Am1241

TMX (Sigma T5648) was dissolved into corn oil and was injected intraperitoneally (i.p.) once every other day, a total of five times, 2 mg/day (10 mg total dose) (Feil et al., 2009 (link); Anastassiadis et al., 2010 (link)). Subsequent verification and CCI modeling were performed 14 days after the final injection.
AM1241 (Sigma A6478) is a CB2 agonist, and AM630 (Sigma SML0327) is a highly specific CB2 antagonist. The drug was dissolved in dimethyl sulfoxide (DMSO), Tween 80, and normal saline (the ratio is 1:2:7). To determine the relationship between neuropathic pain and CB2, 36 WT mice were randomly divided into WT CCI + vehicle (DMSO:Tween 80:normal saline = 1:2:7), WT CCI + 0.1, 1, 3 mg/kg AM1241 (Sigma A6478; Wu et al., 2021 (link); Lozano-Ondoua et al., 2010 (link); Ma et al., 2021 (link)) groups, and WT CCI + 1, 3 mg/kg AM630 (Sigma SML0327; Malan et al., 2001 (link); Buffon et al., 2020 (link); Sultana et al., 2021 (link)) groups (n = 6 at each group). Fifteen CB1cKO mice were randomly divided into CB1cKO CCI + vehicle and CB1cKO CCI + 3, 5 mg/kg AM630 (n = 5 at each group). AM1241/AM630 or its vehicle (0.1 ml) was injected i.p. daily from 12th to 17th day after CCI surgery.

PEG-DTT hydrogels were prepared by the Michael addition reaction using poly(ethylene glycol) diacrylate (PEGDA) (molecular weight: 700, Sigma-Aldrich, United States) and DTT (Sigma-Aldrich, United States) over the sodium tetraborate catalyst. AM1241 (0, 1, 2.5, 5, 10, 20, 40, 80, 120, and 200 µM) (Sigma-Aldrich, United States) was then immediately added to this solution and mixed to prepare a finished AM1241-loaded PEG-DTT hydrogel. First, 100 mg of PEGDA (0.14 mM) and 22 mg of DTT (0.14 mM) were dissolved in 0.5 ml of water. Then, 0.5 ml of 0.1 mol/L borax (Sigma-Aldrich, United States) solution was added to the mixture and stirred vigorously for 10 s. Keep the solution at room temperature (25°C) for a while and check the gelation time by tube inversion.