presenting NIHSS were significant predictors of mRS 0-2 and death at 90 days, whereas age did not reach significance in multivariate analysis. Conversely, for short-term NIHSS improvement, AIV and age were significant predictors, unlike presenting NIHSS. The interaction between age and NIHSS was similar to the interaction of AIV and atrophy for mRS 0-2 at 90 days. Conclusion: Combinations of automated software-based imaging analysis and clinical data can be useful for predicting short-term neurological outcome and may improve long-term prognostication in EVT. These results provide a basis for future development of predictive tools built into decision-aiding software in stroke.

Growth Differentiation Factor-11 Causes Neurotoxicity During Ischemia in vitro.

Age-related neuronal dysfunction can be overcome by circulating factors present in young blood. Growth differentiation factor-11 (GDF-11), a systemic factor that declines with age, can reverse age-related dysfunction in brain, heart and skeletal muscle. Given that age increases susceptibility to stroke, we hypothesized that GDF-11 may be directly protective to neurons following ischemia. Primary cortical neurons were isolated from E18 Wistar rat embryos and cultured for 7-10 days. Neurons were deprived of oxygen and glucose (OGD) to simulate ischemia. Neuronal death was assessed by lactate dehydrogenase, propidium iodide or CellTox™ green cytotoxicity assays. 40 ng/mL GDF-11 administration during 2 h OGD significantly increased neuronal death following 24 h recovery. However, GDF-11 pre-treatment did not affect neuronal death during 2 h OGD. GDF-11 treatment during the 24 h recovery period after 2 h OGD also did not alter death. Real-time monitoring for 24 h revealed that by 2 h OGD, GDF-11 treatment had increased neuronal death which remained raised at 24 h. Co-treatment of 1 µM SB431542 (ALK4/5/7 receptor inhibitor) with GDF-11 prevented GDF-11 neurotoxicity after 2 h OGD and 24 h OGD. Transforming growth factor beta (TGFß) did not increase neuronal death to the same extent as GDF-11 following OGD. GDF-11 neurotoxicity was also exhibited following neuronal exposure to hydrogen peroxide. These results reveal for the first time that GDF-11 is neurotoxic to primary neurons in the acute phase of simulated stroke through primarily ALK4 receptor signaling.

Rapamycin in ischemic stroke: Old drug, new tricks?

The significant morbidity that accompanies stroke makes it one of the world's most devastating neurological disorders. Currently, proven effective therapies have been limited to thrombolysis and thrombectomy. The window for the administration of these therapies is narrow, hampered by the necessity of rapidly imaging patients. A therapy that could extend this window by protecting neurons may improve outcome. Endogenous neuroprotection has been shown to be, in part, due to changes in mTOR signalling pathways and the instigation of productive autophagy. Inducing this effect pharmacologically could improve clinical outcomes. One such therapy already in use in transplant medicine is the mTOR inhibitor rapamycin. Recent evidence suggests that rapamycin is neuroprotective, not only via neuronal autophagy but also through its broader effects on other cells of the neurovascular unit. This review highlights the potential use of rapamycin as a multimodal therapy, acting on the blood-brain barrier, cerebral blood flow and inflammation, as well as directly on neurons. There is significant potential in applying this old drug in new ways to improve functional outcomes for patients after stroke.

The role of the endoplasmic reticulum stress response following cerebral ischemia.

Background Cornu ammonis 3 (CA3) hippocampal neurons are resistant to global ischemia, whereas cornu ammonis (CA1) 1 neurons are vulnerable. Hamartin expression in CA3 neurons mediates this endogenous resistance via productive autophagy. Neurons lacking hamartin demonstrate exacerbated endoplasmic reticulum stress and increased cell death. We investigated endoplasmic reticulum stress responses in CA1 and CA3 regions following global cerebral ischemia, and whether pharmacological modulation of endoplasmic reticulum stress or autophagy altered neuronal viability . Methods In vivo: male Wistar rats underwent sham or 10 min of transient global cerebral ischemia. CA1 and CA3 areas were microdissected and endoplasmic reticulum stress protein expression quantified at 3 h and 12 h of reperfusion. In vitro: primary neuronal cultures (E18 Wistar rat embryos) were exposed to 2 h of oxygen and glucose deprivation or normoxia in the presence of an endoplasmic reticulum stress inducer (thapsigargin or tunicamycin), an endoplasmic reticulum stress inhibitor (salubrinal or 4-phenylbutyric acid), an autophagy inducer ([4'-(N-diethylamino) butyl]-2-chlorophenoxazine (10-NCP)) or autophagy inhibitor (3-methyladenine). Results In vivo, decreased endoplasmic reticulum stress protein expression (phospho-eIF2α and ATF4) was observed at 3 h of reperfusion in CA3 neurons following ischemia, and increased in CA1 neurons at 12 h of reperfusion. In vitro, endoplasmic reticulum stress inducers and high doses of the endoplasmic reticulum stress inhibitors also increased cell death. Both induction and inhibition of autophagy also increased cell death. Conclusion Endoplasmic reticulum stress is associated with neuronal cell death following ischemia. Neither reduction of endoplasmic reticulum stress nor induction of autophagy demonstrated neuroprotection in vitro, highlighting their complex role in neuronal biology following ischemia.

