Recently, PRIM&R hosted a webinar titled Identifying and Preventing Distress in Laboratory Animals, which was presented by Mollie A. Bloomsmith, PhD, and Eric Hutchinson, DVM, DACLAM.

Stress and distress can negatively impact the welfare of laboratory animals and have adverse consequences for research. Minimizing distress in animals is mandated by federal regulations and remains an ethical obligation for research and compliance staff. To take measures to minimize distress, it is essential to understand its causes and manifestations among the various species of laboratory animals.

This webinar provided strategies for detecting signs of distress, identifying research procedures likely to lead to distress, and understanding how to prevent or minimize distress in laboratory animals.

After the webinar, Drs. Bloomsmith and Hutchinson answered some of the questions from webinar attendees that time didn’t allow us to answer live. We’re pleased to share those answers with the readers of Ampersand. If you’re interested in listening to the entire webinar, you are able to purchase the on-demand version.

Q: How does stress cause dermatitis?
Eric Hutchinson (EH): Ulcerative dermatitis (UD) has two important features that are relevant to this discussion of its relationship to anxiety and stress. First, UD is a pathological description of the end-result of a disorder that has a necessary (or close to it) and sufficient behavioral component. That is to say, UD is how we describe the skin lesions that result from excessive scratching, and excessive scratching by itself is enough to cause ulcerative dermatitis. So when we talk about the many known or suspected causes of UD we are essentially discussing the causes of this scratching. The association between anxiety and itching or scratching is well established in primates (including the upright kind), conventionally accepted in dogs and cats, and an area of ongoing study in mice, with Amano et al. (2008)¹ demonstrating that psychological distress alone is enough to establish UD in some mice, and several others in the literature showing that treatment with anti-anxiety medications can ameliorate or prevent UD. Second, UD (and the scratching that causes it) can become self-perpetuating because of the positive feedback of the itch/scratch cycle, the tissue damage that often results, and the bacterial invasion that often results from that. Stress is certainly not the only or even most common cause of itch/scratch in mice, with allergens and parasites being other leading contenders, but as discussed above, any initial trigger of the itch/scratch cycle is enough to lead to the complicated set of lesions we recognize as UD. 

Also, it should be noted that genetic strain predilections for UD or any other disorder do not rule out its being caused by psychological distress.

Q: What would be the best way to treat ulcerative dermatitis without causing more stress?
EH: As you may suspect, the first answer is to do what you can to prevent UD. Elimination of dermal parasites, selection of strains that are less prone to the disorder, and good stewardship of psychological well-being are all possible ways to reduce the incidence of UD.

Once UD is established in an animal, however, treatment for the secondary problems that arise is often necessary in order to clear up the lesion. Treatments to promote tissue healing and treat microbial infection are common, but are typically applied topically and thus may perpetuate anxiety-related itch/scratch. If logistically feasible, though, there are treatments that can be added to feed to accomplish either of these goals, including Vitamin E as reported in Lawson et al. (2005)², or any of the multitude of commercially available antimicrobial mouse feeds. Administration in the feed would reduce handling, which would reduce anxiety. Another potential treatment is to trim the animals’ toenails, which may interrupt the progression from itch/scratch to the self-perpetuating tissue damage and bacterial infection. This requires handling the mice, but much less frequently than for topical ointment application. 

It is interesting to speculate whether the notorious failure rate of UD treatments may partially be explained by the stress caused by the application of topical treatment. Still, the trade-off between treatment and anxiety pretty clearly favors treating, even if it’s necessary to handle animals frequently to do so, since untreated UD is highly likely to progress

Q: How often do you recommend rodent cage changes to prevent stress?
EH: It is impossible to have a prescription that could be applied to every situation. Many factors should and do influence the decision of how often to change cages, included caging type (e.g., ventilated or static), disease status (e.g., pinworm infection) and even disease model (e.g., diabetic or other polyuric models), and stress should be considered as one of those factors. Regardless, cages need to be changed eventually, so facilities should consider ways to mitigate the stress caused by this process (such as by transfer of nesting material) and limit its consequences (such as by delaying cage change for new litters). ^{3, 4}

