5
Control of Pain

The goal of this chapter is to provide guidance to investigators and veterinarians in the selection of suitable pharmacologic agents and management strategies for the prevention or alleviation of pain in laboratory animals. The first section provides a rational basis for the pharmacologic control of pain. It discusses the pharmacology of general anesthesia in laboratory animals and describes the major classes of drugs used to achieve the clinical goals of analgesia, sedation, and restraint (see Table 5-1). Discussion of the major drug classes is organized for the reader to extract information along three dimensions—clinical use, pharmacologic effects, and dose recommendations—each related to particular species. The second part addresses the nonpharmacologic control of pain and deals with the use of hypothermia, tonic immobility, and acupuncture.

The first part of this chapter focuses on experimental paradigms in which pain is an integral part of the study, but the concepts presented should also be considered applicable to pain of clinical, surgical, or inapparent sources.

An important idea regarding laboratory animals in pain involves an anthropomorphic analogy that animals should not be exposed to pain greater than human beings would tolerate (Bowd, 1980) and that, "unless the contrary is established, investigators should consider that procedures that cause pain and distress in human beings may cause pain or distress in other animals" (IRAC, 1985). Human subjects in pain studies are exposed only to painful stimuli that they will tolerate, and they can remove a painful stimulus at any time. Low levels of pain (near the pain threshold) induce minimal discomfort and stress and are well tolerated by animals or human subjects. Most human pain is of this type, and it is part of our daily



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Recognition and Alleviation of Pain and Distress in Laboratory Animals 5 Control of Pain The goal of this chapter is to provide guidance to investigators and veterinarians in the selection of suitable pharmacologic agents and management strategies for the prevention or alleviation of pain in laboratory animals. The first section provides a rational basis for the pharmacologic control of pain. It discusses the pharmacology of general anesthesia in laboratory animals and describes the major classes of drugs used to achieve the clinical goals of analgesia, sedation, and restraint (see Table 5-1). Discussion of the major drug classes is organized for the reader to extract information along three dimensions—clinical use, pharmacologic effects, and dose recommendations—each related to particular species. The second part addresses the nonpharmacologic control of pain and deals with the use of hypothermia, tonic immobility, and acupuncture. The first part of this chapter focuses on experimental paradigms in which pain is an integral part of the study, but the concepts presented should also be considered applicable to pain of clinical, surgical, or inapparent sources. An important idea regarding laboratory animals in pain involves an anthropomorphic analogy that animals should not be exposed to pain greater than human beings would tolerate (Bowd, 1980) and that, "unless the contrary is established, investigators should consider that procedures that cause pain and distress in human beings may cause pain or distress in other animals" (IRAC, 1985). Human subjects in pain studies are exposed only to painful stimuli that they will tolerate, and they can remove a painful stimulus at any time. Low levels of pain (near the pain threshold) induce minimal discomfort and stress and are well tolerated by animals or human subjects. Most human pain is of this type, and it is part of our daily

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Recognition and Alleviation of Pain and Distress in Laboratory Animals experience. Only when pain becomes severe (approaching pain tolerance levels) is our behavior dominated by attempts to avoid or escape it. That degree of pain needs to be alleviated. In pain studies, giving animals control over the source of pain is an effective way to let them minimize it. In that situation, escape-avoidance behavior is an appropriate adaptive response. An animal displaying such behavior might be experiencing discomfort, but is not yet in distress (see Table 1-3). However, if the animal is denied control of the stimulus and it approaches or exceeds the limit of tolerance, maladaptive behaviors will appear and the animal should be considered to be in distress. In distressed animals, maladaptive behaviors can include persistent attacks on the perceived source of the pain (e.g., electrified grid floors or other parts of the apparatus), prolonged immobilization or ''freezing," self-mutilation of the area of the body receiving the stimulus, or a state of learned helplessness in which the animal gives up and no longer attempts to escape, avoid, or control the stimulus. In most experiments involving the study of pain, the animal or the investigator can set a limit on the magnitude and duration of the stimulus. The