Neuroprotection in stroke: the importance of collaboration and reproducibility.

Acute ischaemic stroke accounts for 6.5 million deaths per year, and by 2030 will result in the annual loss of over 200 million disability-adjusted life years globally. There have been considerable recent advances in the gold standard of acute ischaemic stroke treatment, some aspects of which-aspirin to prevent recurrence, and treating patients in specialized stroke wards-are widely applicable. Recanalization of the occluded artery through thrombolysis and/or endovascular thrombectomy is restricted to only a small proportion of patients, due to contra-indications and the costs associated with establishing the infrastructure to deliver these treatments. The use of neuroprotective agents in stroke has been a notable failure of translation from medical research into clinical practice. Yet, with the advent of endovascular thrombectomy and the ability to investigate patients in much greater detail through advanced imaging modalities, neuroprotective agents can and should be re-examined as adjunct therapies to recanalization. In parallel, this requires appropriate planning on behalf of the preclinical stroke research community: there is a need to reinvestigate these therapies in a more collaborative manner, to enhance reproducibility through reduced attrition, improved reporting, and adopting an approach to target validation that more closely mimics clinical trials. This review will describe some of the novel strategies being used in stroke research, and focus on a few key examples of neuroprotective agents that are showing newfound promise in preclinical models of stroke therapy. Our primary aim is to give an overview of some of the challenges faced by preclinical stroke research, and suggest potential ways to improve translational success.

Inflammatory Stroke Extracellular Vesicles Induce Macrophage Activation.

BACKGROUND AND PURPOSE: Extracellular vesicles (EVs) are protein-lipid complexes released from cells, as well as actively exocytosed, as part of normal physiology, but also during pathological processes such as those occurring during a stroke. Our aim was to determine the inflammatory potential of stroke EVs. METHODS: EVs were quantified and analyzed in the sera of patients after an acute stroke (<24 hours; OXVASC [Oxford Vascular Study]). Isolated EV fractions were subjected to untargeted proteomic analysis by liquid chromatography mass-spectrometry/mass-spectrometry and then applied to macrophages in culture to investigate inflammatory gene expression. RESULTS: EV number, but not size, is significantly increased in stroke patients when compared to age-matched controls. Proteomic analysis reveals an overall increase in acute phase proteins, including C-reactive protein. EV fractions applied to monocyte-differentiated macrophage cultures induced inflammatory gene expression. CONCLUSIONS: Together these data show that EVs from stroke patients are proinflammatory in nature and are capable of inducing inflammation in immune cells.

Novel method to study pericyte contractility and responses to ischaemia in vitro using electrical impedance.

Pericytes are contractile vascular mural cells overlying capillary endothelium, and they have been implicated in a variety of functions including regulation of cerebral blood flow. Recent work has suggested that both in vivo and ex vivo, ischaemia causes pericytes to constrict and die, which has implications for microvascular reperfusion. Assessing pericyte contractility in tissue slices and in vivo is technically challenging, while in vitro techniques remain unreliable. Here, we used isolated cultures of human brain vascular pericytes to examine their contractile potential in vitro using the iCelligence electrical impedance system. Contraction was induced using the vasoactive peptide endothelin-1, and relaxation was demonstrated using adenosine and sodium nitroprusside. Endothelin-1 treatment also resulted in increased proliferation, which we were able to monitor in the same cell population from which we recorded contractile responses. Finally, the observation of pericyte contraction in stroke was reproduced using chemical ischaemia, which caused a profound and irreversible contraction clearly preceding cell death. These data demonstrate that isolated pericytes retain a contractile phenotype in vitro, and that it is possible to quantify this contraction using real-time electrical impedance recordings, providing a significant new platform for assessing the effects of vasoactive and vasculoprotective compounds on pericyte contractility.

Multi-modal assessment of neurovascular coupling during cerebral ischaemia and reperfusion using remote middle cerebral artery occlusion.

