Building Consensus

A team approach to animal-use protocol development and animal management is valuable for meeting research objectives while maximizing attention to animal care. The team approach relies on the idea of shared responsibilities. Participants include neuroscientists and their laboratory personnel; veterinarians; animal-husbandry staff; the IACUC chair, members, and staff; and the research institution.

The institution must establish a culture of respect for the animals and maintain a commitment to following the Guide, PHS Policy, and the AWRs. However, the Guide, PHS Policy, and the AWRs are not intended to be barriers to research. Working as a team, the principal investigator (PI), the IACUC, and the veterinarian should be able to devise a means of accomplishing the research goal while addressing the needs of the animals. The “performance standard” encouraged by the Guide (p. 3) is a powerful tool for IACUCs and veterinarians to use in developing strategies to promote both animal well-being and good science.

Developing an animal care and use protocol is a negotiation among the PI, the veterinarian, and the IACUC to balance animal well-being with the experimental goals. By involving all parties early in the protocol-development process, particularly for protocols that involve extensive experimental manipulation or difficult to maintain animal models, the PI can help to prevent misunderstandings and delays while facilitating IACUC review and approval.

Development of Protocols

The PI and research staff develop the scientific concept that underlies an animal protocol. The PI is the expert in the scientific goals and the experimental design and is often highly knowledgeable about animal use and well-being. The veterinarian is the trained expert in the latter subject and can advise the PI and the IACUC on performance-standard development and implementation. However, the IACUC has the final responsibility and authority for evaluating the protocol outcome and approving exceptions to guidelines and regulations if any are necessary. The veterinarian should be involved in animal-use protocol development, preferably before the protocol is submitted to the committee for official review. Indeed, according to the AWRs, the veterinarian must be consulted for any procedure that may cause more than momentary or slight pain or distress (AWR 2.31(d)(1)(iv)(B)). In some cases, veterinary input is gathered primarily during the final protocol-review process, and this delay can complicate protocol review and prolong the approval process.

The IACUC staff and/or chair can solicit additional information and perform a prereview of the animal-use protocol to help the PI recognize portions of the protocol that require clarification or additional information. The IACUC must consider sample size, pain and/or distress, and experimental and humane endpoints among other considerations. The IACUC may request direct observation of a procedure (particularly a new or unusual procedure), pilot studies, and particular experimental measurements or monitoring procedure to evaluate and ensure animal well-being.

Execution of Protocols

The research team has the primary responsibility for animal assessment and intervention. However, researchers and the animal-care staff must coordinate their efforts to provide appropriate animal care and monitoring. Unanticipated adverse effects of the research that are or may be a threat to the health or safety of the animal must be reported to the IACUC immediately. As an animal-use protocol must describe any anticipated adverse effects, if an unanticipated adverse effect that was or could be a threat to the health or safety of the animal were to occur, then the protocol does not accurately reflect the animal-use activity and must be modified accordingly and reapproved.

The veterinary staff has the legal responsibility for animal care. Veterinary medical care is best administered with consideration for the scientific goals of the study. However, the veterinarian must have institutional authority to make decisions on behalf of the animal in critical situations.

The husbandry staff has day-to-day responsibility for assessment of animal well-being, regardless of experimental use. The caretaker staff is in a unique position to observe large numbers of animals and to understand the animals both individually and as a species or strain. They observe eating, drinking, urination, defecation, behavioral abnormalities, and subtle signs of problems. Because they observe large numbers of normal animals, the animal-care staff can be key participants in determining the phenotype of knockout and transgenic rodents.

The IACUC, after review and approval of the animal care and use protocol, has the responsibility to ensure that procedures are carried out in accordance with the protocol. The IACUC may request periodic reports from the PI or from the veterinarian monitoring the research. During semiannual facility inspection, the IACUC can verify that procedures are consistent with the approved protocol.

