Female mice display sex-specific differences in cerebrovascular function and subarachnoid haemorrhage-induced injury.

BACKGROUND: In male mice, a circadian rhythm in myogenic reactivity influences the extent of brain injury following subarachnoid haemorrhage (SAH). We hypothesized that female mice have a different cerebrovascular phenotype and consequently, a distinct SAH-induced injury phenotype. METHODS: SAH was modelled by pre-chiasmatic blood injection. Olfactory cerebral resistance arteries were functionally assessed by pressure myography; these functional assessments were related to brain histology and neurobehavioral assessments. Cystic fibrosis transmembrane conductance regulator (CFTR) expression was assessed by PCR and Western blot. We compared non-ovariectomized and ovariectomized mice. FINDINGS: Cerebrovascular myogenic reactivity is not rhythmic in females and no diurnal differences in SAH-induced injury are observed; ovariectomy does not unmask a rhythmic phenotype for any endpoint. CFTR expression is rhythmic, with similar expression levels compared to male mice. CFTR inhibition studies, however, indicate that CFTR activity is lower in female arteries. Pharmacologically increasing CFTR expression in vivo (3 mg/kg lumacaftor for 2 days) reduces myogenic tone at Zeitgeber time 11, but not Zeitgeber time 23. Myogenic tone is not markedly augmented following SAH in female mice and lumacaftor loses its ability to reduce myogenic tone; nevertheless, lumacaftor confers at least some injury benefit in females with SAH. INTERPRETATION: Female mice possess a distinct cerebrovascular phenotype compared to males, putatively due to functional differences in CFTR regulation. This sex difference eliminates the CFTR-dependent cerebrovascular effects of SAH and may alter the therapeutic efficacy of lumacaftor compared to males. FUNDING: Brain Aneurysm Foundation, Heart and Stroke Foundation and Ted Rogers Centre for Heart Research.

Effect of mobile application types on stroke rehabilitation: a systematic review.

BACKGROUND: Stroke is a significant contributor of worldwide disability and morbidity with substantial economic consequences. Rehabilitation is a vital component of stroke recovery, but inpatient stroke rehabilitation programs can struggle to meet the recommended hours of therapy per day outlined by the Canadian Stroke Best Practices and American Heart Association. Mobile applications (apps) are an emerging technology which may help bridge this deficit, however this area is understudied. The purpose of this study is to review the effect of mobile apps for stroke rehabilitation on stroke impairments and functional outcomes. Specifically, this paper will delve into the impact of varying mobile app types on stroke rehabilitation. METHODS: This systematic review included 29 studies: 11 randomized control trials and 18 quasi-experimental studies. Data extrapolation mapped 5 mobile app types (therapy apps, education apps, rehab videos, reminders, and a combination of rehab videos with reminders) to stroke deficits (motor paresis, aphasia, neglect), adherence to exercise, activities of daily living (ADLs), quality of life, secondary stroke prevention, and depression and anxiety. RESULTS: There were multiple studies supporting the use of therapy apps for motor paresis or aphasia, rehab videos for exercise adherence, and reminders for exercise adherence. For permutations involving other app types with stroke deficits or functional outcomes (adherence to exercise, ADLs, quality of life, secondary stroke prevention, depression and anxiety), the results were either non-significant or limited by a paucity of studies. CONCLUSION: Mobile apps demonstrate potential to assist with stroke recovery and augment face to face rehabilitation, however, development of a mobile app should be carefully planned when targeting specific stroke deficits or functional outcomes. This study found that mobile app types which mimicked principles of effective face-to-face therapy (massed practice, task-specific practice, goal-oriented practice, multisensory stimulation, rhythmic cueing, feedback, social interaction, and constraint-induced therapy) and education (interactivity, feedback, repetition, practice exercises, social learning) had the greatest benefits. Protocol registration PROPSERO (ID CRD42021186534). Registered 21 February 2021.

Circadian Rhythmicity in Cerebral Microvascular Tone Influences Subarachnoid Hemorrhage-Induced Injury.

