Establishment of an optimized murine asthma model forHumulus pollen allergy
Forty mice were randomly divided into four groups. Two different routes and doses were used for sensitization, based on studies by Ya-li et al.[10] and Conejero et al.[15] (Fig. 1 A). Briefly, mice in the SC sensitization group (SC.HSE, n=10) were sensitized via SC injection on the back of the neck with 25 μg of HSE adsorbed to alum adjuvant (Imject Alum; Pierce, Rockford, IL,USA) in a volume of 200 μL. Mice in the SC control group (SC.PBS, n=10) received SC injections of PBS adsorbed to alum adjuvant (200 μL). Mice in the IP sensitization group (IP.HSE, n=10) were sensitized via IP injection—at approximately 1 cm from the intersection point of a line running between the thighs and the midline—with 300 μg of HSE adsorbed to alum adjuvant in a volume of 200 μL. Mice in the IP control group (IP.PBS, n=10) received IP injections of PBS adsorbed to alum adjuvant (200 μL). Sensitization injections were administered at weekly intervals for 3 weeks. To induce asthma, beginning on day 21, sensitized and control mice were placed in a plastic box and challenged for 30 min daily via the airways with aerosolized 1% HSE or an equal volume of saline (control group) using a jet nebulizer (BARI Co., Ltd., Germany), for 7 consecutive days. Airway hyperresponsiveness (AHR) to methacholine (Mch, Sigma-Aldrich, St. Louis, MO, USA) was measured 24 h after the last challenge in all animals. On day 29, mice were anesthetized with 2% sodium pentobarbital; sera were then obtained for measurement of specific IgE levels and of IgE profiles binding to HSE. Additionally, bronchoalveolar lavage fluid (BALF) was prepared from the right lung for inflammatory cell enumeration and cytokine quantitation. Paraffin sections from the left lung were used for histological assessment. The spleen was homogenized for cytokine measurements.