investigator must determine the intensity and duration of the pain that the animal experiences and minimize it. Four general approaches are available to minimize pain (Dubner, 1987): use of general anesthesia or neurosurgery, use of local anesthesia and analgesia, training of animals to control the stimulus, and control of the stimulus by the investigator. When an invasive procedure is performed on an appropriately anesthetized animal, there is little concern about pain as long as the animal remains anesthetized. The anesthetic state can be monitored by assessing pupillary size, palpebral and toepinch response, stability of heart rate and blood pressure, and electroencephalographic activity. In a study in which anesthetic agents would confound the data, surgical lesions of the central nervous system can be made under anesthesia, the animal allowed to recover from anesthesia, and data collected on a functional decerebrate with no possibility of conscious* sensation. An alternative to anesthesia or neurosurgery in some studies, and often a valuable adjunct to anesthesia, is the administration of local anesthetic or analgesic agents to eliminate or reduce pain. Some studies are concerned with pain mechanisms and the relationship between pain-related behavior and neurobiologic processes and require that animals be exposed to actual or potential tissue damage. In others, pain might be a consequence of techniques and methods whose purposes are unrelated to the nature or magnitude of the pain produced. In most such investigations with conscious animals, the animals can be taught to avoid or escape painful stimuli. An important distinction should be made between reflexes associated with nociception and the maladaptive behaviors associated with distress when pain *   The terms conscious and consciousness are used throughout this text to refer to the awake state, the state in which stimuli can be perceived (e.g., "The conscious animal is aware of its surroundings"). Unconsciousness refers to the state in which stimuli are not perceived (e.g., "The anesthetic should produce a loss of consciousness").

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Recognition and Alleviation of Pain and Distress in Laboratory Animals exceeds the limit of tolerance. A nociceptive reflex can occur without the perception of pain. In studies in which animals experience pain, methods that allow them to control the intensity and duration of pain include withdrawal responses elicited by stimulating their paws or tails so that they move their paws or tails away from the source of aversive stimulation (Dubner, 1987). In tasks that use such reflexive measures, animals often need to be placed under considerable restraint, and that might produce stress that can influence the outcome of an experiment (Gärtner et al., 1980; Pare and Glavin, 1986). Such stress is minimized by the use of unrestrained escape-avoidance tasks, in which animals are taught to control the stimulus. The teaching uses operant-conditioning procedures and closely mimics conditions under which humans participate in experimental pain studies: animals choose to participate by initiating trials (e.g., to obtain food), they determine the magnitudes of aversive stimulation that they will accept by refusing to obtain food and escaping intolerable stimuli, and they can withdraw from the experiment by ceasing to initiate new trials. The most difficult types of experiments in which pain should be minimized include those in which animals cannot control the magnitude of the painful stimulus. In such experiments, the investigator or veterinarian must assess the pain (see Chapter 4). That should be done first by knowledgeable assessment of the intensity of the stimulus and second, when possible, by having the investigator receive the maximal stimulus that it is possible to deliver to the animal in the protocol being used. The committee believes that that practice can yield a useful estimate of stimulus potency. Others believe that so little might be known about the stimulus characteristics that it would be difficult to know what to match when the same stimulus is applied to another animal; if this is the case, it can lead to an underestimation of the pain that a given animal might experience. Whenever an animal is in an experimental situation and after its return to its home cage, its behavior should be carefully monitored. While it is in an experimental trial, does it attack the experimental apparatus or parts of its own body? Does it control the stimulus through smoothly coordinated behaviors? When returned to its home cage, does it have an abnormal gait and appear unusually aggressive? Does it look at or groom the injured area excessively? Does it have an unusual posture and guard an injured limb excessively? Does it have normal food and water intake and maintain its body weight in comparison with control animals? Does it exhibit normal social adjustment when placed in a cage with other animals of the same species and respond normally to its handler? Is its sleeping-waking cycle normal? Does it exhibit abnormal vocalization, such as whimpering or crying? Has its species-typical behavior changed? Questions like those underlie the importance of knowing the normal behavioral characteristics of a particular species and the intensity of aversive stimuli. Although the answer to any one of the above questions alone does not necessarily indicate that an animal is in pain or distress, the presence of changes in several elements of behavior does suggest that pain is intolerable. The experimenter and a veterinarian should determine whether the pain can be reduced and whether the experiment can be performed with less pain.