The institution provides the animal-research infrastructure in the form of core facilities for animal care, mandates and resources for training, and the occupational health and safety program. The institutional official (IO) is the individual who is authorized to legally commit on behalf of the research facility that the requirements of 9 CFR parts 1, 2, and 3 will be met (AWR 1.1). The IO is generally the senior administrator with authority to commit institutional resources to ensure compliance with governing regulations and guidelines. The IO should empower the appropriate people to intervene on behalf of the animals, provide adequate facilities and staff, and take the lead in creating a compliant and responsible institutional culture.

Regulatory Oversight

Extensive guidelines, policies, and regulations govern the management of pain and distress in laboratory animals. The US Government Principles (IRAC, 1985) state:

Proper use of animals, including the avoidance or minimization 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 may cause pain or distress in other animals [Principle IV].

Procedures with animals that may cause more than momentary or slight pain or distress should be performed with appropriate sedation, analgesia, or anesthesia. Surgical or other painful procedures should not be performed on unanesthetized animals paralyzed by chemical agents [Principle V].

The AWRs, PHS Policy, and the Guide require the IACUC to ensure that animal-use protocols include strategies for minimizing pain and distress in animals. Specifically, USDA (through the AWR 2.31 (d)(ii) and (e) and APHIS/AC Policy 12) requires the investigator to consider alternatives to procedures that may cause more than momentary or slight pain or distress and to provide a written narrative description of the methods and sources used to determine that alternatives to the procedure were not available. As noted in the Institutional Animal Care and Use Committee Guidebook (ARENA-OLAW, 2002), the animal-use protocol must provide sufficient information for the IACUC to evaluate the pain and/or distress potentially resulting from the study and the appropriateness of the methods proposed to minimize it. The attending veterinarian is an important team member, working with the researcher and the IACUC to ensure animal welfare when there is the potential for pain and/or distress.

Assessment of Pain

According to the Guide, “fundamental to the relief of pain in animals is the ability to recognize its clinical signs in specific species” (p. 64). Pain can be assessed by evaluating behavioral measures such as eating, socializing, and withdrawal reflexes, and physiologic measures such as heart rate and respiration rate (see Table 2-1). However, species, and even strains and individuals of the same species, may vary widely in their perception of and response to pain (NRC, 1992; Wixon, 1999). Even for an individual animal, pain sensitivity varies among different tissues and organs (Baumans et al., 1994), and pain sensitivities can be altered by pathologic processes or experimental procedures (Carstens and Moberg, 2000). For example, during the initial phase of lipopolysaccharide-induced fever, rats exhibit hyperalgesia, whereas they exhibit hypoalgesia during the later stages of the illness (Carstens and Moberg, 2000). The existence of these differences underscores the point that pain and distress exist as a continuum of experience. In addition, some animals may hide signs of pain; for example, it has been suggested that rats may mask pain during the dark-cycle hours to avoid displaying abnormal activity and increasing their risk of predation (Roughan and Flecknell, 2000).

AALAS (AALAS, 2000) suggests that the magnitude of the pain that the animal is expected to experience be categorized in the protocol and monitored and that there be an opportunity to adjust the pain category once the study is under way. It is important that researchers and animal-care staff have a solid knowledge of the normal and abnormal physiology, behavior, and appearance of the animals in their care (Anil et al., 2002; NRC, 1992).

Acceptable levels of noxious stimulation are those that are well tolerated and do not result in maladaptive behaviors. Acceptable levels range from an animal's pain threshold to its pain tolerance level. Pain threshold is the stimulus level at which pain is first perceived, while pain tolerance is the highest intensity of painful stimulation that an animal will voluntarily accept. As the intensity of a stimulus approaches the pain tolerance level, an animal's behavior will become dominated by attempts to avoid or escape the stimulus, and this degree of pain must be alleviated (Dubner, 1987).

It is important to note that it is usually incorrect to infer that an animal's pain tolerance level is signaled by the onset of avoidance or escape behavior, as some avoidance-escape behavior is an appropriate adaptive response. It is only when the animal's behavior is dominated by avoidance-escape attempts that the behavior becomes maladaptive, signaling unacceptable levels of pain (NRC, 1992).

