F5881

Mouse models were induced using modified protocols as previously reported [7 (link)–9 ]. Mice were randomly grouped and sensitized on day 1 and day 8. For the OVA/Alum group of asthma, 25 µg OVA (A5503, Sigma Aldrich, St. Louis, MO, USA) dissolved in 100 µl 0.9% saline was mixed 1:1 with Imject Alum (Pierce, Rockford, IL, USA) by intraperitoneal (IP) injection. For the OVA/CFA group of asthma, 25 µg OVA dissolved in 100 µl 0.9% saline was mixed 1:1 with CFA (F5881, Sigma Aldrich, St. Louis, MO, USA) by IP. injection. For the OVA/LPS group of asthma, mice were lightly anesthetized with isoflurane, and intratracheally injected using a combination of 25 µg OVA with 0.1 or 10 µg LPS (L2630, Sigma Aldrich, St. Louis, MO, USA) in a total volume of 40 µl, with 0.9% saline as the diluent. For the OVA/CFA/0.1 LPS group, the model of sensitization is a combination of the OVA/CFA group and OVA/LPS group. After sensitization, the above-mentioned groups were challenged with aerosolized 1% OVA for 30 min on days 15-17. For the OVA/CFA/0.1 LPS + DNase I group or the OVA/CFA/0.1 LPS + CI-amidine group, on the basis of the OVA/CFA/0.1 LPS group, intravenous injection of DNase I (5 mg per kg body weight) or intraperitoneal injection of Cl-amidine (10 mg per kg body weight) was performed 1 h before each challenge. Mice sensitized and challenged with 0.9% saline were used as controls.

Inflammation and pain were induced over a 48-h period by intraplantar injection of CFA (10 μl, F5881, Sigma-Aldrich) into the left hind paw. Control mice were injected with 10 μl saline. The effects of incarvillateine on hyperalgesia were determined at different times after administration by measuring the thermal sensitivity. To determine the effects of incarvillateine on inflammation, the ventral-dorsal paw thickness was measured with a digital caliper before CFA injection and 30 min after incarvillateine administration, and paw edema rate was calculated as a percentage of change from baseline paw thickness. Mice were then sacrificed, and the injected paws were cut and homogenized in 0.01 M PBS. After centrifugation at 10000 g/min for 5 min, 100 μl supernatant was used for the determination of IL-1β levels by enzyme-linked immunosorbent assay (ELISA).

The KLH–peptide complex antigens were dissolved in saline at 2 mg/ml and filtered through 0.22-μm sterile filters. The antigens (0.5 ml) were emulsified with an equal volume of Freund’s complete adjuvant (F-5881, Sigma, Darmstadt, Germany) and injected intradermally at multiple sites along the backs of male New Zealand white rabbits. Two rabbits were injected with one kind of antigen. Booster doses were made in Freund’s incomplete adjuvant (F-5506, Sigma, Darmstadt, Germany) at 2-week intervals with 500-μg doses of the same antigen, totally two times with intervals of 2 weeks. Rabbit serum was collected 14 days after the second booster injection. Antibody titers were detected by indirect ELISA using the respective antigen as coating antigen and the pre-immunization serum of each rabbit as the negative control.

Protein antigens were prepared in PBS or in PBS-A at 1 mg/mL, and the volume corresponding to the desired amount of protein was increased to an injectable volume with PBS or PBS-A. This volume was then mixed 1:1 (v/v) with either Freund’s adjuvant, complete (F5881, Sigma-Aldrich), or Sigma adjuvant system (S6322, Sigma-Aldrich). For viscous adjuvants, the solution was mixed by repeated passage through a syringe until a smooth emulsion was formed (over approximately 30 min on ice). Injections were performed on 6-week-old male and female Jcl:Wistar rats (Clea, Tokyo, Japan) using a 1 mL glass syringe and a 27-gauge needle. Prime and boost injections of 250 µg protein were injected intraperitoneally every two weeks. Final boosts of 250 µg of protein were delivered intravenously without adjuvant via the tail vein. Volumes varied depending on the injection route and experimental requirements and were in accordance with the relevant JP Home Office animal license for the procedure. The experimental protocol was approved by the Committee of the Institute for Animal Experimentation of Tottori University (17-Y-28).

An injury was induced by injection using an insulin needle (0.3 ml BD Micro-Fine) of 50 μl undiluted Complete Freund’s Adjuvant (CFA; F5881, Sigma) unilaterally in the intraplantar surface of the right hind paw, leaving the contralateral left hind paw as an internal control of the pain threshold of the animal. Intraplantar, as well as intrathecal injections were performed while the animal was under isoflurane anesthesia (2%) for maximum of 60 s.