BACKGROUND AND PURPOSE: Circadian rhythms influence the extent of brain injury following subarachnoid hemorrhage (SAH), but the mechanism is unknown. We hypothesized that cerebrovascular myogenic reactivity is rhythmic and explains the circadian variation in SAH-induced injury. METHODS: SAH was modeled in mice with prechiasmatic blood injection. Inducible, smooth muscle cell-specific Bmal1 (brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1) gene deletion (smooth muscle-specific Bmal1 1 knockout [sm-Bmal1 KO]) disrupted circadian rhythms within the cerebral microcirculation. Olfactory cerebral resistance arteries were functionally assessed by pressure myography in vitro; these functional assessments were related to polymerase chain reaction/Western blot data, brain histology (Fluoro-Jade/activated caspase-3), and neurobehavioral assessments (modified Garcia scores). RESULTS: Cerebrovascular myogenic vasoconstriction is rhythmic, with a peak and trough at Zeitgeber times 23 and 11 (ZT23 and ZT11), respectively. Histological and neurobehavioral assessments demonstrate that higher injury levels occur when SAH is induced at ZT23, compared with ZT11. In sm-Bmal1 KO mice, myogenic reactivity is not rhythmic. Interestingly, myogenic tone is higher at ZT11 versus ZT23 in sm-Bmal1 KO mice; accordingly, SAH-induced injury in sm-Bmal1 KO mice is more severe when SAH is induced at ZT11 compared to ZT23. We examined several myogenic signaling components and found that CFTR (cystic fibrosis transmembrane conductance regulator) expression is rhythmic in cerebral arteries. Pharmacologically stabilizing CFTR expression in vivo (3 mg/kg lumacaftor for 2 days) eliminates the rhythmicity in myogenic reactivity and abolishes the circadian variation in SAH-induced neurological injury. CONCLUSIONS: Cerebrovascular myogenic reactivity is rhythmic. The level of myogenic tone at the time of SAH ictus is a key factor influencing the extent of injury. Circadian oscillations in cerebrovascular CFTR expression appear to underlie the cerebrovascular myogenic reactivity rhythm.

Cerebral Autoregulation in Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) is a devastating stroke subtype with a high rate of mortality and morbidity. The poor clinical outcome can be attributed to the biphasic course of the disease: even if the patient survives the initial bleeding emergency, delayed cerebral ischemia (DCI) frequently follows within 2 weeks time and levies additional serious brain injury. Current therapeutic interventions do not specifically target the microvascular dysfunction underlying the ischemic event and as a consequence, provide only modest improvement in clinical outcome. SAH perturbs an extensive number of microvascular processes, including the "automated" control of cerebral perfusion, termed "cerebral autoregulation." Recent evidence suggests that disrupted cerebral autoregulation is an important aspect of SAH-induced brain injury. This review presents the key clinical aspects of cerebral autoregulation and its disruption in SAH: it provides a mechanistic overview of cerebral autoregulation, describes current clinical methods for measuring autoregulation in SAH patients and reviews current and emerging therapeutic options for SAH patients. Recent advancements should fuel optimism that microvascular dysfunction and cerebral autoregulation can be rectified in SAH patients.

Role of perivascular and meningeal macrophages in outcome following experimental subarachnoid hemorrhage.

The distribution and clearance of erythrocytes after subarachnoid hemorrhage (SAH) is poorly understood. We aimed to characterize the distribution of erythrocytes after SAH and the cells involved in their clearance. To visualize erythrocyte distribution, we injected fluorescently-labelled erythrocytes into the prechiasmatic cistern of mice. 10 minutes after injection, we found labelled erythrocytes in the subarachnoid space and ventricular system, and also in the perivascular spaces surrounding large penetrating arterioles. 2 and 5 days after SAH, fluorescence was confined within leptomeningeal and perivascular cells. We identified the perivascular cells as perivascular macrophages based on their morphology, location, Iba-1 immunoreactivity and preferential uptake of FITC-dextran. We subsequently depleted meningeal and perivascular macrophages 2 days before or 3 hours after SAH with clodronate liposomes. At day 5 after SAH, we found increased blood deposition in mice treated prior to SAH, but not those treated after. Treatment post-SAH improved neurological scoring, reduced neuronal cell death and perivascular inflammation, whereas pre-treatment only reduced perivascular inflammation. Our data indicate that after SAH, erythrocytes are distributed throughout the subarachnoid space extending into the perivascular spaces of parenchymal arterioles. Furthermore, meningeal and perivascular macrophages are involved in erythrocyte uptake and play an important role in outcome after SAH.

