Recognition and Alleviation of Distress in Laboratory Animals (2008)

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2 Stress and Distress: Definitions Introduction The various views and language used in the discussion of stress and distress lead to confusion in the scientific, regulatory, and animal welfare communities. Indeed, the animal welfare literature itself does not distin- guish stress from distress in any systematic fashion, and the term distress rarely appears in the biomedical sciences literature. In addition, the U.S. Government Principles and the Animal Welfare Act (see below) use both terms without definitions. Moreover, the general public often uses “stress” and “distress” interchangeably, and frequently in conjunction with the term “suffering,” thus blurring distinctions between these concepts. Because there is in fact good scientific evidence for both an adaptive stress response and a state of distress, it is important to distinguish these terms. Even though this chapter attempts to clarify these terms as much as possible, the avail- able scientific information—while useful—is far from complete, and distress remains a complex and still poorly understood phenomenon. This chapter, therefore, is an amalgam of current scientific information, along with the Committee members’ perspectives, best professional judgment, and expert opinion. While there is general agreement that pain and distress usually have a direct impact on animal welfare and quality of life, the descriptions of these conditions have evolved from different views and terminologies. The U.S. Animal Welfare Act (AWA 1990) uses the words “pain and distress”, whereas in the European Union’s Directive on the Protection of Animals Used for Experimental and Other Scientific Purposes (EEC 1986) the equiva- 13 

14 RECOGNITION AND ALLEVIATION OF DISTRESS IN LABORATORY ANIMALS lent phrase is “pain, suffering, distress and lasting harm.” Distress can be used to describe a state in which an animal, unable to adapt to one or more stressors, is no longer successfully coping with its environment and its well- being is compromised. Generally, a state of distress develops over a relatively long period of time; however, short, intense stressor(s) can also compromise animal well- being and induce acute distress. Thus, an animal may be in distress even if it appears to recover rapidly after the removal of the stressor or the conclu- sion of the procedure. What Is Stress? Stress is an inferred internal state. Because no single biological ­parameter can adequately inform on a stressful condition and no single stress response is present in all stress-related situations, there are many definitions of stress based primarily on metrics used to test hypothetical models of this state. A general distillation of the literature suggests that stress denotes a real or perceived perturbation to an organism’s physiological homeostasis or p ­ sychological well-being. In its stress response the body uses a constellation of behavioral or physiological mechanisms to counter the perturbation and return to normalcy. Events that precipitate stress (called stressors) elicit any of a number of coping mechanisms or adaptive changes, including behav- ioral reactions, activation of the sympathetic nervous system and adrenal medulla, secretion of stress hormones (e.g., glucocorticoids and prolactin), and mobilization of the immune system. Stress responses may involve at least one and perhaps several of the above systems, although none of them is by itself necessary or sufficient to denote stress. Furthermore, the absence or presence of any of these responses does not include or preclude the identification of a stressful state (for a comprehensive review see Moberg 2000). Stress responses have several key attributes: • They serve to promote physiological and psychological adaptation and are, therefore, beneficial and desirable. For example, activation of the sympathoadrenomedullary (SAM) system rapidly increases blood flow to the musculature and raises circulating glucose ­levels, resulting in an enhanced capacity to flee or fight (the “fight or flight” response). Over a longer time frame, glucocorticoid produc- tion in response to infection helps restrict the immune system, thus preventing deleterious effects of inflammatory factors on tissues (Gillis et al. 1979; Munck et al. 1984). • Apparent stress reactions can occur in situations unrelated to stress, and therefore their presence alone is not sufficient to indicate stress. For example, the diurnal rhythm of glucocorticoid secretion in most 

