NF-κB Signaling-Mediated Activation of WNK-SPAK-NKCC1 Cascade in Worsened Stroke Outcomes of Ang II-Hypertensive Mice.

BACKGROUND: Worsened stroke outcomes with hypertension comorbidity are insensitive to blood pressure-lowering therapies. In an experimental stroke model with comorbid hypertension, we investigated causal roles of ang II (angiotensin II)-mediated stimulation of the brain WNK (with no lysine [K] kinases)-SPAK (STE20/SPS1-related proline/alanine-rich kinase)-NKCC1 (Na-K-Cl cotransporter) complex in worsened outcomes. METHODS: Saline- or ang II-infused C57BL/6J male mice underwent stroke induced by permanent occlusion of the distal branches of the middle cerebral artery. Mice were randomly assigned to receive either vehicle dimethyl sulfoxide/PBS (2 mL/kg body weight/day, IP), a novel SPAK inhibitor, 5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-hydroxybenzamide (ZT-1a' 5 mg/kg per day, IP) or a NF-κB (nuclear factor-κB) inhibitor TAT-NBD (transactivator of transcription-NEMO-binding domain' 20 mg/kg per day, IP). Activation of brain NF-κB and WNK-SPAK-NKCC1 cascade as well as ischemic stroke outcomes were examined. RESULTS: Stroke triggered a 2- to 5-fold increase of WNK (isoforms 1, 2, 4), SPAK/OSR1 (oxidative stress-responsive kinase 1), and NKCC1 protein in the ang II-infused hypertensive mouse brains at 24 hours after stroke, which was associated with increased nuclear translocation of phospho-NF-κB protein in the cortical neurons (a Pearson correlation r of 0.77, P<0.005). The upregulation of WNK-SPAK-NKCC1 cascade proteins resulted from increased NF-κB recruitment on Wnk1, Wnk2, Wnk4, Spak, and Nkcc1 gene promoters and was attenuated by NF-κB inhibitor TAT-NBD. Poststroke administration of SPAK inhibitor ZT-1a significantly reduced WNK-SPAK-NKCC1 complex activation, brain lesion size, and neurological function deficits in the ang II-hypertensive mice without affecting blood pressure and cerebral blood flow. CONCLUSIONS: The ang II-induced stimulation of NF-κB transcriptional activity upregulates brain WNK-SPAK-NKCC1 cascade and contributes to worsened ischemic stroke outcomes, illustrating the brain WNK-SPAK-NKCC1 complex as a therapeutic target for stroke with comorbid hypertension.

A Novel Na+-K+-Cl- Cotransporter 1 Inhibitor STS66* Reduces Brain Damage in Mice After Ischemic Stroke.

Background and Purpose- Inhibition of brain NKCC1 (Na+-K+-Cl- cotransporter 1) with bumetanide (BMT) is of interest in ischemic stroke therapy. However, its poor brain penetration limits the application. In this study, we investigated the efficacy of 2 novel NKCC1 inhibitors, a lipophilic BMT prodrug STS5 (2-(Dimethylamino)ethyl 3-(butylamino)-4-phenoxy-5-sulfamoyl-benzoate;hydrochloride) and a novel NKCC1 inhibitor STS66 (3-(Butylamino)-2-phenoxy-5-[(2,2,2-trifluoroethylamino)methyl]benzenesulfonamide), on reducing ischemic brain injury. Methods- Large-vessel transient ischemic stroke in normotensive C57BL/6J mice was induced with 50-min occlusion of the middle cerebral artery and reperfusion. Focal, permanent ischemic stroke in angiotensin II (Ang II)-induced hypertensive C57BL/6J mice was induced by permanent occlusion of distal branches of middle cerebral artery. A total of 206 mice were randomly assigned to receive vehicle DMSO, BMT, STS5, or STS66. Results- Poststroke BMT, STS5, or STS66 treatment significantly decreased infarct volume and cerebral swelling by ≈40% to 50% in normotensive mice after transient middle cerebral artery occlusion, but STS66-treated mice displayed better survival and sensorimotor functional recovery. STS5 treatment increased the mortality. Ang II-induced hypertensive mice exhibited increased phosphorylatory activation of SPAK (Ste20-related proline alanine-rich kinase) and NKCC1, as well as worsened infarct and neurological deficit after permanent distal middle cerebral artery occlusion. Conclusions- The novel NKCC1 inhibitor STS66 is superior to BMT and STS5 in reducing ischemic infarction, swelling, and neurological deficits in large-vessel transient ischemic stroke, as well as in permanent focal ischemic stroke with hypertension comorbidity.

