Rivaroxaban versus aspirin for prevention of covert brain infarcts in patients with embolic stroke of undetermined source: NAVIGATE ESUS MRI substudy

Background Covert brain infarcts are associated with important neurological morbidity. Their incidence in patients with embolic stroke of undetermined source (ESUS) is unknown. Aims To assess the incidence of covert brain infarcts and cerebral microbleeds using MRI in a prospective substudy of the NAVIGATE ESUS randomized trial and to evaluate the effects of antithrombotic therapies. Methods At 87 sites in 15 countries, substudy participants were randomly assigned to receive rivaroxaban 15 mg daily or aspirin 100 mg daily and underwent brain MRI near randomization and after study termination. The primary outcome was incident brain infarct (clinical ischemic stroke or covert brain infarct). Brain infarcts and microbleeds were ascertained centrally by readers unaware of treatment. Treatment effects were estimated using logistic regression. Results Among the 718 substudy participants with interpretable, paired MRIs, the mean age was 67 years and 61% were men with a median of 52 days between the qualifying ischemic stroke and randomization and a median of seven days between randomization and baseline MRI. During the median (IQR) 11 (12) month interval between scans, clinical ischemic strokes occurred in 27 (4%) participants, while 60 (9%) of the remaining participants had an incident covert brain infarct detected by MRI. Assignment to rivaroxaban was not associated with reduction in the incidence of brain infarct (OR 0.77, 95% CI 0.49, 1.2) or of covert brain infarct among those without clinical stroke (OR 0.85, 95% CI 0.50, 1.4). New microbleeds were observed in 7% and did not differ among those assigned rivaroxaban vs. aspirin (HR 0.95, 95% CI 0.52–1.7). Conclusions Incident covert brain infarcts occurred in twice as many ESUS patients as a clinical ischemic stroke. Treatment with rivaroxaban compared with aspirin did not significantly reduce the incidence of covert brain infarcts or increase the incidence of microbleeds, but the confidence intervals for treatment effects were wide. Registration: https://www.clinicaltrials.gov . Unique identifier: NCT 02313909

Carotid plaque morphology-An indicator for brain infarcts

Cerebrovascular adverse events are one of the most common causes for morbidity and mortality worldwide. Thromboembolism being the culprit behind a significant number of such events. An attempt has been made to ascertain the association of carotid plaque morphology in the occurrence of ischemic stroke. Heterogeneous plaques (Type 2 & 3) weremostly observed to be ipsilateral to the side of brain infarct, as compared to the contralateral side. Detection of heterogeneous plaques and plaque ulceration in patients may indicate future development of stroke. This study included 75 consecutive patients having anterior circulation infarct in MRI of the brain and atherosclerotic changes in extra cranial carotid system on Carotid Duplex scan. Plaque morphology ipsilateral to the brain infarct was compared to that on the contralateral side. Significantly higher number of heterogeneous plaques (Type 2 & 3) were present ipsilateral to the side of brain infarct, as compared to the contralateral side (p-value 0.004). Plaque ulceration was also observed to be present more frequently on the side of infarct (22.6% on the ipsilateral side and 5.4% on the contralateral side). Moreover, Diabetes mellitus and dyslipidemia were the most prevalent risk factors (78.2% and 65.2%, respectively) in patients with these types of plaques. Detection of heterogeneous plaques and plaque ulceration in patients may indicate future development of stroke, necessitating prompt and appropriate management protocols.Periodic screening of such patients with Carotid Duplex Ultrasonography is expected to be very helpful.

Eagle syndrome presenting as a neurological emergency: A case report

Background: Eagle syndrome, due to the elongation of the styloid process as well as the calcification of the stylohyoid ligament, rarely presents itself with a major neurological disorder such as a brain infarct. Case Description: Authors describe the case report of a previously healthy 64-year-old Caucasian male that complained of inability to control his right upper and lower extremity of an acute nature. Imaging at the emergency department (magnetic resonance of the brain and computerized angiography) showed the presence of elongated styloid process bilaterally with clear predomination at the left side. The brain ischemia (left temporal brain infarct) was due to carotid artery dissection, and the left internal carotid artery was not visualized during the contrast-enhanced angiography. The patient was hospitalized at a neurological facility and thereafter referred to surgery for styloidectomy. Conclusion: The present case underscores the need for a prompt diagnosis and an enhanced awareness of this syndrome, especially among emergency department professionals.

