Stress: it’s everywhere – higher gas prices, longer work hours, deadlines. Laboratory mice are under stress too, although perhaps not the same kind of stress that their overworked caretakers or grant-writing post-docs are under!
Handling mice is an unavoidable part of an animal caretaker’s routine, and the stress this causes to laboratory mice is an often overlooked factor that may affect experimental outcomes. Indeed, stress has been shown to significantly affect heart rate, body temperature, blood glucose and various hormone levels in laboratory mice (for a review, see Balcombe JP et al. 2004. Contemp Top Lab Anim Sci. 43: 42-51 [PMID: 15669134;"]). Enforced stress also decreases plasma insulin and increases plasma glucose levels in genetically obese C57BL/6- ob/ob mice (B6.V-Lepob/J – Stock # 000632; see Surwit RS et al. 1984. Diabetes 33: 616-18 [PMID: 6376217]).
So, what can we do to limit the effects of stress on mice and experimental results? Habituation is the key. We typically recommend that investigators allow their mice to acclimate to their new environment for at least a week before using them in experiments. Longer periods may be even more beneficial. Acclimatization should go beyond just leaving the mice on the shelf for 7-10 days before using them. For example, if blood will be drawn from or something will be injected into the mice, simulate those procedures as part of the habituation routine.
In a paper published late last year in Nature Methods, Jane Hurst and Rebecca West at the University of Liverpool report that how mice are handled can affect their fear and anxiety responses (Hurst JL, West RS. Nature Meth. 7: 825-826 [PMID: 20835246]). Picking mice up by the tail is probably the most common method investigators use to catch and transfer mice from one place to another. Hurst and West compared the fear and anxiety responses of mice handled by this method to the fear and anxiety of mice caught either by scooping them up in a “cupped” hand or by allowing them to walk into a clear acrylic tunnel before handling them. After a nine-day habituation period in which mice were handled by the various methods for a total of 60 seconds per day, mice caught by the tail had the highest incidences of the most common anxiety indicators – urination and defecation.
Generally, they also had higher levels of anxiety in an elevated plus maze. In contrast, mice that were caught and handled by “cupping” and (especially) in a tunnel, generally scored lower in these anxiety tests and were more willing to interact voluntarily with the handler. (If you have the required subscription, check out the supporting online movies at the Nature Methods website.) Moreover, restraining the tunnel- and cupping-handled mice by neck skin “scruffing” did not alter their willingness to interact with the handler.
These observations suggest that habituating mice to handling by either the cupping or tunnel method minimizes handling-related stress. As Hurst and West point out, however, such handling methods may not be best if a strong anxiety response is required. Additionally, strains such as some wild-derived or other aggressive mice may not respond as well to the non-restraining methods as the C57BL/6, BALB/c and ICR mice that Hurst and West studied. Nevertheless, this interesting study emphasizes that habituation can minimize handling stress in mice and that mice should be handled as consistently as possible to minimize stress and its effects on experimental variability.