Q: How does social buffering apply to surgical procedures?
Mollie Bloomsmith (MB): The concept of social buffering is that having a compatible partner ameliorates (or buffers) distress when animals experience a stressful life event. As Gilbert & Baker (2010)⁵ demonstrated, rhesus macaques exposed to the restraint/anesthesia of other monkeys, showed reduced abnormal behavior when they were pair-housed than when they were housed singly. With regard to a surgical procedure, animals may benefit from having a compatible partner nearby when they are recovering from a surgical procedure; in some cases this may require housing that only allows visual contact between the partners.

Q: What type of nesting material do you suggest?
EH: Any kind of material out of which the mice can build a true nest structure, with a domed ceiling that doesn’t collapse with each exit and entry. For a specific example, see Hess SE, Rohr S, Dufour B, Gaskill BN, Pajor EA, Garner JP. 2008. Home improvement: C57BL/6J mice given more naturalistic nesting materials build better nests. J Am Assoc Lab Anim Sci 47: 25–31

Q: How do you balance the potential risk for injury with social housing via contact panels or full access? Similarly, how do you weigh the distress induced by repeated separation of social partners, including mothers and infants, with the potential benefit of social housing?
MB: When primates are housed socially with full access to one another, there is a higher risk of injury than when they are housed with limited social access via perforated panels. However, we also know from recent studies that they benefit more from full social access than when they only have access through panels. For example, rhesus macaques who are fully paired show less abnormal behavior, less tension-related behavior, more affiliation, and more activity than when those same monkeys were housed with protected contact panels (Baker et al, 2012a; 2014)⁶. In addition, the risk of wounding may be less than some believe. For example, in one report on over 1,000 introductions of unfamiliar macaques, only 6% of the introductions had some type of wound in the first two weeks, and the majority of those wounds were minor; only 4% of the pairs were later separated permanently for social incompatibility (Truelove et al, in press)⁷. So when balancing effects on the monkeys, it seems that at least for rhesus macaques, strong behavioral benefits outweigh the relatively low risk of injury. However, it seems that female cynomolgus macaques show a different response–their behavior in full contact and protected contact is quite similar (Baker et al, 2012b)⁸, so there may be significant species differences when weighing the pros and cons of housing options.

Even though we know that social separations are distressing for nonhuman primates, it does seem that they can adjust to repeated separations, at least in some circumstances. A study comparing intermittent pair housing (rhesus monkeys were separated for two 24‐hour periods per week) found that this frequent separation of pairs did not detract from the behavioral benefits of pair housing (Baker et al, 2014)⁹. I would expect that frequent separations between mothers and infants would be more difficult, so if this was to be attempted, I would suggest close monitoring for both mothers and infants.

Q: Can you shape rats to be handled (before a procedure)?
EH: Yes! Rats are highly trainable and are amenable to positive human contact, especially when handled frequently and at a young age in a positive manner. I really like this guide by Kenneth McCarson, but there are many other good how-tos available on the internet (often by pet rat enthusiasts).

Q: What are your criteria for intervention or humane euthanasia for animals experiencing self-induced physical harm (ex: as illustrated by your mouse with extensive necrotic skin lesions)? Is it humane to not intervene?
MB: I think each primate that has expressed self-injurious behavior should be treated. These treatments might include providing a social companion, additional enrichment, additional cage space, using positive reinforcement training to address fear or to reduce distress, altering research or management procedures, and/or administering psychoactive drugs. If treatments are not effective, then euthanasia should be considered. Since self-injurious behavior can be very difficult to successfully treat, and since it is so concerning, it is common to continue treatments and behavioral monitoring for the remainder of the primate’s lifetime. Each institution should determine their own guidelines for treatments of self-injurious behavior, and for the timeline for improvement. It is useful for many of those working with a self-injurious primate (animal care staff, behavior staff, veterinary staff, research staff) to work closely together when trying to address this complex behavioral problem.