High-Fat Diet Aggravates Cerebral Infarct, Hemorrhagic Transformation and Neuroinflammation in a Mouse Stroke Model

Background: Stroke in context of type 2 diabetes (T2D) is associated with a poorer outcome than in non-diabetic conditions. We aimed at creating a new reproducible mouse model of stroke in impaired glucose tolerance conditions induced by high-fat diet. Methods: Adult C57BL6 mice were fed for 2 months with either normal diet (ND) or high-fat diet (HFD). We used a model of Middle Cerebral Artery Occlusion (MCAO) for 90 min. Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT) were used to assess pre-diabetic status. Brain infarct volume, hemorrhagic transformation (HT) as well as systemic and cerebral inflammatory markers were evaluated. Results: HFD was associated with an increased body weight and glycemia following OGTT. The HFD group presented a significant increase in brain infarct volume (38.7 (IQR 30–46.7%) vs. 28.45 (IQR 21–30%); p = 0.016) and HT (HFD: 2 (IQR 1–5) vs. ND: 0 (IQR 0–1); p = 0.012) and higher levels of IL-6 and MCP-1 in infarcted hemisphere compared to the ND group. Conclusion: Two months of HFD in adult mice were sufficient to alter the lipid profile and the control of hyperglycemia. These metabolic perturbations were significantly associated with increased infarct volume and hemorrhagic complications.

Inhibition of Acyl-CoA Synthetase Long-Chain Family Member 4 Facilitates Neurological Recovery After Stroke by Regulation Ferroptosis

BackgroundIschemic stroke is the main cause of disability worldwide, leading to a serious socioeconomic burden. Ferroptosis is a non-apoptotic form of programmed cell death and is related to various diseases. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is considered a target of ferroptosis, but its specific role in ischemic stroke remains unclear. In this study, we investigate whether the inhibition of ACSL4 promotes the recovery of neurological function in a way that prevents ferroptosis.MethodsA transient cerebral ischemia model was established for mice by middle cerebral artery occlusion (MCAO); glutathione peroxidase 4 (GPx4), ACSL4 and cyclooxygenase 2 (COX2) were detected by Western blot, and changes to mitochondria were observed by a transmission electron microscope. A kit was used to determine iron levels and lipid peroxide indicators, such as glutathione peroxidase (GPx), reduced glutathione (GSH), total glutathione/oxidized glutathione (GSH/GSSG), lipid peroxidation, reactive oxygen species, superoxide and malonaldehyde. Following MCAO, a ferroptosis inhibitor, liproxstatin-1, was administered intranasally immediately at a concentration of 10 mg/kg. Rosiglitazone was used to inhibit ACSL4 and was administered intravenously 1 h before MCAO at a concentration of 0.4 mg/kg. Brain injury was determined by neurological deficit scores, neuroscore (28-point), corner test and gait analyses, at 24 and 72 h after stroke. Brain infarct volume was determined by 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining at 72 h after stroke.ResultsAfter MCAO, GPx4 protein expression decreased, ACSL4 and COX2 protein expression increased, GPx activity decreased and iron accumulation. Transmission electron microscopy confirmed that the outer mitochondrial membrane of neurons had ruptured and mitochondrial cristae had decreased or disappeared. Liproxstatin-1 could significantly attenuate the decrease of GPx4 and the increase of COX2 after MCAO, dramatically reducing iron accumulation and decreasing GPx activity, accompanied by a marked reduction in changes in lipid peroxidation indicators. The use of rosiglitazone to inhibit ACSL4 could significantly improve neurological function and reduce the brain infarct volume at 72 h after stroke. Importantly, inhibiting ACSL4 could significantly attenuate the decline of GPx4 after MCAO and markedly attenuate iron accumulation and a decrease in GPx activity. Additionally, changes in lipid peroxidation indicators were also significantly inhibited.ConclusionThis study indicates that inhibiting ACSL4 can promote the recovery of neurological function after stroke by suppression of ferroptosis.

