Abstract Body: Introduction: Nitro-oleic acid (OA-NO₂) is an endogenous peroxisome proliferator-activated receptor-γ (PPARγ) ligand and can activate this receptor under both physiological and pathological conditions. In this study, we explore the role and molecular mechanisms of OA-NO₂ in maintaining blood-brain barrier (BBB) integrity and enhancing neurovascular function during ischemic stroke, with a particular emphasis on the activation of PPARγ signaling pathways.
Methods: EC-selective PPARγ conditional knockout (EC-PPARγ cKO) and wild-type (WT) mice underwent 1-hour middle cerebral artery occlusion (MCAO) with 1 to 7 days of reperfusion. Mice were treated with oleic acid (OA) or OA-NO₂ (10 mg/kg) via tail vein 2 hours after MCAO. Sensorimotor function was assessed using the rotarod, foot fault, and adhesive tape removal tests. BBB integrity was measured with fluorescein-dextrans, and brain infarct was analyzed with anti-MAP2 immunostaining. Peripheral immune cell infiltration was examined by immunofluorescent methods. Total RNA and proteins were extracted from brain tissue. BBB tight junction mRNAs, proteins, and inflammatory factors were quantified using qPCR and Western blotting.
Results: OA-NO₂ treatment significantly enhanced sensorimotor function in stroke mice compared to the OA group. However, this beneficial effect was lost in the EC-PPARγ cKO mice. Compared to OA controls, intravenous administration of OA-NO₂ led to reduced BBB leakage in ischemic brains, as indicated by a significant decrease in the extravasation of BBB tracers. This reduction in BBB leakage was also abolished in the EC-PPARγ cKO mice. Furthermore, OA-NO₂ treatment reduced brain infarction in stroke mice, but this protective effect was completely reversed in the EC-PPARγ cKO mice. Interestingly, OA-NO₂ treatment promoted a shift towards an anti-inflammatory microglial phenotype (M2) in the peri-infarct regions of EC-PPARγ WT mice, but not in EC-PPARγ cKO mice. Mechanistically, OA-NO₂ increased levels of major endothelial tight junction mRNAs and proteins, including Claudin 5 and ZO-1, in EC-PPARγ WT mice but not in EC-PPARγ cKO mice following ischemic stroke.
Conclusions: Treatment with OA-NO₂ effectively reduces BBB leakage-triggered peripheral immune cell infiltration, brain infarction, and long-term neurobehavioral deficits in mice following ischemic stroke. The neurovascular protection conferred by OA-NO₂ primarily operates through PPARγ-dependent signaling pathways.