STRESS AND DISTRESS: DEFINITIONS 15 animals results in glucocorticoid levels at the diurnal peak that can rival those measured following stressor exposure (Dallman et al. 1987). Thus, no single parameter can serve as a litmus test for stress and diagnosis of stress based on a single metric can be misleading. • Stressors may not necessarily be unpleasant (defined by the ani- mal’s willingness to terminate the stressor); they can be ­pleasurable (Selye’s “eustress” concept; Selye 1975), as defined by an organism’s willingness to obtain the stressor. For example, naturally rewarding behaviors, such as exercise, increase sympathetic ­activity and cir- culating glucocorticoids in a profile very similar to that seen follow- ing aversive stressors (Droste et al. 2003). The self-administration of a drug, such as cocaine, similarly fits the definition of a stressor because multiple physiological systems are recruited to adapt to and oppose the drug’s action. • Physiological and behavioral responses are stressor-specific and so the processes engaged to restore homeostasis or well-being also differ. Thus, the following are all considered stressors, although they elicit variable behavioral and physiologic responses: viral or bacterial infection, threat of physical harm, drugs, exercise, sexual activity, high altitude, restraint, hunger, and thirst. Many of the above elicit “useful” or “good” stress, which is beneficial to the ani- mal in the long term. For example, while caloric restriction might be stressful or unpleasant because chronic hunger is involved, it promotes longevity and good health (Kemnitz et al. 1994; Lawler et al. 2005; Messaoudi et al. 2006). • Responses to stressors are variable due both to individual (some individuals are better able to cope than others) and intraspecies dif- ferences. For example, strain differences in inbred mice may result in dramatically different physiological or behavioral responses to stress (Crawley 2000; Hedrick and Bullock 2004; Silver 1995). What Is Distress? Distress has many definitions (see, for example, various dictionaries). Most definitions characterize distress as an aversive, negative state in which coping and adaptation processes fail to return an organism to physiologi- cal and/or psychological homeostasis (Carstens and Moberg 2000; Moberg 1987; NRC 1992). Progression into the maladaptive state may be due to a severe or prolonged stressor or multiple cumulative stressful insults with   etailed information on behavioral and physiological data of various subsets of murine D inbred strains is available at the Mouse Phenome Database at the Jackson Laboratory; http:// aretha.jax.org/pub-cgi/phenome/mpdcgi. 

16 RECOGNITION AND ALLEVIATION OF DISTRESS IN LABORATORY ANIMALS d ­ eleterious effects on the animal’s welfare. Distress can follow both acute and chronic stress, provided that the body’s biological functions are suffi- ciently altered and its coping mechanisms overwhelmed (Moberg 2000). The transition of stress to distress depends on several factors. Of clear importance are stressor duration and intensity, either of which is likely to produce behavioral or physical signs of distress. For example, short-term restraint does not cause marked problems in adaptation, whereas pro- longed restraint can result in behavioral or physiological distress some- times expressed by vocalization or gastric ulcers (Ushijima et al. 1985). In addition, predictability and controllability (i.e., the ability of the animal to control its environment) are important determinants in the transition of stress to distress. Numerous studies indicate that, in animals that can predict the onset of a stressful stimulus or control its duration, the behavioral and physi- ological impacts of stressor exposure are attenuated. Notable among these studies are findings that rats exposed to inescapable shock develop clear signs of distress, whereas yoked rats that can terminate shock exposure do not, despite subjection to the same intensity and duration of shock experi- ence (Maier and Watkins 2005). Furthermore, the stress response may induce insufficient or inappropri- ate changes in the behavioral and physiologic control systems (noted above) or inadequate or undesirable responses to their output signals. For example, chronic social subordination has been shown to elicit behavioral with- drawal, prolonged alterations in the hypothalamic-pituitary-adrenal (HPA) axis output, and subsequent immunosuppression (Blanchard et al. 2001), all of which preclude effective coping and adaptation. Further ­studies have shown that in chronic distress states, such as depression, the gluco­corticoid feedback systems fail (Carroll et al. 1976). Thus, if stress responses them- selves fail to appropriately cope or produce successful adaptation they may be not merely ineffective but actively deleterious. For example, while cortico­steroid responses are essential for the adaptation process, marked or prolonged hypersecretion can produce pronounced metabolic and immune dysfunction (Munck et al. 1984). Should an animal have the option to behaviorally express a choice in response to a stressful condition and thus exercise some control over its environment, then its adaptive behaviors should be distinguished from maladaptive ones displayed in distress (NRC 2003a, page 22; Mench 1998). However, a cause and effect relationship between various abnormal behav- iors and distress or the operationalization and validation of the degree of abnormality associated with distressed states has not yet been established. Distress may not always manifest itself with recognizable “maladaptive behaviors, such as abnormal feeding or aggression” (NRC 2003a, page 16) but instead with subclinical pathological changes, such as hypertension and immunosuppression, which are not behaviorally identifiable. As Moberg 