In pain studies, giving animals control over the source of pain by allowing them to withdraw from a painful stimulus is an effective way to minimize pain and the distress associated with it. If an animal is denied control of the stimulus and it approaches the tolerance limit, maladaptive behaviors will appear, and the animal should be presumed to be in distress. Maladaptive behaviors include persistent attacks on the perceived source of the pain, self-mutilation at the injured or stimulated site, and a state of learned helplessness in which the animal gives up and no longer attempts to escape, avoid, or control the stimulus. To avoid the development of maladaptive behaviors and to minimize pain during experimental manipulations where the animal is denied control of the stimulus, it is critical that the neuroscientist attempt to define the level of pain produced by the stimulus (Dubner, 1987), and ensure that the level imposed by the stimulus is below that which causes the development of maladaptive behaviors. In most cases, previous experimental or published data will indicate the level of pain produced by the stimulus; lacking this information, a pilot study to identify the level of stimulus that produces maladaptive behavior could be useful.

Pain assessment will vary with the pain scale or scoring system used. Scoring systems involve assigning a numeric score to constellations of behavioral, physical, and physiologic observations, and this process can be subjective. There are no generally accepted objective criteria for assessing the degree of pain that an animal is experiencing, and different species or strains can vary in their response to pain. Physiologic measures include heart rate, blood pressure, and respiration rate, but obtaining most of the measures requires some degree of intervention, which may not be feasible or desirable (Baumans et al., 1994).

Recent studies on pain in animals include methods for quantifying specific motor behaviors as indirect measures of responses to mechanical, thermal, or chemical injury (Dubner and Ren, 1999). Animals will withdraw an injured body part from a stimulus, where different levels of stimulation affect the latency or force of withdrawal. This withdrawal response is considered a measure of pain, which correlates highly with more integrative nocifensive behaviors (behaviors in response to pain), such as licking of the injured body part and guarding behavior.

Some behavioral signs are usually associated with pain (Soma, 1987). Animals often communicate through posture. They may exhibit guarding behavior in an attempt to protect the injured part. Vocalizations are important indicators of pain in several species (Anil et al., 2002). Animals in pain may lick, bite, scratch, shake, or rub the site of injury. Restlessness may also be observed, including pacing, lying down and getting up, and shifting weight. Animals in pain may stay in one place for abnormal lengths of time and be reluctant to move or rise. They may withdraw from contact with other animals. They may become listless and refuse to eat or reduce their eating and drinking. They may avoid being handled. These are all possible signs of pain, but none alone is sufficient to determine the presence or level of pain. For example, many animals vocalize intensely when they are handled even if they are not in pain (e.g., Stafleu et al., 1992). Multiple criteria should therefore be assessed (Bayne, 2000; Wallace et al., 1990).

An important step in determining that an animal is in pain is recognition of a departure from normal behavior and appearance (Dubner, 1987; Kitchen et al., 1987; Morton and Griffiths, 1985; NRC, 1992). But as Bayne (2000) indicates, assessments vary with the scale used, and the scales can be very subjective. Flecknell and Silverman (2000) noted that preprocedural scoring is necessary to obtain an appropriate baseline so that confounding variables (such as behavioral effects produced by analgesics) can be identified. For example, some of the consequences of surgery in rats, such as loss of body weight and reduction in food and water intake (signs frequently interpreted to indicate pain or distress), can also be produced in normal, unoperated-on rats by administering opioid analgesics.

Recent evidence indicates that some signs of pain may not be perceived by personnel, such as the ultrasonic vocalizations of infant mice (Nastiti et al., 1991), but are detectable with appropriate equipment. Several excellent references discuss species-specific behaviors that are indicative of pain (Carstens and Moberg, 2000; Hawkins, 2002; Morton and Griffiths, 1985; NRC, 1992; Roughan and Flecknell, in press; Soma, 1987; Wallace et al., 1990).

