Sleeping through anything: The effects of unpredictable disruptions on mouse sleep, healing, and affect

Abstract

Many aspects of the laboratory environment are not tailored to the needs of rodents, which may cause stress. Unpredictable stressors can cause ulcers, prolonged pituitary-adrenal activation, and anhedonia. Similarly, pain has been demonstrated to slow wound healing, and mice experiencing pain exhibit altered behavior. However it is unknown how husbandry, which occurs when the mice are inactive, and lack of analgesia, specifically in a punch biopsy procedure, effects animal physiology, behavior, and welfare, particularly as it relates to sleep fragmentation. We hypothesized that sleep fragmentation, induced by unpredictable husbandry and lack of pain management will slow wound healing. Two main treatments were tested in a factorial design in C57BL/6 mice of both sexes (64 mice total); 1) analgesia (carprofen and saline) and 2) sleep disruptions (random and predictable). Mice were singly housed in a non-invasive sleep monitoring apparatus on arrival (Day -4). Disruption treatments were applied from Day -3 to 2. All mice received a punch biopsy surgery (Day 0) with topical lidocaine gel and their analgesic treatment prior to recovery, and on Days 1 and 2. Nesting behavior was assessed daily and a sugar cereal consumption test, as a measure of anhedonia, was conducted on Days -1 to 2. On Day 3, mice were euthanized and wound tissue and adrenal glands were collected. We found that the disruption predictability had no effect on mouse sleep, wound healing, or adrenal cortex:medulla ratio. It's possible that the disruption period was not long enough to induce chronic stress. However, male mice who received analgesia slept more than their female counterparts; this may be related to sex differences in pain perception. Overall, it does not appear that the predictability of disturbance effects sleep fragmentation or stress responses, indicating that husbandry activities do not need to occur at set predictable times to improve welfare.

Fig 1. Sleep apparatus viewed from above (A) and a close up side view of an individual mouse cage (B).

Sugary cereal used for the sucrose preference test can be seen in B.

Fig 2. Experimental timeline.

Lists all measurements made on each day of experiment. Day -1 is considered baseline. Mice arrive on Day -4. Abbreviations: BWT—bodyweight; Sleep—sleep monitoring; TINT—Time to Integrate Nesting Material Test; Food—food consumption; Sucrose—sucrose consumption; Analgesia—analgesia treatment.

Fig 3. Average percentage of time spent sleeping by light phase and day of experiment.

Different letters indicate significant (Tukey, P < 0.05) differences within categories. Data presented are LSM and SE.

Fig 4. Average sleep bout length by lights on/off and day of experiment.

Different letters indicate significant (Tukey, P < 0.05) differences within categories, bars indicate differences between categories. Data presented are LSM and SE.

Fig 5. Mean activity level by light phase and day of experiment.

Different letters indicate significant differences within categories, bars with asterisks indicate differences between categories (Tukey, P < 0.05). Data were square root transformed for analysis; y-axis is back-transformed. Activity level is a linear measurement from 0 to 3; higher values indicate higher levels of activity. Data presented are LSM and SE.

Fig 6. Activity level by day of experiment, sex, and analgesia treatment.

Bars with asterisks indicate differences between categories (Tukey <0.05). Data were square root transformed for analysis; y-axis is back-transformed. Activity level is a linear measurement from 0 to 3; higher values indicate higher levels of activity. Data presented are LSM and SE.