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals PHARMACOLOGIC CONTROL OF PAIN In studies in which pain is not the focus, pharmacologic control of pain is usually required. The use of drugs requires consideration of several interacting factors (Short and Van Poznak, 1992). • What is the clinical goal of drug administration? Common clinical goals include general anesthesia for surgical procedures, lighter general anesthesia for experimental studies that cannot be conducted in awake animals, sedation and analgesia for minor surgical and diagnostic procedures, management of postsurgical pain and pain associated with disease, sedation and tranquilization for the relief of non-pain-induced stress or distress, and temporary restraint. • What are the pharmacologic actions of the drugs being considered? That is important if they will be used before an experimental study. Knowledge of the pharmacology, pharmacodynamics, duration of action, species-typical actions, and specific actions on the organ systems under study is important for proper interpretation of experimental data. Drugs that are least likely to influence the systems under study should be chosen. Pain and pain-induced distress can be adequately alleviated pharmacologically, but drug actions often confound interpretation of experimental results, because every drug has actions in addition to the one for which it is used (e.g., pain relief). Nevertheless, pharmacologic management is efficacious, often necessary, and usually experimentally acceptable. Knowledge of the so-called side-effects of drugs, however, is important both to the well-being of animals and to an understanding of their potential contributions to experimental outcomes. • How do species vary in their responses to the drugs being considered, and how are responses affected by other factors? Actions and doses in one animal species might not be relevant in another species. Not only do dosages vary by species, breed, and strain, but other factors—such as sex, environment, age, nutrition, and health status—play a major role; the very young, old, or obese present a greater anesthetic challenge. For example, male mice sleep longer than female mice after the administration of pentobarbital. Environmental factors have been shown to affect hepatic drug-metabolizing enzyme systems (microsomal liver enzymes). Softwood bedding, such as pine or cedar, contains aromatic amines that induce the hepatic microsomal enzymes, thereby increasing barbiturate metabolism and reducing sleeping time. In contrast, hardwood bedding or the absence of bedding (as in the use of wire-bottom cages) allows hepatic enzymes to remain at a resting state, so sleeping times might be longer. A dirty environment might inhibit drug-metabolizing enzymes and thus prolong anesthesia. Anesthetics given repeatedly at short intervals can induce hepatic microsomes and thus increase anesthetic metabolism and decrease sleeping time. Fasting is important especially in herbivorous species, in which ingesta can account for a substantial portion of an animal's body weight; the gastrointestinal tracts of herbivores are not empty after a 24-hour fast.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals Drugs categorized as general anesthetics, sedatives, hypnotics, ataractics, anxiolytics, tranquilizers, nonsteroidal anti-inflammatory drugs, and opioids all have been used to prevent or minimize pain and distress produced by painful and nonpainful procedures and to produce unconsciousness and analgesia for surgical procedures. Barbiturates and inhalational anesthetics are considered general anesthetics and are commonly used as total anesthetic agents, that is, drugs that produce unconsciousness and muscle relaxation sufficient for surgical intervention. Barbiturates are also considered hypnotics and are used in veterinary medicine as general anesthetics. General anesthetics produce a total loss of awareness and of responsiveness to painful stimuli during surgical procedures. Other drugs are also used to alleviate pain and distress. Neuroleptanalgesics, dissociative anesthetics, and continuously infused potent opioids in people or animals premedicated with low doses of hypnotics or benzodiazepines are used for producing surgical anesthesia. Opioids are used because of their cardiovascular-sparing aspects and lack of profound depression of the central nervous system (CNS); they should therefore be considered for studies involving the CNS and cardiovascular system. The opioid agonists, which include the morphine-like drugs (e.g., fentanyl and