Eligibility for Endovascular Trial Enrollment in the 6- to 24-Hour Time Window: Analysis of a Single Comprehensive Stroke Center.

BACKGROUND AND PURPOSE: The results of the DAWN trial (Diffusion-Weighted Imaging or Computerized Tomography Perfusion Assessment With Clinical Mismatch in the Triage of Wake Up and Late Presenting Strokes Undergoing Neurointervention With Trevo) support the benefit of endovascular therapy in patients presenting beyond the 6-hour time window with anterior circulation large vessel occlusions. The impact of these results with respect to additional number of eligible patients in clinical practice remains unknown. METHODS: A retrospective review of ischemic stroke admissions to a single DAWN trial-participating comprehensive stroke center was performed during the DAWN enrollment period (November 2014 to February 2017) to identify patients meeting criteria for DAWN and DEFUSE-3 (Endovascular Therapy Following Imaging Evaluation for Ischemic Stroke-3) eligibility. Patients presenting beyond 6 hours were further investigated to clarify reasons for trial exclusion. RESULTS: Of the 2667 patients with acute ischemic stroke admitted within the study period, 30% (n=792) presented within the 6- to 24-hour time window, and 47% (n=1242) had a National Institutes of Health Stroke Scale ≥6. Further clinical trial-specific selection criteria were applied based on the presence of large vessel occlusion, baseline modified Rankin Scale score, core infarct, and perfusion imaging (when available). There were 45 patients who met all DAWN trial criteria and 47 to 58 patients who would meet DEFUSE-3 trial criteria. Thirty-three percent of DAWN-eligible patients are DEFUSE-3 ineligible. CONCLUSIONS: Of all patients with acute ischemic stroke presenting to a single comprehensive stroke center, 1.7% of patients qualified for DAWN clinical trial enrollment with an additional 0.6% to 1% qualifying for the DEFUSE-3 trial. These data predict an increase in thrombectomy utilization with important implications for comprehensive stroke center resource optimization and stroke systems of care.

Clinically distinct electroencephalographic phenotypes of early myoclonus after cardiac arrest.

OBJECTIVE: We tested the hypothesis that there are readily classifiable electroencephalographic (EEG) phenotypes of early postanoxic multifocal myoclonus (PAMM) that develop after cardiac arrest. METHODS: We studied a cohort of consecutive comatose patients treated after cardiac arrest from January 2012 to February 2015. For patients with clinically evident myoclonus before awakening, 2 expert physicians reviewed and classified all EEG recordings. Major categories included: Pattern 1, suppression-burst background with high-amplitude polyspikes in lockstep with myoclonic jerks; and Pattern 2, continuous background with narrow, vertex spike-wave discharges in lockstep with myoclonic jerks. Other patterns were subcortical myoclonus and unclassifiable. We compared population characteristics and outcomes across these EEG subtypes. RESULTS: Overall, 401 patients were included, of whom 69 (16%) had early myoclonus. Among these patients, Pattern 1 was the most common, occurring in 48 patients (74%), whereas Pattern 2 occurred in 8 patients (12%). The remaining patients had subcortical myoclonus (n = 2, 3%) or other patterns (n = 7, 11%). No patients with Pattern 1, subcortical myoclonus, or other patterns survived with favorable outcome. By contrast, 4 of 8 patients (50%) with Pattern 2 on EEG survived, and 4 of 4 (100%) survivors had favorable outcomes despite remaining comatose for 1 to 2 weeks postarrest. INTERPRETATION: Early PAMM is common after cardiac arrest. We describe 2 distinct patterns with distinct prognostic significances. For patients with Pattern 1 EEGs, it may be appropriate to abandon our current clinical standard of aggressive therapy with conventional antiepileptic therapy in favor of early limitation of care or novel neuroprotective strategies. Ann Neurol 2016;80:175-184.