A new hypertonic saline assay for analgesic screening in mice: effects of animal strain, sex, and diurnal phase

Purpose There exists a pressing need for the identification of novel analgesics. We recently reported on a new preclinical assay for rapid analgesic screening based on intraplantar (i.pl.) injection of 10% hypertonic saline (HS) in female outbred (CD-1) mice. Herein, we characterized the HS assay’s performance in inbred (C57BL/6) mice, sensitivity to sex differences, and effects of diurnal rhythm phase. Methods In randomized, controlled, blinded in vivo animal experiments, we studied nociceptive responses induced by i.pl. HS in C57BL/6 ( vs CD-1) mice of both sexes ( n = 240) and determined diurnal rhythm phase effects in female animals. We established the HS assay’s sensitivity to morphine by constructing dose-response curves and calculating half-maximal inhibitory doses (ID 50 s). Results The injection of i.pl. HS produced nociceptive (licking and biting) responses in all C57BL/6 mice tested. In both C57BL/6 and CD-1 mice, the mean (95% confidence interval [CI]) response magnitudes were greater in females vs males (C57BL/6: 87 sec [64 to 110] vs 45 sec [29 to 61]; difference in means, 42 sec; 95% CI, 17 to 68; P < 0.001; n = 10/group; CD-1: 110 sec [95 to 126] vs 53 sec [32 to 74]; difference in means, 57 sec; 95% CI, 34 to 79; P < 0.001; n = 10/group). The mean (95% CI) nociceptive responses were greater at 24:00 hr than at 12:00 hr in C57BL/6 mice (64 sec [40 to 88] vs 37 sec [24 to 51]; difference in means, 27 sec; 95% CI, 7 to 47; P = 0.007; n = 10/group), but not in CD-1 mice ( P = 0.97). Intravenous morphine dose-dependently attenuated nociceptive responses of both C57BL/6 and CD-1 mice (ID 50 , 0.6 and 2.5 mg·kg −1 , respectively; P = 0.41). Conclusion These findings in inbred and outbred mice solidify the utility of the HS assay as an effective, rapid, robust, and versatile preclinical tool for analgesic screening.