I agree for mice and other animals as well – self-induced harm necessitates intervention, though that may take many forms. Specifically for the example of UD in the mice, I think early treatment and progress tracking is key; if you are treating UD aggressively and it is still progressing, humane endpoints shouldn’t wait until there is more lesion than mouse.

Q: Is it less stressful to house rodents singly before surgery to condition them to be single housed after surgery? Or keeping them group housed upon arrival until surgery date?
EH: Rodents likely benefit from social buffering just as primates and dogs do, so first I would be sure that the proposed surgery actually requires single housing afterward. Assuming that review has been done, the question as posed has yet to be examined, to my knowledge. Undoubtedly, the separation from one’s social partner is a stressor, as is surgery, so whether it is better to have both of those imposed at once or in a staggered manner is certainly a valid question. However, even if it turns out that the staggered version causes more transient or lesser-in-degree stress than the all-at-once version, I would caution that social isolation is known to have cumulative effects in many animals (i.e. more days spent alone leads to more disturbance in the long-term). So if the choice is between housing a rat together or alone in advance of a surgery that is in the distant or indeterminate future, I would side on keeping them together as long as possible until the literature says otherwise.

Q: What is the best age to wean rats to avoid separation anxiety?
EH: In the wild, rats are weaned at between 4 and 5 weeks of age (vs. the typical 3 weeks in the laboratory). If normal social development is the only concern, then I would suggest waiting those extra 1-2 weeks.

PRIM&R would like to thank Drs. Bloomsmith and Hutchinson for sharing their expertise on this important topic.

If you were unable to attend this webinar and are interested in purchasing the recording, you may do so here.

References Cited:

1. Amano, H., Negishi, I., Akiyama, H., Ishikawa, O. (2008)   Psychological stress can trigger atopic dermatitis in NC/Nga mice: An inhibitory effect of corticotropin-releasing factor. Neuropsychopharmacology, 33 (3), pp. 566-573.
2. Lawson GW, Sato A, Fairbanks LA, Lawson PT. 2005. Vitamin E as a treatment for ulcerative dermatitis in C57BL/6 mice and strains with a C57BL/6 background. Contemp Top Lab Anim Sci 44:18–21.
3. Rasmussen S, Miller MM, Filipski SB, Tolwani RJ. (2011) Cage change influences serum corticosterone and anxiety-like behaviors in the mouse. J Am Assoc Lab Anim Sci.;50:479–483.
4. Van Loo, P. L.P. et al. Modulation of aggression in male mice: influence of cage cleaning regime and scent marks. (2000) Animal Welfare. 9, 281-295.
5. Gilbert, M. H. and Baker, K. C. (2011). Social buffering in adult male rhesus macaques (Macaca mulatta): Effects of stressful events in single vs. pair housing. Journal of Medical Primatology 40: 71–78.
6. Baker KC, Bloomsmith MA, Oettinger B, et al. (2012a). Benefits of pair housing are consistent across a diverse population of rhesus macaques. Applied Animal Behaviour Science 137:148–156.
7. Truelove, M.A., Martin, A.L., Perlman, J.E., Wood, J.S., Bloomsmith, M.A. (in press). Pair housing of macaques: A review of partner selection, introduction techniques, monitoring for compatibility, and methods for long-term maintenance of pairs. American Journal of Primatology. Article first published online: 30 SEP 2015; http://onlinelibrary.wiley.com/doi/10.1002/ajp.22485/abstract
8. Baker KC, Crockett CM, Lee GH, et al. (2012b). Pair housing for female longtailed and rhesus macaques in the laboratory: behavior in protected contact versus full contact. Journal of Applied Animal Welfare Science 15:126–143.
9. Baker, K.C., Bloomsmith, M.A., Oettinger, B., Neu, K., Griffis, C., Schoof, V. (2014). Comparing options for pair housing rhesus macaques using behavioral welfare measures. American Journal of Primatology 76:30–42.