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Unit Three – Pain and Distress

Humane care and use of animals relies on an understanding of animal pain and distress, from biological, ethical, and legal perspectives.

 

Readings

An excellent text is Recognition and Alleviation of Pain and Distress in Laboratory Animals (Committee on Pain and Distress in Laboratory Animals, Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, 1992). There is much of value throughout the book, but start by reading Chapter 1 (Introduction), Chapter 2 (The Basis of Pain), and Chapter 3 (The Basis of Stress and Distress Not Induced by Pain). Note that the Institute of Laboratory Animal Resources (ILAR) is now the Institute for Laboratory Animal Research.

You may wish to peruse a useful follow up to this report, another ILAR/NRC publication, Definition of Pain and Distress and Reporting Requirements for Laboratory Animals, proceedings of a workshop held in 2000.

The Humane Society of the United States has a useful list of suggested resources for understanding pain and distress. Other good sites are Altweb, the Alternatives to Animal Testing Web Site, and IACUC and Pain-Related Internet Resources.

 

Ethical and Legal Obligations

While the abolitionist argument can be made that no use of animals by humans is justifiable (see Unit One), most people support traditional uses of animals, including their use in research. There are certainly both ethical and legal limitations to how animals are used, including three U.S. Government Principles that deal with the central concept of minimization of pain and distress:

IV. 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.

V. 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.

VI. Animals that would otherwise suffer severe or chronic pain or distress that cannot be relieved should be painlessly killed at the end of the procedure or, if appropriate, during the procedure.

The regulations, including the Guide for the Care and Use of Laboratory Animals, contain numerous provisions to help implement these principles, some of which were discussed in Unit Two. Examples include searching for alternatives to painful procedures, consulting with a veterinarian in the planning of studies with potential for pain or distress, and specifically justifying to the IACUC when it is necessary to withhold pain-relieving medications. Although this discussion focuses on the animal welfare aspects of minimizing pain and distress, it is important to recognize that, unless one is specifically studying pain and distress, their presence will affect the animal physiologically and can seriously disrupt the interpretation of experimental results.

One specific requirement in the AWARs is that “A proposal to conduct an activity involving animals, or to make a significant change in an ongoing activity involving animals, must contain [among other things] a description of procedures designed to assure that discomfort and pain to animals will be limited to that which is unavoidable for the conduct of scientifically valuable research, including provision for the use of analgesic, anesthetic, and tranquilizing drugs where indicated and appropriate to minimize discomfort and pain to animals…” Similar language is in the PHS Policy with regard to information that must be in grant applications to PHS agencies. This language is clearly utilitarian, and highlights the cost-benefit approach that must be taken by both researchers and regulators, including the IACUC; there is an ethical cost of inflicting pain or distress on laboratory animals that must be outweighed by the potential benefit of the research being conducted.

Another regulatory requirement unique to the AWARs is the filing by the institution of an annual report that accounts for numbers of animals used, by species and category of pain or distress (see text-block). Many IACUCs require that this information be provided in the protocol form, and the IACUC may provide additional guidelines for determining the appropriate category. (Some institutions use a different system for categorizing pain and distress.) This determination is not always straightforward, as illustrated below in the Study Questions. Difficulties in reporting animal numbers have prompted an effort by USDA to revise its policies (see Policy #11 in the Animal Care Policy Manual). This effort was the basis for a 2000 workshop convened by the Institute for Laboratory Animal Research (see Readings, above).

According to the AWARs, an annual report must be filed with the USDA. Among other things, the report must:

• State the common names and the numbers of animals being bred, conditioned, or held for use in teaching, testing, experiments, research, or surgery but not yet used for such purposes (Category B).