oxymorphone), produce analgesia and sedation through the activation of opioid receptors in the CNS. The dissociative anesthetic agent ketamine has both analgesic and anesthetic properties, but should be combined with other drugs for major procedures. The anti-inflammatory action of the nonsteroidal anti-inflammatory drugs is a major factor in their analgesic effects. It should be emphasized that many of the drugs to be discussed here, with the exception of the opioids, are CNS depressants and might not be good analgesic. Tranquilizers, ataractics, and hypnotics can be used as adjuncts in the treatment of non-pain-induced stress and distress, but not for the treatment of pain. Many combinations of drugs have been used in various species for analgesia, sedation, restraint, and general anesthesia. Table 5-1 shows clinical uses of drugs in various classes included in this chapter. GENERAL ANESTHESIA The aim of general anesthesia is to produce unconsciousness as rapidly and as smoothly as possible and to maintain a depth of anesthesia appropriate for the objectives of the surgery or study. The depth of anesthesia might have to be varied on the basis of the intensity of the stimulation and changing physiologic conditions, as the anesthetist attempts to balance adequate anesthesia and analgesia with physiologic function. The level of anesthesia required can vary from moderate for surgical intervention to light for the study period that follows. Considerable information is available on the pharmacologic effects of anesthetic agents on organ functions in some species (Merin, 1975; Covino et al., 1985; Miller, 1990). General anesthesia can affect all physiologic functions; for inhalational anesthetics, the magnitude of the affects is related to the alveolar concentration of the specific drug.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals TABLE 5-1 Use of Drugs Category Analgesia Anxiolysis Sedation Anesthesia Inhalational anesthetic — — — PA Barbiturates — PA PA PA Cyclohexamines PA — — PA Neuroleptanalgesics PA — PA SA-C Benzodiazepines — PA — C Tranquilizers — PA PA C Opioid agonists PA — PA C Opiod agonist-antagonists PA — SA — Nonsteroidal anti-inflammatory drugs PA — — — NOTE: PA = primary action; SA = secondary action; C = combined with other drugs. Research animals undergoing general anesthesia differ somewhat from animals normally encountered in clinical veterinary practice. Research animals usually are not ill or traumatized and generally are of the same species as the other animals within the study and have similar characteristics. Once a research model has been selected, consistency between animals is very important. Whether recovery and postoperative care are part of the experimental protocol or recovery is not intended, the concern for the management of anesthesia and care during the procedures should be identical, because it should be assumed that general anesthesia will affect whatever system is under study. The investigator should perturb the organ system under study only during a steady state of anesthesia (Soma and Klide, 1987; Some et al., 1988a); this minimizes the effect of changing depths of anesthesia on the animal and allows interpretation of data from the standpoint of a known, established baseline. Data from different laboratories often are not easily compared, and differences in methods of anesthesia can contribute to the difficulty. Ultimate goals of clinical and investigative anesthesia might differ, but the basic principles of good anesthetic techniques do not: maintain a depth of anesthesia that minimizes changes in physiologic function, blocks response to stimulation, and produces unconsciousness. Inhalational Anesthesia The development of inhalational anesthesia for both companion and laboratory animals has progressed a great deal in the last 20 years. It is beyond the scope of this section to describe the various methods and equipment that can be used for the administration of inhalational anesthesia to a broad range of animals, but information is readily available (Soma, 1971; Lumb and Jones, 1984; Hartsfield, 1987; Klide, 1989). Inhalational anesthesia can be administered by mask for a short procedure in most animals, but for longer periods of anesthesia the trachea should

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Recognition and Alleviation of Pain and Distress in Laboratory Animals be intubated for better control of the airway and depth of anesthesia. The ease of tracheal intubation varies from difficult in rodents and ruminants to easy in carnivores (Lumb and Jones, 1984; Short, 1987). The advantages of inhalational anesthesia for both the preparative and study periods include the ability to define and vary the depth of anesthesia and estimate the alveolar concentration. The end-tidal alveolar concentration of an anesthetic at which 50% of animals (ED50) will not respond to a specified painful stimulus is the minimal alveolar concentration (MAC) (Eger et al., 1965; Eger, 1978) (Table 5-2). At 1.0 MAC, animals are unconscious and under anesthesia, but 50% will respond to a noxious stimulus. Adequate surgical anesthesia