Assessment of pain should not be influenced by the biases of the observer (Sanford et al., 1986), and the observer should be well trained in both normal and abnormal behaviors of the species in question. Variability among observers can have a substantial effect on the interpretation of assessment data (Holton et al., 1998).

Chronic or persistent pain differs from acute pain because it may not be associated with any obvious pathologic condition and does not serve any protective function. Signs of chronic pain can be subtle and difficult to detect in that an animal's behavior may change slowly and incrementally. Chronic or persistent pain is also more likely to lead to distress and maladaptive behavior. Signs of chronic or persistent pain include decrease in appetite, weight loss, reduction in activity, sleep loss, irritability, and decrease in mating behavior and reproductive performance (Soma, 1987). Alterations in urinary and bowel activities and lack of grooming are often associated with persistent pain. Severe chronic pain can reduce body temperature, cause a weak and shallow pulse, and depress respiration. As noted above, animals cannot control chronic or persistent pain, and it is important to assess the intensity of the pain by using behavioral measures.

Assessment of Distress

Some of the husbandry and experimental procedures that animals experience in neuroscience and behavioral research have the potential to cause distress. Distress can be either transient or prolonged and can range from mild to severe. Determining when stress becomes distress, and thus an animal-welfare concern that requires amelioration, is difficult. Our understanding of the relationship between the (measurable) physiologic changes that occur during an acute stress response and ensuing adverse psychologic states is generally poor. An animal's mental state can be inferred only indirectly, and many factors can influence whether an animal responds in an adaptive or maladaptive fashion to a particular stressor. Those factors include genetic predisposition, experience, age, sex, species, and the social context in which the stressor occurs.

It is even more difficult to assess the effects of chronic and intermittent stressors that are likely to be experienced by animals as part of routine husbandry and housing in the research setting. There is evidence that animals can successfully adapt physiologically to most stressors experienced as part of routine husbandry and housing (Line et al., 1989; Sharp et al., 2002a,b); however, animals may not be able to physiologically adapt to all such stressors, and their ability to adapt is likely dependent on the aversiveness, duration, and frequency of the stressor (Line et al., 1989). Yet, even when animals can adapt physiologically to such chronic stressors, they may not exhibit behavioral adaptation (Ladewig, 2000). In some cases of chronic stress (for example, in severe depression), the physiologic stress system may eventually stop responding normally to challenges, even after the source of the chronic stress is removed (Sapolsky, 1998).

Nevertheless, it is imperative to evaluate and (where possible) ameliorate distress in the research environment (NRC, 2000). Several general schemes have been proposed for recognizing distress, including a measurable shift in biologic resources (Moberg, 1999), such as a change in metabolic function, or evidence of maladaptive behavior (NRC, 1992). The physiologic and behavioral changes that accompany some states of distress have been fairly well characterized. For example, common manifestations of fear or anxiety are motor tension (shakiness and jumpiness), hyperactivity of the sympathetic nervous system (sweating, increased respiration and heart rate, and frequent urination), and hyperattentiveness (increased vigilance and scanning) (Rowan, 1988).

One problem in assessing stress and distress has been that measurement techniques that involve handling, blood sampling, or tissue collection may themselves be stressors and cause physiologic changes. However, many noninvasive or less invasive methods for physiologic monitoring can now be used in the research setting. They include implanted radio transmitters to measure autonomic nervous function, microdialysis techniques for sample collection, remote blood sampling methods, biosensors for recording central nervous system responses in freely moving animals, and measurement of hormones in hair, feces, and urine (Cook et al., 2000; Goode and Klein, 2002; Koren et al., 2002).