Abstract P781: Ischemic Postconditioning Protects Against Hemorrhagic Transformation Induced by Hyperglycemia in Ischemic Stroke

Background: Hyperglycemia occurs in over 40% of ischemic stroke patients, which induces hemorrhagic transformation (HT) and worsens stroke outcomes. The management of hyperglycemia with insulin did not show favorable outcomes. Thus, strategies for managing hyperglycemia-exacerbated stroke injury are urgently needed. We previously demonstrated that ischemic postconditioning (IPostC) (repeated transient interruption of cerebral blood flow during reperfusion) can reduce brain infarct size and improve neurological outcomes. In this study, we hypothesized that IPostC can reduce HT in ischemic stroke with acute hyperglycemia. Method: Male mice were subjected to middle cerebral artery occlusion (MCAO) for 1 hour, followed by reperfusion to mimic ischemic stroke. Glucose was injected before MCAO to induce hyperglycemia. IPostC was initiated upon reperfusion with 3 cycles of 30-second reperfusion followed by 10 seconds of MCA occlusion. Brain infarct was visualized by TTC staining and quantitated using Image J. Hemorrhagic transformation was evaluated by hemorrhagic scores. Result: Acute hyperglycemia significantly increased the brain infarct size (by 25%, p<0.01), brain edema (p<0.001) and hemorrhagic transformation (HT) (average HT scores: 0.75 in MCAO group vs 15.6 in MCAO plus hyperglycemia group, p<0.001), Mice with hyperglycemia also exhibited more severe neurological deficit and higher mortality rate at 24 hours after MCAO. 2) IPostC treatment significantly reduced brain infarct size (p<0.01), brain edema (p<0.05) and attenuated HT (p<0.001). Neurological deficit and mortality rate was reduced with IPostC treatment. Conclusion: Our findings suggest that IPostC can counteract the effects of acute hyperglycemia and reduce brain injury, edema and HT after stroke. Grant/Other Support: NIH Grant R01NS064136C

Abstract P759: Microglial EZH2 Inhibition Leads to Neuroprotection After Stroke in Aged Mice

Introduction: Ischemic stroke results in activation of microglia, which may polarize towards a pro-inflammatory (M1) phenotype and/or an anti-inflammatory (M2) phenotype. Enhancer of zeste homolog (EZH) 2 is a histone-lysine N-methyltransferase enzyme. Accumulating evidence has suggested EZH2 is key modulator of microglia polarization by epigenetically regulating gene expression. We here investigated whether microglial-specific deletion of EZH2 leads to a beneficial protective effect in stroke in vivo . We further tested the therapeutical potential of an EZH2 inhibitor after stroke. Methods: Aged male mice were subjected to 60-minutes middle cerebral artery stroke. Tamoxifen administration was started 30 days prior to stroke to induce genetic deletion of microglial EZH2 in CX3CR1-creER/EZH2-floxed mice. EZH2 floxed mice were used as controls. EZH2 inhibitor GSK343 was I.P. injected (once a day for two consecutive days), starting three hours after stroke onset. Mice were sacrificed for immunohistochemistry and crystal violet staining (brain infarct assay) after behavior tests at 3 days after stroke. Results: The expression of microglial EZH2 was significantly abrogated in KO mice compared to the control floxed mice (144±15.43 vs. 50.65±4.99 cells/mm 2 , N=5/each group, P<0.01). EZH2 deletion reduced brain infarct volume (29.27±2.23% vs. 6.07±0.88%, N=7/each group, P<0.001) and improved functional outcome assayed by adhesive removal test (59±13.1 sec, N=7 in floxed control vs. 26.28±4.1 sec, N=12 in KO, P<0.01). Mechanistically, microglial EZH2 deletion led to a decrease in expression of M1 marker iNOS (170±14.78 vs. 76.65±11.38 cells/mm 2 , N=4/each group, P<0.05), an increase in M2 marker Arg1 (96.64±11.48 vs. 203.3±22.02 cells/mm 2 , N=4/each group, P<0.05) co-stained in microglia (Iba1). Finally, GSK343 treatment robustly reduced infarct volume (37.1±2.97% vs. 21.9±2.92%) and increased latency to fall in hang-wire test (23.5±3.5 vs. 45.9±7.1 sec) (N=8/group, p<0.05) 3 days after stroke. Conclusions: Both genetic deletion of EZH2 in microglia and pharmacological inhibition of EZH2 improved stroke outcome in aged. The effect may be due to limiting microglial M1 polarization and enhancing M2 polarization.

