4.2.3. Environmental Impacts

If the grimace scales are to be used as a practical tool they need to be repeatable across time and conditions, and not subject to extraneous influences. This requirement also relates to their face validity as reliable indicators of pain. In common with the other factors that may influence the scales, there has been limited research in this sphere.

*Animals* **2020**, *10*, 1838

Miller and Leach in 2015 [58] performed a comprehensive evaluation of some of the factors that might be predicted to influence grimace responses. One factor that may have an impact is the circadian cycle and whether di fferences in score occur across the day. For both live scoring and retrospective scoring, there were largely no di fferences seen between scores dependent on whether scoring took place in the morning, lunchtime or at the end of the day. There were some exceptions to this with BALB/c mice showing a greater live MGS score at noon compared to am and C57BL/6 mice showing higher retrospective MGS scores in both afternoon time points in comparison to the morning. It should be noted that in this, as in the majority of the studies examining grimace scores, animals were scored during the light phase of the circadian cycle when they would be expected to be inactive. There is some evidence that grimace scores may not be comparable between dark and light conditions with the finding that MGS was higher in the dark than in bright light in CD1 mice treated with a peptide believed to induce pain and migraine symptoms [83]. Analysis of the action units showed that the transition to light caused a significant decrease in orbital tightening and nose bulge [83]. Given that this finding was also observed in vehicle controls it appears unrelated to the migraine symptoms, and may be an aspect of normal biology needing consideration. Alternately, Matsumiya et al. 2012 found no di fference in baseline MGS scores between morning and evening but did find that in operated animals scores were higher in the dark cycle, implying that pain was greater in the mice active phase [84]. However, in consideration of the use of the scales as a practical tool the reality is that most scoring will occur during the working day, in the light cycle, and therefore the findings of Miller and Leach in 2015 [58] provide confidence that time of scoring should not influence the score. A further important outcome from Miller and Leach [58] was that there was no effect of repeatedly being placed in the photography boxes on grimace score i.e., a habituation e ffect over the three occasions used [58]. The later study by Jirkof et al. [85] in 2020 supports this finding. This provides assurance that longitudinal monitoring post-procedure could occur throughout the day without the need to account for time of day or habituation to the box. However, as discussed earlier the need to remove animals to a separate box does impede the practical application of the test. Further study should consider time of day e ffects in the non-stimulated home environment.

There is further evidence of an impact of the external environment on grimace scores. Sorge et al. [59] compared grimace responses of mice and rats recorded after a painful insult, in the presence of a male compared to a female. Significant decreases in grimace response were recorded compared to the situation with no observer in the room. Females did not induce such a change. The findings therefore sugges<sup>t</sup> that olfactory cues from human males lead to a physiological stress response, and associated stress-induced analgesia.

There is also some evidence of inter-laboratory variation in the outputs obtained from behavioral testing, to include MGS scores. In a multicenter study, Jirkof et al. [85] in 2020 demonstrated some quantitative di fferences in scores, although they were qualitatively comparable (direction of e ffect). However, variability between research centers in the MGS, especially when presented as a median score, was less pronounced than in burrowing behavior readouts [84]. This inter-lab variability was recognized across the spectrum of preclinical research pursuits, arising as a result of environmental variables leading to stress [86]. While this issue may be a concern when considering basic-to-clinical translation and reproducibility, it is less likely to be of concern for clinical application of the grimace scales. As a clinical tool, provided good inter and intra-observer, and thus intra-site agreemen<sup>t</sup> is obtained, grimace scores may be relied on locally for welfare determination subject to some of the other caveats discussed in this paper.
