ABSTRACT: Male (age 10-16 weeks) C57BL/6 (wild-type) (Charles River Laboratories), α7 nicotinic receptor deficient (C57BL/6 background, B6.129S7-Chrna7tm1Bay/J, Stock No. 003232 from The Jackson Laboratory) and Alox15-deficient (C57BL/6 background, B6.129S2-Alox15tm1Fun/J, Stock No. 002778 from The Jackson Laboratory) mice were used. After 1 hour of recovery from vagus nerve stimulation or sham surgery, 0.1 mg zymosan (Sigma-Aldrich, MO, USA) was administered intraperitoneally. Mice were euthanized by carbon dioxide asphyxiation at 4, 12, 24, and 48 hours after zymosan challenge. Peritoneal exudates were collected by injecting 4 mL of PBS intraperitoneally, shaking the abdomen, and recovering approximately 3 mL of exudate.
Samples were extracted and lipid mediators were identified and quantified. In brief, 2 volumes of ice-cold methanol containing deuterated internal standards (d8-5S-HETE, d4-LTB4, d5-LXA4, d4-PGE2, and d5-RvD2, d5-MaR1, d5-MaR2, d5-RvD3, d5-RvE1 and d5-17R-RvD1) representing each chromatographic region of identified LM were added to inflammatory peritoneal exudates. Following protein precipitation (-20°C for a minimum of 45 min), supernatants were extracted on an ExtraHera instrument (Biotage, Uppsala, Sweden) using solid-phase extraction with Isolute C18 500 mg columns (Biotage Uppsala, Sweden). Methyl formate fractions were collected, brought to dryness and resuspended in phase (methanol/water, 1:1, vol/vol) for injection on a Shimadzu LC-20AD HPLC and a Shimadzu SIL-20AC autoinjector, paired with a QTrap 6500+ (Sciex, Stockholm, Sweden). An Agilent Poroshell 120 EC-C18 column (100 mm × 4.6 mm × 2.7 μm) was kept at 50°C and mediators eluted using a mobile phase consisting of methanol/water/acetic acid of 20:80:0.01 (vol/vol/vol) that was ramped to 50:50:0.01 (vol/vol/vol) over 0.5 min and then to 80:20:0.01 (vol/vol/vol) from 2 min to 11 min, maintained till 14.5 min and then rapidly ramped to 98:2:0.01 (vol/vol/vol) for the next 0.1 min. This was subsequently maintained at 98:2:0.01 (vol/vol/vol) for 5.4 min, and the flow rate was maintained at 0.5 ml/min. QTrap 6500+ was operated in negative mode using a multiple reaction monitoring method. Each lipid mediator was identified and quantified using the following criteria: 1) presence of a peak at a retention time corresponding with the relevant standard (2) ≥ 8 data points and (3) signal-to-noise ratio (S/N) >5 (25,26). Calibration curves were obtained for each using synthetic compound mixtures at 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100, and 200 pg that gave linear calibration curves with an r2 values of 0.98–0.99.