CFTR Therapeutics Normalize Cerebral Perfusion Deficits in Mouse Models of Heart Failure and Subarachnoid Hemorrhage.

Heart failure (HF) and subarachnoid hemorrhage (SAH) chronically reduce cerebral perfusion, which negatively affects clinical outcome. This work demonstrates a strong relationship between cerebral artery cystic fibrosis transmembrane conductance regulator (CFTR) expression and altered cerebrovascular reactivity in HF and SAH. In HF and SAH, CFTR corrector compounds (C18 or lumacaftor) normalize pathological alterations in cerebral artery CFTR expression, vascular reactivity, and cerebral perfusion, without affecting systemic hemodynamic parameters. This normalization correlates with reduced neuronal injury. Therefore, CFTR therapeutics have emerged as valuable clinical tools to manage cerebrovascular dysfunction, impaired cerebral perfusion, and neuronal injury.

Robust effects of genetic background on responses to subarachnoid hemorrhage in mice.

Outcome varies among patients with subarachnoid hemorrhage but known prognostic factors explain only a small portion of the variation in outcome. We hypothesized that individual genetic variations influence brain and vascular responses to subarachnoid hemorrhage and investigated this using inbred strains of mice.Subarachnoid hemorrhage was induced in seven inbred and a chromosome 7 substitution strain of mouse. Cerebral blood flow, vasospasm of the middle cerebral artery, and brain injury were assessed. After 48 h of subarachnoid hemorrhage, mice showed significant middle cerebral artery vasospasm that correlated positively with reduction in cerebral blood flow at 45 min. Mice also had increased neuronal injury compared to sham controls; A/J and C57BL/6 J strains represented the most and least severe, respectively. However, brain injury did not correlate with cerebral blood flow reduction at 45 min or with vasospasm at 48 h. Chromosome 7 substitution did not influence the degree of vasospasm or brain injury.Our data suggested that mouse genetic background influences outcome of subarachnoid hemorrhage. Investigations into the genetic factors causing these inter-strain differences may provide insight into the etiology of the brain damage following subarachnoid hemorrhage. These findings also have implications for animal modeling of disease and suggest that genetic differences may also modulate outcome in other cardiovascular diseases.

Therapeutically Targeting Tumor Necrosis Factor-α/Sphingosine-1-Phosphate Signaling Corrects Myogenic Reactivity in Subarachnoid Hemorrhage.

BACKGROUND AND PURPOSE: Subarachnoid hemorrhage (SAH) is a complex stroke subtype characterized by an initial brain injury, followed by delayed cerebrovascular constriction and ischemia. Current therapeutic strategies nonselectively curtail exacerbated cerebrovascular constriction, which necessarily disrupts the essential and protective process of cerebral blood flow autoregulation. This study identifies a smooth muscle cell autocrine/paracrine signaling network that augments myogenic tone in a murine model of experimental SAH: it links tumor necrosis factor-α (TNFα), the cystic fibrosis transmembrane conductance regulator, and sphingosine-1-phosphate signaling. METHODS: Mouse olfactory cerebral resistance arteries were isolated, cannulated, and pressurized for in vitro vascular reactivity assessments. Cerebral blood flow was measured by speckle flowmetry and magnetic resonance imaging. Standard Western blot, immunohistochemical techniques, and neurobehavioral assessments were also used. RESULTS: We demonstrate that targeting TNFα and sphingosine-1-phosphate signaling in vivo has potential therapeutic application in SAH. Both interventions (1) eliminate the SAH-induced myogenic tone enhancement, but otherwise leave vascular reactivity intact; (2) ameliorate SAH-induced neuronal degeneration and apoptosis; and (3) improve neurobehavioral performance in mice with SAH. Furthermore, TNFα sequestration with etanercept normalizes cerebral perfusion in SAH. CONCLUSIONS: Vascular smooth muscle cell TNFα and sphingosine-1-phosphate signaling significantly enhance cerebral artery tone in SAH; anti-TNFα and anti-sphingosine-1-phosphate treatment may significantly improve clinical outcome.

