Intratracheal SP-D and BaP Exposure in Mice

All animal experiments (approval number: A19-021-2) were carried out under the Kyushu University Guidelines for Animal Experiments and the ARRIVE guidelines. Eight-week-old female C57BL/6J mice were obtained from Japan SLC (Shizuoka, Japan) and were housed under specific pathogen-free conditions for 1 week before administration of recombinant human SP-D (5 μg in 50 μl of PBS) and BaP (1 mg in 50 μl of tricaprylin) by intratracheal instillation. The mice were sacrificed and analyzed 48 h later. At intratracheal instillation and terminal procedure, the mice were anesthetized with a mixture of medetomidine hydrochloride (0.3 mg/kg), midazolam (4 mg/kg) and butorphanol tartrate (5 mg/kg) intraperitoneally. The mice were monitored for a surgical plane of anesthesia determined by loss of reflexes (lack of response to toe and tail pinch), muscle relaxation, and deep, rhythmic breathing. At termination, the mice were euthanized by exsanguination. The right lung lobes were fixed by intratracheal instillation of 10% neutral-buffered formalin at a pressure of 20 cm H₂O and paraffine-embedded for histology, then the left lung lobes were taken for protein extraction.