• State the common names and the numbers of animals upon which teaching, research, experiments, or tests were conducted involving no pain, distress, or use of pain-relieving drugs. Routine procedures (e.g., injections, tattooing, blood sampling) should be reported with this group (Category C);

• State the common names and the numbers of animals upon which experiments, teaching, research, surgery, or tests were conducted involving accompanying pain or distress to the animals and for which appropriate anesthetic, analgesic, or tranquilizing drugs were used (Category D);

• State the common names and the numbers of animals upon which teaching, experiments, research, surgery, or tests were conducted involving accompanying pain or distress to the animals and for which the use of appropriate anesthetic, analgesic, or tranquilizing drugs would have adversely affected the procedures, results, or interpretation of the teaching, research, experiments, surgery, or tests. An explanation of the procedures producing pain or distress in these animals and the reasons such drugs were not used shall be attached to the annual report (Category E).

 

 

Study Questions

1. To what USDA Category should animals in the following be assigned?

• Guinea pigs will be euthanized for collection of liver tissue. Animals are sedated prior to euthanasia in a carbon dioxide chamber.
• Dogs are used to optimize conditions for radiography. The procedure is not painful, but animals must be anesthetized to prevent movement.
• Rabbits will be used to study the course of a bacterial infection. The infection causes illness after 24 hours, but the PI will euthanize animals 12 hours after being infected and expects no clinical signs.
• In a follow up to the above study with rabbits, the PI wants to evaluate the infection at a later stage. However, the rabbits will be euthanized at the first sign of infection.

2. What is meant by “death as an endpoint,” and what are the regulatory expectations for justifying this type of experiment?

3. What is “multiple major survival surgery,” and what are the regulatory expectations for justifying this type of experiment?

 

Discussion Questions

1. Do you think the reporting of animal numbers by pain category helps promote the minimization of pain and distress? What improvements in the category definitions would you propose?

2. The Humane Society of the United States has made a detailed response to USDA about how to revise the reporting requirements for pain and distress. Does this proposal address appropriately the concerns with this reporting mechanism?

 


Definitions

Before addressing more fully how to minimize pain and distress, we discuss what these terms actually mean – not by any means an easy task.

According to the AWARs, a “painful procedure as applied to any animal means any procedure that would reasonably be expected to cause more than slight or momentary pain or distress in a human being to which that procedure was applied, that is, pain in excess of that caused by injections or other minor procedures.” Policy #11 goes on to provide several examples of a painful procedure (e.g., surgery or ocular irritancy testing), and refers to IACUC responsibilities to ensure that alternatives to such procedures have been considered appropriately by the PI; the search for alternatives was introduced in Unit Two and will be discussed further below. Policy #11 does not define distress, but again provides several examples of procedures that might cause more than momentary or slight distress, e.g., food or water deprivation.

The definitions provided in the reading assignment (pp. 3-7) are perhaps more useful.

Stress is the effect produced by external (i.e., physical or environmental) events or internal (i.e., physiologic or psychologic) factors, referred to as stressors, which induce an alteration in an animal’s biologic equilibrium…Stress might not be harmful to an animal; it might evoke responses that neither improve nor threaten an animal’s well-being…Some stress probably is necessary for well-being, if adaptation occurs with a reasonable expenditure of energy.”

Distress is an aversive state in which an animal is unable to adapt completely to stressors and the resulting stress and shows maladaptive behaviors. It can be evident in the presence of various experimental or environmental phenomena, such as abnormal feeding, absence or diminution of postprandial grooming, inappropriate social interaction with conspecifics or handlers…and inefficient reproduction…”

Pain results from potential or actual tissue damage. Pain can be considered a potent source of stress, that is, a stressor. It can also be considered a state of stress itself, however, and can lead to distress and maladaptive behaviors.”

An important distinction is made between pain and nociception. Nociception refers to the processing by the nervous system of information related to tissue damage, and it is “…qualitatively different from the perception of pain, i.e., in the interpretation of sensory information as unpleasant.” This distinction is captured in the definition of pain adopted in 1986 by the International Association for the Study of Pain: “an unpleasant sensory or emotional experience associated with actual or potential tissue damage.”