One of the most important noninvasive methods for assessing distress is observation of animal behavior. At least when measured in situations where animals have behavioral flexibility and choice, behaviors can provide information about what animals prefer or avoid, and hence are indicators of emotional states (Mench, 1998). Behavior is a part of an animal's adaptive repertoire for responding to stressors, so it is important to distinguish adaptive behaviors from maladaptive behaviors, such as self-mutilation, unresponsiveness to important signals, hyperactivity, and excessive response to stimulation. Useful observation requires knowledge of the natural history and perceptual capacities of the particular species or strain of animal (Bayne, 1996), the usual frequencies and intensities of particular behaviors, and the causes and functions of the behaviors (Mench, 1998; Rushen, 2000). Because of individual variability, a baseline behavioral profile of an animal should be established if changes in behavior are going to be used to monitor the animal for distress. As with the assessment of pain, personnel assessing behavior should be knowledgeable and skilled in the interpretation of behavior, and assessments should not be influenced by the observers' biases (Bayne, 2000).

Alleviation of Pain and/or Distress

Four general approaches are available to minimize pain (Dubner, 1987): the use of general anesthesia, the use of local anesthesia and/or analgesia, the training of animals to avoid situations that produce pain (escape-avoidance behavior), and control of the intensity and/or duration of the stimulus by the neuroscientist.

The selection of a general anesthetic should reflect professional judgment as to which anesthetic best meets the clinical and humane requirements without compromising the scientific aspects of the research protocol (NRC, 1996). That sufficient anesthesia has been provided can be ensured by monitoring reflexive responses to painful stimuli, respiration, pupil size, stability of heart rate and blood pressure or electroencephalographic activity. Occasionally in neuroscience research, surgical lesions are created producing a functional decerebration and thereby eliminating all possibility of pain and the need for general anesthesia.

The use of local anesthesia and/or analgesia is also a widely used technique for alleviating pain and/or distress. Vainio et al. (2002) provide a useful description of the clinical efficacy and adverse effects of opioids and nonsteroidal anti-inflammatory drugs (NSAIDS) for analgesia in laboratory animals, as well as the dose, route, and frequency of administration of the common opioids and NSAIDS.

For a discussion of the training of animals to avoid painful stimulation and of how investigators can control the intensity or duration of painful stimulation, see Chapter 4. In some cases, euthanasia may be the most appropriate means of alleviating pain (see Chapter 2—“Humane Endpoints”).

Distress is usually an undesirable outcome of an experiment, so strategies for avoiding or minimizing it should be identified during the planning of the study. When distress arises from unintended sources, identification and elimination of the cause is the most obvious course of action. Distress can also be alleviated pharmacologically and nonpharmacologically. Two of the major causes of distress are lack of predictability and control of stimuli (Sapolsky, 1998), and it can be useful to condition animals to experimental or husbandry procedures that they will experience or to allow them to have some control over the stimuli imposed, for example, by providing a way to escape from staged aggressive encounters (as discussed in Chapter 7) or by providing nesting material so that a thermally challenged animal can better control its body temperature. Environmental modifications that can make an animal more comfortable, including changing the ambient temperature, increasing ease of access to food and water, and determining whether social contact would ameliorate or accentuate the stress burden. Appropriate enrichment of the social and structural environment can decrease distress by decreasing boredom and fearfulness, and facilitating coping (Bayne et al., 2002; Carlstead and Shepherdson, 2000; Mench, 1998). If possible, pain-related distress should be managed pharmacologically. However, if pain is the object of the study, pharmacologic options for reducing distress may be few or unavailable. In some studies, the use of sedatives, anxiolytics, dissociative anesthetics, and/or analgesics will not conflict with study goals; IACUCs should require that these options be discussed in animal-use protocols for neuroscience or behavioral research.

In all cases the veterinarian should be consulted regarding methods to minimize pain and distress. Accepted best practices for managing unrelieved pain and distress should be incorporated into the protocol design unless there is a scientific reason to do otherwise. The neuroscientist must provide assurance that unrelieved pain or distress will not continue past the point necessary to achieve the scientific goals of the study (ARENA-OLAW, 2002). Additionally, a mechanism for prompt reporting (for example, to the veterinarian or the IACUC) of animals that have been unexpectedly distressed or pained by the study should be developed and implemented (Bayne, 2000) and should be inherent in the animal-use protocol design.