Platelet-mediated changes to neuronal glutamate receptor expression at sites of microthrombosis following experimental subarachnoid hemorrhage.

OBJECT: Glutamate is important in the pathogenesis of brain damage after cerebral ischemia and traumatic brain injury. Notably, brain extracellular and cerebrospinal fluid as well as blood glutamate concentrations increase after experimental and clinical trauma. While neurons are one potential source of glutamate, platelets also release glutamate as part of their recruitment and might mediate neuronal damage. This study investigates the hypothesis that platelet microthrombi release glutamate that mediates excitotoxic brain injury and neuron dysfunction after subarachnoid hemorrhage (SAH). METHODS: The authors used two models, primary neuronal cultures exposed to activated platelets, as well as a whole-animal SAH preparation. Propidium iodide was used to evaluate neuronal viability, and surface glutamate receptor staining was used to evaluate the phenotype of platelet-exposed neurons. RESULTS: The authors demonstrate that thrombin-activated platelet-rich plasma releases glutamate, at concentrations that can exceed 300 µM. When applied to neuronal cultures, this activated plasma is neurotoxic, and the toxicity is attenuated in part by glutamate receptor antagonists. The authors also demonstrate that exposure to thrombin-activated platelets induces marked downregulation of the surface glutamate receptor glutamate receptor 2, a marker of excitotoxicity exposure and a possible mechanism of neuronal dysfunction. Linear regression demonstrated that 7 days after SAH in rats there was a strong correlation between proximity to microthrombi and reduction of surface glutamate receptors. CONCLUSIONS: The authors conclude that platelet-mediated microthrombosis contributes to neuronal glutamate receptor dysfunction and might mediate brain injury after SAH.

Mechanisms, treatment and prevention of cellular injury and death from delayed events after aneurysmal subarachnoid hemorrhage.

INTRODUCTION: Subarachnoid hemorrhage (SAH) patients often develop brain injury as a result of a number of delayed complications, resulting in significant morbidity and mortality. Many of these complications arise due to delayed cerebral ischemia, which occurs secondary to the hemorrhage. AREAS COVERED: The mechanisms of the delayed injury are reviewed, including angiographic vasospasm, cortical spreading ischemia, small arteriolar constriction, microthromboemboli, free radical injury and inflammation. Some current and prospective therapies for SAH are discussed, in the context of these complications. Statins have been particularly promising in experimental studies. EXPERT OPINION: Multiple mechanisms are involved in the pathogenesis of the delayed insult after SAH. New drugs may need to target multiple pathways to injury. Trials aiming to treat complications after SAH could benefit from taking into account the multifactorial pathogenesis of delayed insults.

Molecular alterations in the hippocampus after experimental subarachnoid hemorrhage.

Patients with aneurysmal subarachnoid hemorrhage (SAH) frequently have deficits in learning and memory that may or may not be associated with detectable brain lesions. We examined mediators of long-term potentiation after SAH in rats to determine what processes might be involved. There was a reduction in synapses in the dendritic layer of the CA1 region on transmission electron microscopy as well as reduced colocalization of microtubule-associated protein 2 (MAP2) and synaptophysin. Immunohistochemistry showed reduced staining for GluR1 and calmodulin kinase 2 and increased staining for GluR2. Myelin basic protein staining was decreased as well. There was no detectable neuronal injury by Fluoro-Jade B, TUNEL, or activated caspase-3 staining. Vasospasm of the large arteries of the circle of Willis was mild to moderate in severity. Nitric oxide was increased and superoxide anion radical was decreased in hippocampal tissue. Cerebral blood flow, measured by magnetic resonance imaging, and cerebral glucose metabolism, measured by positron emission tomography, were no different in SAH compared with control groups. The results suggest that the etiology of loss of LTP after SAH is not cerebral ischemia but may be mediated by effects of subarachnoid blood such as oxidative stress and inflammation.