MicroRNA-210 downregulates TET2 and contributes to inflammatory response in neonatal hypoxic-ischemic brain injury

Background Neonatal hypoxic-ischemic (HI) brain injury is a leading cause of acute mortality and chronic disability in newborns. Our previous studies demonstrated that HI insult significantly increased microRNA-210 (miR-210) in the brain of rat pups and inhibition of brain endogenous miR-210 by its inhibitor (LNA) provided neuroprotective effect in HI-induced brain injury. However, the molecular mechanisms underpinning this neuroprotection remain unclear. Methods We made a neonatal HI brain injury model in mouse pups of postnatal day 7 to uncover the mechanism of miR-210 in targeting the ten eleven translocation (TET) methylcytosine dioxygenase 2 that is a transcriptional suppressor of pro-inflammatory cytokine genes in the neonatal brain. TET2 silencing RNA was used to evaluate the role of TET2 in the neonatal HI-induced pro-inflammatory response and brain injury. MiR-210 mimic and inhibitor (LNA) were delivered into the brain of mouse pups to study the regulation of miR-210 on the expression of TET2. Luciferase reporter gene assay was performed to validate the direct binding of miR-210 to the 3′ untranslated region of the TET2 transcript. Furthermore, BV2 mouse microglia cell line was employed to confirm the role of miR-210-TET2 axis in regulating pro-inflammatory response in microglia. Post-assays included chromatin immunoprecipitation (ChIP) assay, co-immunoprecipitation, RT-PCR, brain infarct assay, and neurobehavioral test. Student’s t test or one-way ANOVA was used for statistical analysis. Results HI insult significantly upregulated miR-210, downregulated TET2 protein abundance, and increased NF-κB subunit p65 acetylation level and its DNA binding capacity to the interleukin 1 beta (IL-1β) promoter in the brain of mouse pups. Inhibition of miR-210 rescued TET2 protein level from HI insult and miR-210 mimic decreased TET2 protein level in the brain of mouse pups, suggesting that TET2 is a functional target of miR-210. The co-immunoprecipitation was performed to reveal the role of TET2 in HI-induced inflammatory response in the neonatal brain. The result showed that TET2 interacted with NF-κB subunit p65 and histone deacetylase 3 (HDAC3), a co-repressor of gene transcription. Furthermore, TET2 knockdown increased transcriptional activity of acetyl-p65 on IL-1β gene in the neonatal brain and enhanced HI-induced upregulation of acetyl-p65 level and pro-inflammatory cytokine expression. Of importance, TET2 knockdown exacerbated brain infarct size and neurological deficits and counteracted the neuroprotective effect of miR-210 inhibition. Finally, the in vitro results demonstrated that the miR-210-TET2 axis regulated pro-inflammatory response in BV2 mouse microglia cell line. Conclusions The miR-210-TET2 axis regulates pro-inflammatory cytokine expression in microglia, contributing to neonatal HI brain injury.

Electroacupuncture Pre-treatment Exerts an Anti-apoptotic Effect in Cerebral Ischaemia and Reperfusion Injury in Rats

Background: Electroacupuncture (EA) pre-treatment can play a significant neuroprotective role in rats with cerebral ischaemia and reperfusion (CIR) injury, but the specific mechanism needs to be further elucidated. <br><br>Objective: To investigate the effects of EA pre-treatment on apoptosis-related proteins Bax, Bcl-2, Cytochrome c (cyt c), cleaved caspase-9 and -3 in rats with CIR injury. Methods: CIR injury was induced using middle cerebral artery occlusion (MCAO) ischaemia-reperfusion. Thirty-six male Specific Pathogen Free (SPF) Sprague-Dawley rats were randomly divided into three groups: Sham (n=12), MCAO (n=12), and the EA+MCAO group (n=12). EA+MCAO group rats received electroacupuncture at DU20 for 2 consecutive weeks before MCAO model preparation. After 24 h of reperfusion, neurological deficits were evaluated by modified neurological severity scores. Brain infarct volumes were examined by 2,3,5-triphenyltetrazolium chloride staining. Expression of Bax, Bcl-2, cyt c, cleaved caspase-9 and -3 in the ischaemic penumbra were detected by Western blotting, and apoptosis evaluated by TUNEL staining. <br><br>Results: After perfusion for 24 h, compared with the MCAO group, the neurological deficit scores and brain infarct volumes in the EA+MCAO group were significantly decreased (P<0.05), as was the level of Bax (P<0.05) or the Bax/Bcl-2 ratio (P<0.05), levels of cyt c, cleaved caspase-9 and -3 also decreased (P<0.05), like the number of TUNEL-positive cells (P<0.05), in contrast, the level of Bcl-2 increased (P<0.05). <br><br>Conclusion: EA pre-treatment exerts an anti-apoptotic effect through Bax-to-Bcl-2 ratio downregulation, blockage of mitochondrial cyt c release to the cytosol, and reduction of caspase-9 and -3 expression in rats with CIR injury.