occurs between 1.25 and 1.50 MAC, and 1.3 MAC is considered the ED95. At 0.4 MAC, humans can respond to verbal commands (Stoelting et al., 1970). The MAC in some animals is slightly higher than that measured in humans (White et al., 1974; Steffey et al., 1977; Eger, 1978; Weiskopf and Bogetz, 1984); this could be a real species difference or could be due to the use of different measurement criteria. Despite some differences, the MAC is roughly consistent between species. It must be emphasized that the MAC represents alveolar concentration and that it is measured as end-tidal concentration. The MAC is agent-specific and can be used to establish a consistent depth of anesthesia and to compare agents. The MAC also defines the potency of inhalational agents; the lower the MAC, the more potent the anesthetic. The selection, measurement, and maintenance of the end-tidal concentration at some multiple of the MAC will allow light anesthesia to be used with neuromuscular blocking agents while ensuring an adequate depth of anesthesia. Under these circumstances, the investigator can be confident that the animal is unconscious. The MAC can be used to establish a consistent depth of anesthesia during a study and between animals. The establishment of the MAC for a study ensures that changes in physiologic functions are fairly consistent and are not due to changes in the depth of anesthesia. TABLE 5-2 MAC Values of Anesthetics in Different Species (% End-Tidal Alveolar Concentration)a Agent Dog Human Cat Rat or Mouse Goldfish Toad Horse Swine Enflurance 2.20 1.68 1.20     2.12     Ether 3.04 1.92 2.10 3.20 2.20 1.63     Halothane 0.87 0.75 0.82 0.95 0.76 0.67 0.88 1.25 Isoflurane 1.50 1.15   1.38   1.31     Methoxyflurane 0.23 0.16 0.23 0.22 0.13 0.22     Nitrous oxideb 200 101 136     190     a Data from Stimpfel and Gershey, 1991; Eger, 1978. Lower MAC values in % end-tidal concentrations indicate greater potency. b MAC values for nitrous oxide are derived from hyperbaric studies to establish potency. Greater than 100% anesthetic is not possible in clinical situations.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals Nitrous oxide is not commonly used as an anesthetic for laboratory animals and probably should not be, for two reasons. Its analgesic potency is low—the MAC (Table 5-2) exceeds 100% end-tidal concentration of the anesthetic for most species (Stimpfel and Gershey, 1991)—and it is potentially toxic to the reproductive system and bone marrow of personnel when used alone or in combination with other volatile agents without appropriate ventilation (Stoelting, 1987). The end-tidal concentration of an inhalational agent is established by collecting a sample of gas from the endotracheal tube at the end of exhalation. Usually, a small needle is placed in the endotracheal tube and sealed to prevent leaks. Sampling should start when a state of equilibrium has been achieved at the concentration being delivered. With modern agents (e.g., halothane and isoflurane), that occurs after the induction of anesthesia when a predetermined level of anesthetic has been maintained for 10–15 minutes (Soma et al., 1988a). End-tidal samples can be collected in animals that weigh 1–2 kg with a 1-ml gastight syringe; small amounts of gas are collected at each breath until the volume of gas necessary for measurement has been collected. In larger animals, a greater volume can be collected at each breath; the volume of gas collected at each breath should be scaled to the size of the animal, to prevent dilution of the end-tidal sample with gas in the anesthetic equipment. Gas samples collected with a syringe can be measured with a gas chromatograph calibrated for the specific anesthetic agent. Quadripole mass spectrometers also have been developed for respiratory-gas and anesthetic-gas measurements; their use allows breath-by-breath analysis of all exhaled gas (Marquette Gas Analysis Corp., St. Louis, Mo.; Brueland Kjaer Institute, Marlborough, Mass.). Gas-specific anesthetic monitors that allow breath-by-breath continuous monitoring of an inhalational agent are available (Puritan Bennett Corp., Kansas City, Mo.; Sensor Medics Corp., Anaheim, Calif.; Biochem International, Waukesha, Wis.). The continuous monitors might not be suitable for animals smaller than cats, because continuous flows of gas are necessary at 50 ml/min to measure end-tidal samples; in these circumstances, continuous flows will dilute the end-tidal sample and produce a concentration lower than the true one. Intravenous Anesthesia Inhalational anesthesia is often preferred for major surgical procedures, but steady-state anesthesia with some drugs can also can be obtained through intravenous administration. The ideal method to establish a steady-state level of anesthesia is to use pharmacokinetic measurements to calculate loading and infusion rates. The pharmacokinetic values used are the disposition and elimination rate constants of a drug in a particular species. Those vary with species, but they have been developed for many drugs in many species (Rigg et al., 1981; Pry-Roberts and Hug, 1984; Soma and Klide, 1987; Ludders, 1992).