Finally, there is the question of suffering. Suffering suggests a quantitatively greater, or perhaps longer, degree of pain or distress, but it is both an imprecise and emotionally charged term. In attempting to define terms useful in this discussion, “The committee [Committee on Pain and Distress in Laboratory Animals; 1992] has chosen not to use the term suffering, which is commonly used to refer to a broad spectrum of behaviors and emotions.” (See the assigned reading, Introduction.)

 

Discussion Question

Does use of the term suffering help in discussion of animal pain and distress? How would you define it?


Anatomy and Physiology of Pain and Distress

The assigned readings do a good job of describing the basic biology of pain and distress. The following study questions will help you focus on the central issues.

 

Study Questions

1. What is a nociceptor?

2. Describe the anatomical components of, and distinction between, nociception and pain.

3. What is hyperalgesia?

4. Pain is a stressor and can lead to distress. Give several examples of stressors that are not painful.

 

Discussion Question

Based on your understanding of pain, as distinct from nociception, do you think animals experience pain in the same way as humans? Do all animals experience pain? Check out the site from graduate students at the University of Edinburgh's Animal Welfare Research Group, which summarizes the controversial issue of whether or not fish experience pain.


Minimization of Pain and Distress

1. Alternatives

The importance of considering alternatives was introduced in Unit Two in the context of protocol preparation and review by the IACUC. According to the AWARs, the IACUC must determine that, “The principal investigator has considered alternatives to procedures that may cause more than momentary or slight pain or distress to the animals, and has provided a written narrative description of the methods and sources, e. g., the Animal Welfare Information Center, used to determine that alternatives were not available.” Additional guidance on this regulation is given in the Animal Care Policy Manual, Policy #12 (see excerpts in text-block).

Alternatives or alternative methods are generally regarded as those that incorporate some aspect of replacement, reduction, or refinement of animal use in pursuit of the minimization of animal pain and distress consistent with the goals of the research. These include methods that use non-animal systems or less sentient animal species to partially or fully replace animals (for example, the use of an in vitro or insect model to replace a mammalian model), methods that reduce the number of animals to the minimum required to obtain scientifically valid data, and methods that refine animal use by lessening or eliminating pain or distress and, thereby, enhancing animal well-being. Potential alternatives that do not allow the attainment of the goals of the research are not, by definition, alternatives.” (From the USDA Animal Care Policy Manual, Policy #12.)

 

A key component in the consideration of alternatives are so-called refinements. This is a broad category that includes not only obvious measures such as the use of anesthetics and analgesics in procedures known to be painful, but also measures that are the joint responsibility of the research, animal care, and veterinary staff, to ensure that animals are housed in an appropriate environment, kept disease-free, and properly cared for and handled. It also includes monitoring of animals by individuals who can recognize behavioral abnormalities that might signal pain or distress. (The assessment of pain and distress is addressed in the next section.) Regulatory expectations for personnel qualifications and training were discussed in Unit Two, but the issue bears repeating in this context. Properly trained personnel at all levels of the research enterprise are one of the most important ways to help ensure that animal pain and distress are minimized.

The Animal Welfare Information Center, part of the National Agriculture Library, was created as part of the 1985 amendments to the AWA, and provides assistance to researchers in the search for alternatives. The AWIC can be contacted directly for assistance, but the web site also has links to useful on-line guidance.

 

2. Assessment of pain and distress

Many procedures performed in research animals can rightly be assumed to be painful, based on their ability to cause pain in humans; this is the language in U.S. Government Principle IV. However, there is also legitimate need to customize pain and distress treatment in individual animals, and this requires tools to recognize signs of pain and distress.

A frequently-cited paper (Morton DB, Griffiths PHM. 1985. Guidelines on the recognition of pain and discomfort in experimental animals and an hypothesis for assessment. Vet Rec 116:431-436) proposed a set of observations for assessing pain and distress; these included change in body weight, external physical appearance, clinical signs, and changes in behavior. A refinement of this approach has been described by E. Carstens and Gary P. Moberg in an article (Recognizing Pain and Distress in Laboratory Animals) in a recent ILAR Journal (Vol 41(2), 2000) focused on “Humane Endpoints for Animals Used in Biomedical Research and Testing.” Carstens and Moberg suggest evaluation of pain in three categories: general behavior (e.g., activity, appetite), appearance (e.g., self-grooming, posture), and physiology (e.g., body temperature, respiratory pattern).

