Tell-tale TINT: Does the Time to Incorporate into Nest Test Evaluate Postsurgical Pain or Welfare in Mice?

Abstract

Identifying early indicators of distress in mice is difficult using either periodic monitoring or current technology. Likewise, poor pain identification remains a barrier to providing appropriate pain relief in many mouse models. The Time to Incorporate to Nest Test (TINT), a binary measure of the presence or absence of nesting behavior, was developed as a species-specific method of identifying moderate to severe distress and pain in mice. The current study was designed to evaluate alterations in nesting behavior after routine surgery and to validate the TINT's ability to measure pain-related behavioral changes. CD1 mice undergoing carotid artery catheterization as part of a commercial surgical cohort were randomly assigned various nesting, surgery, and analgesia conditions. To provide context for the TINT outcomes, we measured other variables affected by pain, such as weight loss, food consumption, and scores derived from the Mouse Grimace Scale (MGS). Mice that had surgery were more likely to have a negative TINT score as compared with controls. All mice were more likely to fail the TINT after receiving postoperative buprenorphine, suggesting that buprenorphine may have contributed to the failures. The TINT, MGS live scoring, and scoring MGS images all loaded strongly on a single component in a principal component analysis, indicating strong convergent validity between these measures. These data indicate that the TINT can provide a quick, objective indicator of altered welfare in mice, with the potential for a wide range of uses.

Figure 1.

Timeline of experimental design. The baseline time point represents Day -2 and Day -1, or the 2 d prior to surgery or anesthesia alone. On Day 0 either surgery or anesthesia only was performed, with measurements being taken at the Post-Sx time point. On Day 1 measurements were taken before (Pre-Rescue) and after (Post-Rescue) buprenorphine administration. The recovery time point represents Days 2–4, following surgery or anesthesia only. NQS: nest scores; MGS: both live and video recorded mouse grimace scale; TINT: Time to integrate into nest test; BWT: mouse body weight; FWT: food weight.

Figure 2.

Effect of the main effect of nesting treatment on TINT performance. The probability of failing the TINT is represented on the y-axis on a back transformed log₁₀ scale. Essentially the data graphed are the log₁₀ LSM ± SE values from the analysis but only the tick marks on the graph have been back transformed to depict values in the real world. Asterisks indicate the significant effect (P < 0.05).

Figure 3.

Effect of experimental time point and surgical condition on performance on the TINT. The probability of failing the TINT is represented on the y-axis on a back transformed log₁₀ scale. Asterisks indicate the significant difference between –Sx and +Sx groups at the Post-Sx and Pre-Rescue time points (P < 0.05). Baseline data is from days -1 and -2 while data from the recovery time point includes days 2, 3, and 4. Data is presented as LSM ± SE.

Figure 4.

Effect of surgical condition on nest quality score. Mice who experienced anesthesia alone (-Sx) had significantly higher NQSs than those mice who received surgery (+Sx). Asterisk indicates the significant effect (P < 0.05). Data is presented as LSM ± SE.

Figure 5.

Effect of time point and surgical condition on change in mouse body weight from the previous day. Different letters over the bars indicate significant differences between pairwise Tukey tests (P < 0.05), thus if 2 letters are the same there is no statistical difference between weight change LSM values. Baseline data is from days -1 and -2 while recovery data includes days 2, 3, and 4. Data is presented as LSM ± SE.