STRESS AND DISTRESS: DEFINITIONS 17 DISTRESS RESERVE RESERVE F1 STRESS F1 F2 F2 Biological Resources F3 F3 F1 F2 F“n” F3 F“n” F“n” BASAL BASAL BASAL FUNCTIONS FUNCTIONS FUNCTIONS Normal Stress Normal Function Function FIGURE 2-1  Reprinted with permission from Macmillan Publishers Ltd: [Lab Animal] (Moberg 1999), copyright (1999). Prolonged or severe stress depletes bodily reserves and affects normal functions thus requiring extended time to revert to homeostasis. During the recovery period, the animal’s well-being and welfare are compromised and the period of distress will last until the biological resources are sufficiently r ­ eplenished (Moberg 2000). The shift in biological resources, such as stunted growth 2-1 in distressed young animals (Moberg 1999), or evidence of maladaptive behaviors (NRC 1992) that occur in this general scheme of transition to and establishment of distress could be useful in recognizing distress (NRC 2003a, page 21). proposed in his 1999 paper “When Does Stress Become Distress”, the use of reserve resources to cope with prolonged or severe stress has a negative impact on other bodily functions (including behavior) and leads to distress. In the hypothetical scheme depicted in Figure 2-1, the “biological cost of distress” requires a prolonged recovery period to revert to homeostasis (Carstens and Moberg 2000). Implications for Animal Welfare Current understanding of animal welfare as a measure of the animals’ quality of life exists in the context of the social and cultural history of animal care and use as well as an expanding knowledge base related to 

18 RECOGNITION AND ALLEVIATION OF DISTRESS IN LABORATORY ANIMALS animal physiology and ethology. As early as 1964 the Brambell Committee acknowledged that “welfare is a wide term that embraces both the physical and mental well being of the animal”. The authors further elaborated that evaluations of animal welfare must take into account the scientific evidence derived from the animals’ structure, functions, and behavior (Brambell 1965; Duncan 2005). Although clinical signs can be used to assess physical well-being, and behavioral studies can provide information about animals’ preferences and cognitive state (for a review of validated animal models for fear and depression see Phelps and LeDoux 2005; also see ­ Bateson and Matheson 2007), the Committee would like to emphasize that no physiologic measures exist to date with which to assess mental well-being directly. Nevertheless, discussions about animal welfare in the laboratory as well as in farm animal communities take into consideration a variety of criteria to assess an animal’s quality of life. It has been proposed that the most important consideration for the assessment of an animal’s welfare is its emotional state (Duncan 2005). Be that as it may, some of these criteria focus on the animals’ ability to experience pleasure and pain (as defined in Bentham 1879), or their higher cognitive capacities (Nuffield Council on Bioethics 2005), while others consider the animals’ housing and husbandry conditions. The latter are of course easier to define and to assess, and are therefore the focus of more scientific research and literature. Housing and husbandry conditions should permit an animal to be physically healthy (i.e., not interfere with its biological functioning), live a natural life, behave more or less normally, and be free of pain and other negative circumstances (that induce negative affective states; Fraser et al. 1997). Concerns for animal welfare are often focused on what the animal may experience (Kirkwood 2007), including its ability to control its envi- ronment or predict the onset of a stressor. In these discussions, the term “ ­ suffer/suffering” is often used, albeit controversially due to lack of con­ sensus with respect to the adverse emotional states to which it may allude, such as pain, distress, boredom, deprivation, fear, frustration, and grief, in which an ­animal may be said to suffer even for only a few minutes. Descriptors of an animal’s welfare are qualitative and range from “poor” to “good” (other adjectives commonly used include “negative”, “compro- mised”, “neutral”, and “positive”). Welfare may be compromised briefly (e.g., during handling, injection, or exposure to a predator) or over longer periods of time (e.g., in the solitary housing of a social species, or in the   For additional discussion of what is normal or natural with regard to laboratory animals, see Chapters 3 and 4.   The Veterinarian’s Oath outlines the moral obligation toward the alleviation of animal s ­ uffering by stating that “. . . I solemnly swear to use my scientific knowledge and skills for the benefit of society through . . . [in part] the relief of animal suffering. . . .” (AVMA, http://www. avma.org/issues/animal_welfare/). 

