Leukotriene B4-BLT1 axis exacerbates neutrophil invasion and motor dysfunction after intracerebral hemorrhage in mice

[Speaker] Masanori Hijioka:1
[Co-author] Yuki Kurauchi:2, Akinori Hisatsune:2, Takahiro Seki:2, Tomoaki Koga:2,3, Yoshihisa Kitamura:1, Takehiko Yokomizo:3, Takao Shimizu:4, Hiroshi Katsuki:1
1:College of Pharmaceutical Sciences, Ritsumeikan University, Japan, 2:Kumamoto University, Japan, 3:Juntendo University, Japan, 4:National Center for Global Health and Medicine, Japan

Background: Intracerebral hemorrhage (ICH) characterized by formation of blood clot into brain parenchyma is most lethal type of stroke, and accompanied with infiltration of numerous leukocytes such as macrophages, monocytes and neutrophils. Among them, recent report has been revealed neutrophils invasion gets worse the prognosis of ICH. In this study, we focused on leukotriene B4 (LTB4), a metabolite of arachidonic acid as a target for ICH therapy.
Methods: Male C57BL/6J mice, BLT1 knockout (KO) mice and wild-type (WT) littermate (8 to 10-week-old) were received the injection of type 7 collagenase (0.025 U) into striatum. LTB4 content in ICH brain was measured by ELISA at 6, 24 and 72 h after ICH induction. Expression levels of mRNAs associate with LTB4 production in ICH brain were measured by Quantitative RT-PCR at 18, 24, 48 and 72 h after ICH induction. Zileuton (3 or 10 mg/kg), an inhibitor of 5-lipoxygenase, was administered by intravenous injection at 15 min, 24 h and 48 h after ICH induction. ONO-4057 (30 or 100 mg/kg), an antagonist of receptor for LTB4, was orally administered at 3, 27, 51 h after ICH induction. Neutrophil invasion was evaluated at 72 h after ICH induction by immunohistochemical analysis with anti-myeloperoxidase antibody. Sensorimotor functions were measured by beam-walking test and limb-placing test.
Results: We found the increase of LTB4 content and the increases of mRNAs encoding 5-lipoxtgenase (5-LOX) and 5-LOX-activating protein in the ICH brain. Inhibition of 5-LOX by the daily treatment with zileuton (3 or 10 mg/kg) prevented ICH-induced increase in LTB4, attenuated neutrophil infiltration into hematoma, and ameliorated sensorimotor dysfunction. In addition, mice deficient for LTB4 receptor BLT1 exhibited lower number of infiltrating neutrophils and lower levels of sensorimotor dysfunction after ICH than wild-type littermates. Similarly, daily administration of ONO-4057 (30 or 100 mg/kg) inhibited neutrophil infiltration and ameliorated sensorimotor dysfunction. ONO-4057 had no effect on hematoma volume and ICH-associated brain edema, but attenuated inflammatory responses of microglia in perihematoma region and axon injury in the internal capsule.
Conclusion: Targeting the LTB4-BLT1 axis is expected to become a novel therapeutic approach to suppress neutrophil invasion and following process.
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