Measuring pain in the (knockout) mouse: big challenges in a small mammal

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Abstract

The increasing popularity of the mouse as a subject in basic science studies of pain can largely be attributed to the development of transgenic ‘knockout’ technology in this species only. To take advantage of this biological technique, many investigators are rushing to adapt to the mouse experimental protocols that were designed for the rat. However, the myriad physiological and behavioral differences between these two rodent species render such adaptations non-trivial and in many cases seriously problematic. In this article we review the basic nociceptive assays used in behavioral pain research (thermal, mechanical, electrical and chemical), and highlight how species differences affect their proper application. In addition, some of the issues specifically pertaining to the interpretation of such data in knockout studies are addressed.

Introduction

Pain plays a clearly adaptive role in protecting against, and recuperating from serious injury, but pain that persists is maladaptive and likely a pathology in its own right. The understanding and treatment of pain has thus long been a high priority. Pain research in humans has always been hampered by its subjective nature and obvious ethical impasses. Animal models have thus been greatly relied on, although they themselves are associated with any number of interpretational challenges. An increasing number of studies of pain employ the laboratory mouse, Mus musculus. Advantages of this mammalian species include relatively inexpensive maintenance and ease of breeding, and the availability of a wide variety of genetic models. Compared with the rat, for example, a far larger number of inbred mouse strains—of which each member is genetically identical—are commercially available (see [24], [75]). Inbred strains have proven invaluable for classical and molecular genetic studies including gene mapping efforts. However, the factor most responsible for the recent acceleration of biomedical studies involving the mouse is the development of transgenic ‘knockout’ technology in this species only. This approach has been enthusiastically applied to pain research (see [56], [62]), such that over 20% of published pain research articles in the past 2 years in which the mouse was the subject species presented or reviewed findings from knockout mice.

Unfortunately, the application of behavioral assays of nociception to transgenic mice has been inconsistent and sometimes of poor quality, leading in some cases to unreliable (i.e. non-replicable) and misleading conclusions. This state of affairs likely results from the small number of laboratories with extensive experience performing behavioral assays of nociception in the mouse. Experiments with knockout mice are also affected in significant ways by issues related to genetic background (‘genotype’), another field where expertise is restricted.

Historically, of course, the typical non-human pain research subject has been the laboratory rat. Though both species belong to the Muridae rodent family and (other than the difference in size) appear superficially similar, mice are not small rats, having diverged evolutionarily over 10 million years ago [31]. Accordingly, adapting behavioral assays from the rat to the mouse is a non-trivial task. The specific challenges of performing behavioral assays of nociception in the mouse, and the impact of genotype on these assays, will be the foci of this review. These same issues are explored in greater depth in an upcoming book chapter, including our recommendations as to optimizing testing protocols for this species [53].

Section snippets

General considerations

Mice are genetically, physiologically, and behaviorally distinct from rats, resulting in differential effects of housing, handling and habituation. All three of these ‘h’ factors impact especially on pain research because of the robust inhibitory effect of stress on nociceptive sensitivity. That is, stress-induced analgesia (SIA) (see [34]) can easily confound measurements of ‘basal’ nociceptive sensitivity, as well as interact with exogenously administered analgesics. Such interactions may be

Nociceptive assays

Pain is not a unitary phenomenon, and thus a wide variety of assays have been developed to model different types of pain. Although acute thermal and mechanical assays exhibit more than adequate empirical validity (i.e. predictive power) for many analgesics [81], acute pain is virtually non-existent as a clinical entity. Thus, a number of animal models of ‘chronic’ pain have been developed (most often in the rat, of course), generally involving an injury of inflammatory or neuropathic (i.e.

Strain differences

As if the practical challenges associated with nociceptive testing in the mouse weren't daunting enough, pain researchers working with knockout mice face a host of additional complications related to the fact that they are comparing genotypes. Mice feature prodigious interindividual differences, and this fact has been exploited in the development of inbred strains. A major focus of our laboratory is in fact to document and ultimately explain the genetic basis of variability in nociceptive and

Is the study of behavioral pain genetics reliable?

A recent study by Crabbe and colleagues in two other laboratories [15] documented the sensitivity of eight mouse genotypes (including the 5-HT1B knockout mouse) on a battery of six behavioral assays. What was intriguing about the study is that the data were collected simultaneously in the three laboratories, and the authors went to extraordinary lengths to control for a large number of environmental factors. Although the data probably did not entirely justify it, the pessimistic conclusion

Acknowledgements

This research is supported by USPHS grants DA11394 and DE12735 (JSM). SGW is supported by a National Research Service Award (MH12544).

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