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. 2018 Jul 9;8:10341. doi: 10.1038/s41598-018-28754-7

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Effects of combining alfaxalone with propofol, or with diazepam on mouse behavior. (A) A 30-min activity test was conducted immediately following i.p. injections of normal saline (20 Units), propofol (50 mg/kg), alfaxalone (30 mg/kg), or the combination of propofol (50 mg/kg) + alfaxalone (30 mg/kg), to determine drug effects on time at rest (absence of movement) as a function of post-injection time (5-min blocks) across the test session (means ± S.E.M.; n = 11–12 mice per treatment group). A repeated measures (rm) ANOVA revealed a significant drug effect (**p = 0.0005) and drug × time interaction (***p < 0.00005), with the propofol + alfaxalone mice exhibiting significantly increased time at rest compared to the single drug/saline control groups on average across the test session. Significant between-groups comparisons are shown for these contrasts: saline vs. propofol + alfaxalone at 5, 10, 15 (*p < 0.003), and 20 (*p = 0.031) min post-injection; alfaxalone vs. propofol + alfaxalone at 5, 10 (^†p < 0.002), and 20 (^†p = 0.038) min; and propofol vs. propofol + alfaxalone at 5, 10 (^#p < 0.0002), and 15 and 20 (^#p < 0.040) min. No significant differences were observed for the contrasts involving the saline vs. propofol or saline vs. alfaxalone groups. (B) A separate study was conducted on an independent cohort of naïve mice to examine the potential sedating drug effects following i.p. injections of normal saline (20 Units), diazepam (4 mg/kg), alfaxalone (30 mg/kg), or the combination of diazepam (4 mg/kg) + alfaxalone (30 mg/kg) on time at rest (n = 10 for each group). An rmANOVA yielded a significant drug effect (**p < 0.00005) and drug × time interaction (***p = 0.0004), with the diazepam + alfaxalone treated mice having significantly greater times at rest compared to the saline, alfaxalone, and diazepam groups on average across the session. Between-groups comparisons conducted within these contrasts showed robust differences between each of the single drug/saline control groups relative to the diazepam + alfaxalone treated mice for every post-injection time (5–30 min) interval (p < 0.0002; for the comparisons involving saline (*), alfaxalone (†), or diazepam (#), respectively). The diazepam group also had significantly increased rest times relative to the saline control mice, with pair-wise comparisons revealing significant differences at 10, 15, 20, and 25-min post-injection (^††p < 0.003).