Inactive-phase neutrophil was linked to poorer collateral perfusion and worse stroke outcomes.
Evidence
This translational preclinical study combined permanent and transient middle cerebral artery occlusion mouse models with pharmacologic and genetic NET interventions plus a 540-patient ischemic stroke biomarker cohort.
Caveat
Human evidence was biomarker association, while direct tests of NET inhibition and clock mechanisms came from mouse models.
Simplified
BACKGROUND: Stroke remains a leading cause of mortality and disability, driven by complex, time-dependent mechanisms that aggravate ischemic injury. Collateral perfusion dictates infarct size, expansion rate, and penumbral preservation, yet its regulation is poorly understood. Beyond structural/genetic factors such as aging or cardiovascular risk, functional influences like circadian immune activity may also affect vascular patency. Neutrophils, key mediators of ischemic injury, exhibit circadian oscillations in phenotype and function that could modulate collateral flow and stroke outcome.
METHODS: We combined permanent and transient middle cerebral artery occlusion models in mice with flow cytometry, single-cell RNA sequencing, confocal microscopy, and laser speckle imaging to investigate time-of-day-dependent neutrophil mechanisms in stroke. Pharmacological (chloramidine, DNase-I) and genetic (Pad4 [peptidyl arginine deiminase 4], Bmal1 [brain and muscle ARNT (aryl hydrocarbon receptor nuclear translocator)-like 1], Cxcr4 [C-X-C chemokine receptor type 4]) interventions were used to define how time-of-day regulation shapes neutrophil function, neutrophil extracellular traps (NETs) formation, and stroke severity. A cohort of 540 patients with ischemic stroke was analyzed for diurnal patterns of NET-related biomarkers and their association with collateral circulation and clinical outcomes. -/ -Neu Neu
RESULTS: Infarct volume and neurological deficits exhibited clear circadian oscillations, with worse outcomes when stroke occurred during the murine inactive phase (Zeitgeber time 5) versus the active phase (Zeitgeber time 13). These fluctuations disappeared after neutrophil depletion or clock disruption. During the inactive phase, neutrophils displayed an activated, NET-prone phenotype, causing microvascular stalling and reduced collateral perfusion. Inhibiting NET formation pharmacologically or through Pad4 deletion restored perfusion and abolished time-of-day effects. In patients, neutrophil and NET-related biomarkers (MPO [myeloperoxidase], elastase, sCD40L [soluble CD40 ligand]) showed diurnal oscillations, peaking during the human inactive phase (evening/night), coinciding with reduced collateral flow and poorer outcomes.
CONCLUSIONS: Time-of-day regulation of neutrophil function critically determines collateral perfusion and stroke severity. Neutrophil-driven during the inactive phase promotes microvascular obstruction and worsens outcomes. Targeting NET formation or timing therapy could enhance collateral efficacy and offer novel chronotherapeutic opportunities for stroke treatment.
Key numbers
2.3×
Increase in infarct volume
Infarct size at ZT5 vs. ZT13 in mouse models.
50%
Reduction in infarct volume
Comparison of infarct volume in treated vs. untreated mice at ZT5.
366
Patient cohort size
Total number of stroke patients analyzed for diurnal variations.
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Circadian Biology