The assessment of distress presents additional difficulty. While there are physiological changes characteristic of distress that could be measured, they often involve obtaining samples (e.g., blood) by methods that may themselves introduce stress. Like pain, there are behavioral correlates of stress, but they are not well characterized; importantly, there is even less known about how to distinguish stress, which is a normal part of life, from distress.

The assessment of pain and distress in animals requires knowledge of both normal behavior and those behaviors that might indicate pain or distress. Those who are responsible for routine animal care, as well as research staff who are involved with potentially painful or distressful experimentation, should seek training to help with this important task. An excellent starting point is Chapter 4 (Recognition and Assessment of Pain, Stress, and Distress) in the Recognition and Alleviation of Pain and Distress in Laboratory Animals.

 

3. Veterinary care

While research staff are expected to be experts in their area of research, and familiar, if not also experts, with their particular animal model, there are regulatory expectations for both the existence and expertise of an institutional laboratory animal veterinarian. As pointed out in Unit Two, veterinary staff must be consulted in the planning of studies involving potential pain or distress; planning may involve both the preparation of the protocol for IACUC submission and training in the experimental methods. Veterinary consultation is valuable for understanding:

• regimens for use of anesthetics, analgesics, and tranquilizers, including monitoring;
• how to recognize species-specific signs of pain and distress;
• surgical approaches and aseptic technique;
• post-procedural monitoring and intensive care.

A good place to read more about the role of the laboratory animal veterinarian is in the Public Statement on Adequate Veterinary Care by the American College of Laboratory Animal Medicine.

 

“ A veterinary-care program is the responsibility of the attending veterinarian, who is certified (see ACLAM, Appendix B) or has training or experience in laboratory animal science and medicine or in the care of the species being used…The veterinarian must provide guidance to investigators and all personnel involved in the care and use of animals to ensure appropriate handling, immobilization, sedation, analgesia, anesthesia, and euthanasia.” (Guide for the Care and Use of Laboratory Animals, 1996, p. 56)

 

The laboratory animal veterinarian may provide guidance on the use of anesthetics, analgesics and tranquilizers in a variety of ways, often including written institutional guidelines; an excellent example is from the University of Minnesota. There are also some excellent texts, including those by Paul A. Flecknell (Laboratory Animal Anaesthesia, 2nd ed. Academic Press, London, 1996) and the comprehensive text produced by the American College of Laboratory Animal Medicine (Kohn, DF et al. (eds). Anesthesia and Analgesia in Laboratory Animals. Academic Press, San Diego, 1997).

Another basic veterinary care issue is surgery, and there are a number of components of experimental surgery that directly impact the minimization of pain and distress. Some of these apply even for terminal procedures, i.e., those in which animals will not recover. The University of Minnesota again does a nice job of discussing these issues. They include:

• adequate acclimation and handling of animals to provide a stress-free anesthetic induction;
• choice of appropriate anesthetic and analgesic regimens to minimize pain both intraoperatively and postoperatively;
• maintenance of physiologic homeostasis during surgery, e.g., by providing fluids and supplemental heat;
• monitoring of anesthesia to ensure both adequate depth and avoid over dosage;
• dedicated facilities and aseptic technique to help prevent infection;
• proper surgical technique, to help minimize postoperative pain, prevent infection, and promote healing;
• postoperative monitoring to ensure smooth recovery, return to normal function, and detect pain.

Appropriate veterinary care of course includes the recognition, treatment and control of animal disease. The goal in modern research animal operations is to avoid all (unplanned) disease in the experimental animal. One reason is perhaps obvious in the current context, i.e., to minimize potential pain and distress associated with overt manifestations of severe disease, i.e., morbidity and mortality. Laboratory animal veterinarians also expend considerable effort in controlling even subclinical disease, because this may contribute to unwanted variation in the experimental study (more in Unit Four). A useful resource for understanding health monitoring of research animals is the Research Animal Diagnostic Laboratory at the University of Missouri.