STRESS AND DISTRESS: DEFINITIONS 19 provision of housing without appropriate enrichment). In order to prevent poor or deteriorating welfare, researchers, animal care staff, and institutions have a responsibility to provide high-quality care for laboratory animals, including ready access to fresh water and a nutritive diet; an environment that ensures shelter and comfort; prevention as well as rapid diagnosis and alleviation (as appropriate) of pain, injury, and disease; species-appropriate space, facilities, and (if appropriate) companionship; and conditions and treatment that do not cause negative emotional states. Fraser and colleagues suggest that good animal welfare implies the absence of pain, fear, and hunger; enables a high level of biological functioning (i.e., normal growth, freedom from disease); and (more controversially) enables animals to expe- rience positive emotional experiences such as comfort and contentment (Fraser et al. 1997). It is possible for an animal to be in a state of poor health that does not impinge on its welfare or emotional state and that may even last for some time without the animal’s conscious awareness. For example, an animal might have a life-threatening aneurysm but be unaware of it and therefore not experience a negative emotional state. In the longer term, however, a breakdown in an animal’s ability to cope with its environment is likely to lead to adverse emotional states and poor welfare. Some of these cases may be quite minor and not give rise to significant ethical concerns; but prolonged or intense circumstances would compromise the animal’s wel- fare enough to warrant concern and also significantly affect the research results. An attempt to graphically depict the relationship between distress and welfare is shown in Figure 2-2. Whereas minor perturbations (e.g., short- term restraint of a rodent) affect an animal’s welfare in terms of its moment- to-moment emotional state, they do not impair its adaptive capacity and thus do not cause distress. In contrast, a major homeostatic disruption (e.g., postsurgical infection), which causes measurable behavioral (withdrawal) and physiological (fever) changes that impair the adaptive capacity of the animal, is considered “distressful” and is indicative of “poor welfare”. Onset of distress can be difficult to recognize. A safe assumption is to follow the fourth principle of the U.S. Government Principles for Uti- lization and Care of Vertebrate Animals used in Testing, Research and T ­ eaching: “Proper use of animals, including the avoidance or minimiza- tion of discomfort, distress, and pain when consistent with sound scientific practices, is imperative. Unless the contrary is established, investigators should consider that procedures that cause pain or distress in human beings   or more information on the effects of housing on brain function or enrichment see Chap- F ter 3. Additional information is contained in articles by the behaviorists Joseph Garner, Hanno Würbel, and Georgia Mason. 

20 RECOGNITION AND ALLEVIATION OF DISTRESS IN LABORATORY ANIMALS DISTRESS Good Maximal Adapting Capacity Animal Welfare Animal Welfare Adapting Capacity Poor None None Stress Level Severe (stressor intensity × time) FIGURE 2-2  Hypothetical depiction of the relationship of stress, distress, adap- tive capacity, and animal welfare. An animal’s quality of life may be progressively d ­ eteriorating while it is still successfully coping with a stressor. The precise moment of transition to the maladaptive state or what precipitates it is unknown, but once the tipping point is attained, the deterioration into a severely sick or debilitated animal occurs fairly quickly. At this point, welfare conditions are very poor and immediate ameliorative action is necessary. fig 2-2 may cause pain or distress in other animals” (IRAC 1985). A degree of critical anthropomorphism, outlined above and in the writings of Morton and colleagues (Morton et al. 1990), coupled with behavioral assessments will likely provide the most direct understanding of an animal’s response to a stressor. Useful indicators include the animal’s choice to continue or stop feeding while in a stressful situation, choice tests that demonstrate how (non)aversive a particular stressor is, or demand studies that titrate the extent of the animal’s attraction or aversion to a potential stressor. These gauges of avoidance or aversion may be complemented by physiological data measuring elevated hypothalamic-pituitary-adrenocortical axis (HPA) or sympatho­adreno­medullary system (SAM) activity (gene or protein activa- tion), elevated hormone levels, or increased activity in target organs (e.g., 

