Remimazolam- and dexmedetomidine-based three-drug mixtures refine injectable anesthesia in mice

The medetomidine-midazolam-butorphanol mixture (MMB) is widely used for injectable anesthesia in mice but is associated with delayed induction, prolonged recovery, impaired oxygenation, and post-anesthetic hypothermia. We investigated whether substituting midazolam with remimazolam, alone or together with replacing medetomidine with dexmedetomidine, improved the anesthetic profile. Male C57BL/6JJcl mice (n=8/group) received intraperitoneal MMB, medetomidine-remimazolam-butorphanol (MRB), or dexmedetomidine-remimazolam-butorphanol (dMRB). Induction was assessed by times to immobilization and loss of the righting reflex (LORR), anesthetic depth by a study-specific 0-5 reflex-loss score, and recovery by time to return of the righting reflex (RORR). Oxygen saturation, heart rate, respiratory rate, and rectal temperature were monitored. Compared with MMB, MRB shortened times to immobilization, LORR, and RORR, whereas dMRB shortened LORR. The reflex-loss score showed no overall group effect or area under the curve (AUC) difference, although a significant interaction between group and time indicated different temporal profiles. No overall group main effects were detected for the physiological variables during anesthesia; however, measured SpO₂ was markedly low in all groups (pooled mean, 61.7 ± 6.6%). During recovery without external heating, rectal temperature differed among groups: the dMRB-MMB AUC comparison was significant, MRB-MMB was borderline, and MRB-dMRB was not significant. MRB improved induction and recovery timing, whereas dMRB improved induction and showed the strongest evidence of better recovery-phase thermoregulation; neither modification corrected the low measured SpO₂.