Introduction: Neurovascular coupling (NVC) becomes impaired upon acute ischemic stroke (AIS). NVC dysfunction is thought to be linked to the evolution of spreading depolarization (SD), that causes vasoconstriction by augmenting the synthesis of 20-HETE. Here, we demonstrate the SD-related disruption of NVC during reperfusion, and block 20-HETE production to improve NVC.Methods: C57BL/6 mice (n=41) were anesthetized with isoflurane. Cerebral blood flow (CBF) variations were captured using laser speckle contrast imaging (LASCA) or laser-Doppler flowmetry. After 10 min baseline recording, transient (45 min) unilateral (1VO) or bilateral (2VO) common carotid artery occlusion produced ischemia. Reperfusion initiated by the release of the occlusions was monitored for 60 min. Subsequently, NVC efficacy was evaluated under isoflurane (0.1%)-medetomidine (0.1mg/kg) anesthesia by whisker stimulation (~2Hz). SHAM operated animals served as control. The CYP450 inhibitor HET0016 (1mg/kg) or its vehicle was administered i.p.Results: SDs regularly occurred after 2VO but no SD was detected in the 1VO and SHAM groups. The relative amplitude of functional hyperemia in the 1VO hemispheres was similar to that seen in the SHAM group (45±35 vs. 40±25%, 1VO vs. SHAM). However, functional hyperemia was impaired in the 2VO SD hemispheres (10±8%). Blocking 20-HETE synthesis with HET0016 at 1 mg/kg concentration did not improve CBF during ischemia, reperfusion or with NVC.Discussion: SD evolution impairs NVC after AIS but the role of 20-HETE IN NVC dysfunction has not been substantiated. Further experiments with higher HET0016 concentration have been scheduled.Acknowledgements: Funding: NKFIH K134377, H2020 No.739593, NAP3.0, TKP2021-EGA-28