Mice in the naïve control group received no treatment for the duration of the experiment. Mice in the inhaled saline group were sensitized intraperitoneally with 200 μl saline on days 1 and 8. On days 15, 16, and 17 after initial sensitization with saline, mice were nebulized with saline for 30 min in a whole-body exposure chamber. Mice in the intranasal saline group were intraperitoneally sensitized as inhaled saline group and then intranasally instilled with 40 μl saline on days 15, 16, and 17. Mice in the inhaled OVA group were intraperitoneally sensitized on days 1 and 8 with 20 μg OVA (Sigma-Aldrich, St. Louis, MO) emulsified in 1 mg aluminum hydroxide (Thermo Fisher Scientific, Waltham, MA) in a total volume of 200 μl. On days 15, 16, and 17 after initial sensitization with OVA, mice were administered aerosolized 5% (v/v) OVA by inhalation for 30 min in a whole-body exposure chamber. Mice in the intranasal OVA group were intraperitoneally sensitized as the inhaled OVA group and then intranasally instilled with 20 μg OVA on days 15, 16, and 17. For intranasal instillation, mice were anesthetized using inhaled anesthesia. Prior to instillation, isoflurane was delivered into induction chamber using small animal portable anesthesia systems (L-PAS-02, LMSKOREA, Inc., Seongnam, Korea) equipped with isoflurane vaporizer. And then, mice were exposed to 2.5% isoflurane delivered in O2 (2 L/min) within induction chamber until a sleep-like state was reached. Mice receiving isoflurane anesthesia were removed from the induction chamber and instillation was performed immediately on board. Intranasal administration of challenge dose (saline or 20 μg OVA diluted in 40 μl saline) was performed by pipetting onto the outer edge of nose of the mice. After instillation, mice moved, fully recovered and transferred to their cage.
Twenty-four hours after the last challenge with OVA, AHR were assessed. And 48 h after the last challenge with OVA, mice were euthanized with an overdose of isoflurane and continuously exposed until 1 min after breathing stops. The sample collections for analysis were performed in sacrificed animals. Lung inflammation, serum IgE production, and protein levels of inflammatory and epithelial cell-derived cytokines were assessed.
Twenty-four hours after the last challenge with OVA, AHR were assessed. And 48 h after the last challenge with OVA, mice were euthanized with an overdose of isoflurane and continuously exposed until 1 min after breathing stops. The sample collections for analysis were performed in sacrificed animals. Lung inflammation, serum IgE production, and protein levels of inflammatory and epithelial cell-derived cytokines were assessed.
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