Fig. 2
S1PR1 antagonists attenuate naloxone-precipitated withdrawal and associated neuroinflammation in mice. Naloxone-precipitated withdrawal behaviors in mice treated with morphine and naloxone (Mor+naloxone; a: n = 4, b: n = 10-11) were attenuated in mice administered oral NIBR-15 (Mor+naloxone +NIBR-15; 3 mg/kg/day; n = 4; a) or FTY720 (Mor+naloxone+FTY720; 0.1 mg/kg/day; n = 12; b). Jumping [a: t(6.0) = 4.1, p = 0.0060, dadj = 1.2; b: t(20) = 2.5, p = 0.023, dadj = 0.85]; front paw shaking [a: t(5.9) = 3.5, p = 0.014, dadj = 1.0; b: t(19) = 3.0, p = 0.0070, dadj = 1.0]; and hunching [a: t(4.8) = 3.2, p = 0.024, dadj = 0.93; b: t(17) = 3.8, p = 0.0014, dadj = 1.3]. Naloxone-precipitated withdrawal was associated with increased levels of IL-1β (c; n = 5), GFAP (d; n = 6), and CD11b (e; n = 6) in mice spinal cords (Mor+Nal) compared to mice treated with morphine alone (Mor; c: n = 7, d: n = 4, e: n = 7). Co-administration with FTY720 blocked these events (Mor+Nal + FTY720; 0.1 mg/kg/day; n = 3; c-e). In b, one animal in the Mor+naloxone group was excluded from the jumping data as a high outlier (Grubb’s test). Images have been cropped and adjustments to brightness and contrast were performed across the blots for clarity by Image J [29]. Results are expressed as mean ± SEM and analyzed by (a-b) two-tailed unpaired Welch’s corrected t test. *P < 0.05 vs. Mor+naloxone or (c-e) two-tailed, one-way ANOVA with Dunnett’s comparisons. [Treatment: (c) F(2, 13) = 14, p = 0.00063, η2 = 0.68; (d) F(2, 13) = 14, p = 0.00060, η2 = 0.68; (e) F(2, 10) = 9.5, p = 0.0049, η2 = 0.65].*p < 0.05 vs. Mor and †p < 0.05 vs. Mor+Nal