 

Study Question

What is the difference between an anesthetic and an analgesic?

 

4. Euthanasia

Most research animals will be euthanized as part of the experiment, and it is important that pain and distress be minimized in this context, for all the reasons discussed above. The principle standards document used to assist in providing optimal euthanasia, from the standpoint of both the animal and the experimentation, is the “2000 Report of the AVMA [American Veterinary Medical Association] Panel on Euthanasia.”

 

“The term euthanasia is derived from the Greek terms eu meaning good and thanatos meaning death. A “good death” would be one that occurs with minimal pain and distress. In the context of this report, euthanasia is the act of inducing humane death in an animal. It is our responsibility as veterinarians and human beings to ensure that if an animal’s life is to be taken, it is done with the highest degree of respect, and with an emphasis on making the death as painless and distress free as possible. Euthanasia techniques should result in rapid loss of consciousness followed by cardiac or respiratory arrest and the ultimate loss of brain function. In addition, the technique should minimize distress and anxiety experienced by the animal prior to loss of consciousness.” (2000 Report of the AVMA Panel on Euthanasia, JAVMA, Vol 218(5), March 1, 2001.)

 

Providing a “good death” (see text-block) requires an understanding of not only the physiology of pain and distress (there is another good discussion of this in the Introduction of the Panel Report), but also the mechanisms of the various euthanasia methods and the proper techniques to perform them. As part of the procedure, it is also essential that death be confirmed, in order to avoid inadvertent recovery of the animal. Finally, there is an important human component to animal euthanasia, which involves both basic safety issues in performing the techniques and the normal psychological responses that we have when killing an animal.

The Panel Report discusses three general mechanisms of inducing death, but the central requirement in all acceptable methods is that, “Euthanasia techniques should result in rapid loss of consciousness followed by cardiac or respiratory arrest and the ultimate loss of brain function.” Methods that work by inducing cardiac or respiratory arrest without rapid loss of consciousness (poisoning is one example) are not acceptable.

Discussion of specific methods in the Panel Report is divided into three categories: inhalant (e.g., inhalant anesthetic overdosage or carbon dioxide), noninhalant pharmaceutical (e.g., injectable anesthetic overdosage orbarbiturate euthanasia solutions), and physical (e.g., cervical dislocation or decapitation). There is also discussion of special considerations, such as euthanasia of nonconventional species (e.g., wildlife, birds, and ectotherms).

Given the basic criteria for ensuring a “good death,” it makes sense that anesthetic overdosage is generally acceptable; assuming that there is proper handling of the animal and administration of the agent, the next step, by definition, is loss of consciousness. However, use of anesthetic drugs may not always be appropriate. One reason is that drugs may interfere with experimental use of organs or tissues after euthanasia. It is, therefore, necessary at times to use physical methods of euthanasia, such as cervical dislocation and decapitation. Both methods are considered to be “conditionally acceptable,” largely because their humaneness depends in large measure on technical skill. When these methods are used in a research setting, they must be justified for scientific reasons and approved by the IACUC.

Carbon dioxide inhalation is used commonly in rodents for euthanasia. It is considered in the Panel Report to be an acceptable method of euthanasia for a variety of species, with certain caveats that are part of the Panel Recommendations. It is worth noting, however, that there is current debate on this issue; unfortunately, the scientific literature is by no means conclusive.

 

Study Questions

1. According to the Animal Care Policy Manual, Policy #12, what specific elements must be included in a literature search for alternatives?

2. What are several specific signs of pain or distress in the mouse?

3. What is meant by aseptic technique?

4. Is injection of potassium chloride (KCl) an acceptable method of euthanasia? Why are cyanide and strychnine considered unacceptable?

 

Discussion Question

According to the regulatory language, the requirement to consider alternatives is for procedures that may cause more than momentary or slight pain or distress to the animals. Should alternatives be considered for all use of animals in research?

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