Hyperacute changes in cerebral blood flow during cerebral ischaemia and reperfusion are important determinants of injury. Cerebral blood flow is regulated by neurovascular coupling, and disruption of neurovascular coupling contributes to brain plasticity and repair problems. However, it is unknown how neurovascular coupling is affected hyperacutely during cerebral ischaemia and reperfusion. We have developed a remote middle cerebral artery occlusion model in the rat, which enables multi-modal assessment of neurovascular coupling immediately prior to, during and immediately following reperfusion. Male Wistar rats were subjected to remote middle cerebral artery occlusion, where a long filament was advanced intraluminally through a guide cannula in the common carotid artery. Transcallosal stimulation evoked increases in blood flow, tissue oxygenation and neuronal activity, which were diminished by middle cerebral artery occlusion and partially restored during reperfusion. These evoked responses were not affected by administration of the thrombolytic alteplase at clinically used doses. Evoked cerebral blood flow responses were fully restored at 24 h post-middle cerebral artery occlusion indicating that neurovascular dysfunction was not sustained. These data show for the first time that the rat remote middle cerebral artery occlusion model coupled with transcallosal stimulation provides a novel method for continuous assessment of hyperacute neurovascular coupling changes during ischaemia and reperfusion, and offers unique insight into hyperacute ischaemic pathophysiology.

The transient intraluminal filament middle cerebral artery occlusion model as a model of endovascular thrombectomy in stroke.

The clinical relevance of the transient intraluminal filament model of middle cerebral artery occlusion (tMCAO) has been questioned due to distinct cerebral blood flow profiles upon reperfusion between tMCAO (abrupt reperfusion) and alteplase treatment (gradual reperfusion), resulting in differing pathophysiologies. Positive results from recent endovascular thrombectomy trials, where the occluding clot is mechanically removed, could revolutionize stroke treatment. The rapid cerebral blood flow restoration in both tMCAO and endovascular thrombectomy provides clinical relevance for this pre-clinical model. Any future clinical trials of neuroprotective agents as adjuncts to endovascular thrombectomy should consider tMCAO as the model of choice to determine pre-clinical efficacy.

A systematic review and meta-analysis of randomized controlled trials of endovascular thrombectomy compared with best medical treatment for acute ischemic stroke.

BACKGROUND: Acute ischemic strokes involving occlusion of large vessels usually recanalize poorly following treatment with intravenous thrombolysis. Recent studies have shown higher recanalization and higher good outcome rates with endovascular therapy compared with best medical management alone. A systematic review and meta-analysis investigating the benefits of all randomized controlled trials of endovascular thrombectomy where at least 25% of patients were treated with a thrombectomy device for the treatment of acute ischemic stroke compared with best medical treatment have yet to be performed. AIM: To perform a systematic review and a meta-analysis evaluating the effectiveness of endovascular thrombectomy compared with best medical care for treatment of acute ischemic stroke. SUMMARY OF REVIEW: Our search identified 437 publications, from which eight studies (totaling 2423 patients) matched the inclusion criteria. Overall, endovascular thrombectomy was associated with improved functional outcomes (modified Rankin Scale 0-2) [odds ratio 1·56 (1·32-1·85), P < 0·00001]. There was a tendency toward decreased mortality [odds ratio 0·84 (0·67-1·05), P = 0·12], and symptomatic intracerebral hemorrhage was not increased [odds ratio 1·03 (0·71-1·49), P = 0·88] compared with best medical management alone. The odds ratio for a favorable functional outcome increased to 2·23 (1·77-2·81, P < 0·00001) when newer generation thrombectomy devices were used in greater than 50% of the cases in each trial. CONCLUSIONS: There is clear evidence for improvement in functional independence with endovascular thrombectomy compared with standard medical care, suggesting that endovascular thrombectomy should be considered the standard effective treatment alongside thombolysis in eligible patients.

Importance of preclinical research in the development of neuroprotective strategies for ischemic stroke.

IMPORTANCE: Preclinical stroke research has had a remarkably low translational success rate, and the clinical need for novel neuroprotective therapeutics has gone largely unmet, especially in light of the severe underuse of thrombolysis in acute ischemic stroke. OBJECTIVE: In this review, we aim to provide a brief overview of the commonly used stroke models, their merits and shortcomings, and how these have contributed to translational failures. We review some recent developments in preclinical stroke, providing examples of how improved study quality and the use of novel methods can facilitate translation into the clinical setting. EVIDENCE REVIEW: This is a narrative review of ischemic stroke neuroprotection based on electronic database searches, references of previous publications, and personal libraries. FINDINGS: The stroke research community has not been complacent in its response to criticism: preclinical stroke studies now demonstrate considerable rigor, standardization, and emphasis on minimization of experimenter bias. In addition, numerous innovative methods and strategies are providing novel avenues for investigating neuroprotection, as well as more extensive characterization of established models. CONCLUSIONS AND RELEVANCE: The improvements in preclinical stroke models and methods will make stroke research a good example for preclinical medicine, in general, and will hopefully instill greater confidence in the clinical community regarding which compounds are worthy of further investigation in a clinical setting.