HbSS mice have constitutively reduced spinal PEA compared with HbAA control mice. (A) HbSS mice exhibited a constitutive reduction in spinal cord PEA, regardless of sex, and female HbSS mice showed reduced OEA compared with HbAA mice, measured with liquid chromatography tandem mass spectrometry. (B) Male HbSS mice had significantly reduced spinal 2-LG compared with HbAA mice. (C-D) Targeting the PEA degradative enzyme, NAAA, with ARN (IP) dose-dependently reduced mechanical and cold hyperalgesia in the 72-hour period after a single treatment in male HbSS mice compared with baseline (BL) recordings and vehicle treatment. Mean ± standard deviation (SD). In panels A-B, data were analyzed with an unpaired Student 2-tailed t test; and in panels C-D, data were analyzed with repeated measures 2-way analysis of variance (ANOVA) and the Tukey post hoc multiple comparisons test. ∗Indicates a difference compared with time-matched vehicle; and †indicates a difference compared with respective BL. ∗,†P < .05; ∗∗,††P < .01; ∗∗∗,†††P < .001; ∗∗∗∗,††††P < .0001. Age: 3.5 to 5.0 months. Panels A-B: male HbAA, n = 7; male HbSS, n = 11; female HbAA, n = 6; female HbSS, n = 6; panels C-D: ARN, HbSS vehicle, n = 9; HbSS 3 mg/kg, n = 4; HbSS 10 mg/kg, n = 5; HbSS 30 mg/kg, n = 5; HbAA vehicle, n = 5; HbAA 30 mg/kg, n = 6. 2-DG, 2-docosahexaenoyl-sn-glycerol; 2-LG, 2-linoleoyl-sn-glycerol; 2-OG, 2-oleoyl-sn-glycerol; BW, body weight; MAG, monoacylglycerols; PWF, paw withdrawal frequency; VF, von Frey.