Pharmacologic reduction of angiographic vasospasm in experimental subarachnoid hemorrhage: systematic review.

Animal models have been developed to simulate angiographic vasospasm secondary to subarachnoid hemorrhage (SAH) and to test pharmacologic treatments. Our aim was to evaluate the effect of pharmacologic treatments that have been tested in humans and in preclinical studies to determine if animal models inform results reported in humans. A systematic review and meta-analysis of SAH studies was performed. We investigated predictors of -translation from animals to humans with multivariate logistic regression. Pharmacologic reduction of vasospasm was effective in mice, rats, rabbits, dogs, nonhuman primates, and humans. Animal studies were generally of poor methodologic quality, and there was evidence of publication bias. Fresh blood injection to simulate SAH (vs. clot placement) and evaluation of vasospasm more than 3 days after SAH were independently associated with successful translation. We conclude that reduction of vasospasm is effective in animals and humans, and that injection of fresh blood and evaluation of vasospasm more than 3 days after SAH may be preferable for preclinical models.

Pharmacologic reduction of angiographic vasospasm in experimental subarachnoid hemorrhage: systematic review and meta-analysis.

Animal models have been developed to simulate angiographic vasospasm secondary to subarachnoid hemorrhage (SAH) and to test pharmacologic treatments. Our aim was to evaluate the effect of pharmacologic treatments that have been tested in humans and in preclinical studies to determine if animal models inform results reported in humans. A systematic review and meta-analysis of SAH studies was performed. We investigated predictors of translation from animals to humans with multivariate logistic regression. Pharmacologic reduction of vasospasm was effective in mice, rats, rabbits, dogs, nonhuman primates (standard mean difference of -1.74; 95% confidence interval -2.04 to -1.44) and humans. Animal studies were generally of poor methodologic quality and there was evidence of publication bias. Subgroup analysis by drug and species showed that statins, tissue plasminogen activator, erythropoietin, endothelin receptor antagonists, calcium channel antagonists, fasudil, and tirilazad were effective whereas magnesium was not. Only evaluation of vasospasm >3 days after SAH was independently associated with successful translation. We conclude that reduction of vasospasm is effective in animals and humans and that evaluation of vasospasm >3 days after SAH may be preferable for preclinical models.

Different effects of clazosentan on consequences of subarachnoid hemorrhage in rats.

One of the major complications after subarachnoid hemorrhage (SAH) is angiographic vasospasm in the large arteries at the base of the brain. However, a clinical trial of clazosentan demonstrated a 65% relative risk reduction in angiographic vasospasm but no effect on mortality or clinical outcome, raising questions about the role of angiographic vasospasm played in outcome after SAH. The purpose of this study was to determine if reducing or reversing angiographic vasospasm with clazosentan reduced other secondary complications such as microthromboembolism, loss of long-term potentiation (LTP) and neuronal cell death in a rat model of SAH. SAH in rats was created by injection of 300 µl non-heparinized autologous blood into the pre-chiasmatic cistern. Clazosentan, 10mg/kg bolus, or vehicle control was administered 1h after SAH intravenously, followed by a continuous infusion (1mg/kg/h) into the jugular vein using an osmotic pump. Rats treated with clazosentan had less large-artery vasospsam compared to vehicle-treated controls. However, clazosentan did not prevent the formation of microthromboemboli, neuronal cell death and degeneration and loss of LTP, suggesting there is a dissociation between large-artery angiographic vasospasm and other secondary complications of SAH. This result suggests that alleviation of angiographic vasospasm alone may not be sufficient to prevent other secondary complications or that off-target drug effects after systemic administration of clazosentan counteract the beneficial effects on angiographic vasospasm.