STRESS AND DISTRESS: DEFINITIONS 21 heart rate, blood pressure, glucose levels). Many authors have pointed to the desirability of using multiple measures to obtain a more comprehensive data set (Rushen 1991). It is important to underscore that reliance on a single measurement of stress may result in erroneous conclusions. Chapter 3 provides more details on distress recognition. Implications for Research There is a rich literature documenting the interference of stress in behavioral and/or physiological endpoints. Strong evidence in rodents has shown that mild stress of 2-3 months duration—a regimen that produces no signs of overt distress—alters the animals’ performance in tests of anxiety, depression, and memory (D’Aquila et al. 1994; Rossler et al. 2000; Song et al. 2006; Willner 1997)������������������������������������������������������ . ���������������������������������������������������� Other findings indicate that rats’ habituation to a test environment can dramatically affect their response to a toxic substance (Damon et al. 1986). On the other hand, in some cases (such as lower a ­ nxiety behavior in the elevated plus maze) the effects of stress may actually be beneficial to the experimental procedure, indicating that prolonged stress may not be uniformly detrimental. Chapter 3 documents the contamination of experi­mental data by unwanted or uncontrolled stress due to inadequate husbandry, noisy environments, olfactory stimuli, or other factors. The impact of distress on both animal welfare and research results is likely even more pronounced than that of stress. Animals exposed to prolonged severe stress experience underlying changes in physiological functions (e.g., gastric lesions [Ushijima 1985] or immunosuppression [Tournier 2001]) that can interfere with experimental manipulations; alter experi­mental variables such as behavior (Morton and Griffiths 1985), drug dosing ­ (Saranteas et al. 2004) and clearance; change the progress of a disease (Johnson et al. 2006); and contribute to morbidity and mortality. A variety of stressors can contribute to unintended distress, from postoperative pain or infection to barren housing conditions or the solitary confinement of an individual of a social species (Gunn and Morton 1995; Morton et al. 1993). Stereotypies, abnormal repetitive behaviors indicative of poor well-being (Garner et al. 2003) that are often observed in distressed animals, are thought to reflect defective brain function (Würbel 2001) and to be a result of poor animal welfare (Mason and Latham 2004). Stereotypies are thus likely to interfere with behavioral, neuroscience, and pharmacological studies. The impact of stress and distress on the quality of scientific research can result in the generation of compromised data, which in turn necessi- tates the use of more animals. This outcome is inconsistent with two of the   t I should be noted that the negative connotations of stereotypies are not universally a ­ ccepted. For further discussion see Chapter 3. 

22 RECOGNITION AND ALLEVIATION OF DISTRESS IN LABORATORY ANIMALS Three Rs: reduction in the number of animals used in an experiment, and refinement of the protocol to minimize or eliminate distress for the animals used ­(Russell and Burch 1959). Conclusions In an effort to reduce the confusion surrounding the definitions of stress and distress and as a basic framework to inform future research in these areas, the Committee offers the following summary of distinctions between the two concepts: • Stress and distress are dissociable concepts, distinguished by an animal’s ability or inability to cope or adapt to changes in its imme- diate environment and experience. • Stress responses are normal reactions to environmental or internal perturbations and can be considered adaptive in nature. Distress occurs when stress is severe, prolonged, or both. • The concepts of stress and distress can be distinguished from that of welfare, in that an adaptive and beneficial stress response may occur against a backdrop of a transient negative emotional state. • Both stress and distress represent potential complications in a wide range of experiments, and should be proactively addressed by good experimental design. REFERENCES AVMA (American Veterinary Medical Association). Issues in animal welfare. Available at www. avma.org/issues/animal_welfare. Accessed February 2, 2007. AWA (Animal Welfare Act). 1990. Available at www.nal.usda.gov/awic/legislat/awa.htm. A ­ ccessed February 6, 2007. Bateson, M. and S. M. Matheson. 2007. Performance on a categorisation task suggests that removal of environmental enrichment induces pessimism in captive European Starlings (Sturnus vulgaris). Anim Welfare 16(Suppl):33-36. Bentham, J. 1879. An Introduction to the Principles of Morals and Legislation. 2005 ed. Boston: Adamant Media Corp. Blanchard, R. J., C. R. McKittrick, and D. C. Blanchard. 2001. Animal models of social stress: Effects on behavior and brain neurochemical systems. Physiol Behav 73(3):261-271. Brambell, F. W. R. 1965. Report of the Technical Committee to Enquire into the Welfare of Animals Kept Under Intensive Livestock Husbandry Systems. London: HMSO. Carroll, B. J., G. C. Curtis, and J. Mendels. 1976. Neuroendocrine regulation in depression, I: Limbic system-adrenocortical dysfunction. Arch Gen Psychiat 33(9):1039-1044. Carstens, E. and G. P. Moberg. 2000. Recognizing pain and distress in laboratory animals. ILAR J 41(2):62-71. Crawley, J. N. 2000. What’s Wrong with My Mouse? Behavioral Phenotyping of Transgenic and Knockout Mice. New York: Wiley-Liss, John Wiley & Sons, Inc. 

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