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Recognition and Alleviation of Pain and Distress in Laboratory Animals BARBITURATES Common Examples Ultra-short-acting methylated oxybarbiturates (methohexital and hexobarbital) and thiobarbiturates (thiopental, thialbarbital, and thiamylal), short-acting oxybarbiturates (pentobarbital), and long-acting oxybarbiturates (phenobarbital). Clinical Use The barbiturates are widely used and versatile. They are classified as sedative-hypnotics on the basis of their use in humans to produce sedation. In veterinary anesthesia, they are used primarily to induce and maintain general anesthesia. An important distinction between the actions of the sedative-hypnotic barbiturates and the inhalational anesthetics and the actions of other drugs described in this chapter is that progressive increases in the dose of barbiturates and inhalational anesthetics produces progressive depression of the CNS and all physiologic functions, which leads to the loss of consciousness and even to death. The barbiturates are classified according to their duration of action. The ultra-short-acting barbiturates are commonly used to induce anesthesia before maintenance with the inhalational anesthetics. The amount necessary is less than that required for surgical intervention; all that is necessary is anesthesia of adequate depth to produce sufficient muscle relaxation for tracheal intubation. A likely dose for this purpose is approximately 8–12 mg/kg. The final dose depends on the degree of sedation produced by drugs used for preanesthetic medication. Deep sedation produced by opioids requires lower induction doses of ultra-short-acting barbiturates than are required when no preanesthetic is used. The ultra-short-acting thiopental, thialbarbital, and thiamylal are used principally to induce anesthesia; they are followed by maintenance with an inhalational agent or used alone for short periods. The long-acting phenobarbital is used primarily in anticonvulsant therapy or for prolonged sedation. Pharmacologic Effects The short duration of the ultra-short-acting thiobarbiturates is the result of a rapid decrease in plasma and therefore brain concentration. The rapid decrease is due to rapid distribution into nonnervous tissue—initially into highly perfused visceral tissues (heart, brain, liver, spleen, etc.) and then into muscle and fat. The final elimination of the drug is through metabolism and renal excretion of metabolites. The ultra-short action of the methylated oxybarbiturate methohexital is attributed to rapid metabolism, rather than redistribution into nonnervous tissues. The short-acting barbiturate pentobarbital is biotransformed in the same way as

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Recognition and Alleviation of Pain and Distress in Laboratory Animals methohexital, but at a lower rate. All clinically used barbiturates are metabolized by the liver, except phenobarbital, which is excreted intact by the kidneys. The barbiturates belong to a group of agents that depress the CNS in a nonselective and dose-dependent manner (Gilman et al., 1990). Barbiturates depress respiratory drive by reducing CNS response to the stimulatory effects of CO2; at high doses, the hypoxic respiratory drive is also suppressed. Barbiturates also depress protective reflexes, such as coughing and laryngeal reflexes. Under light anesthesia, the reflexes are present, and intubation of the trachea stimulates cough and laryngeal closure. Deep anesthesia abolishes those reflexes. Barbiturates, like other general anesthetics, reduce blood pressure, cardiac output, and renal blood flow, but increase or do not change heart rate. Barbiturates are poor analgesics and poor muscle relaxants compared with other general anesthetics, and they require deeper anesthesia for surgery. The effects and dosage of barbiturates are markedly affected by other CNS depressants, and the dose should be modified according to the administration of preanesthetic medication. Dose Recommendations (Table 5-3) The ultra-short-acting thiobarbiturates can be given rapidly intravenously. Their transfer from plasma to brain is very rapid, because of their high lipid solubility, and the effect of an injection is noted quickly. Later doses should be given slowly, carefully, and at short intervals to attain the desired depth of anesthesia. The most commonly used short-acting barbiturate is sodium pentobarbital. The safest method of administration is the intravenous route. Oral, intraperitoneal, intramuscular, and even subcutaneous routes have been used, but they are less reliable for induction, are less consistent in recovery time, and can cause sloughing of tissue when injected perivascularly because of the high pH of their solutions (pH, 10 or above). Sodium pentobarbital is usually supplied as a 6% solution (60 mg/ml). If it is to be administered to smaller species, it should first be diluted (1:1 or 30 mg/ml for intravenous use, and 1:9 or 6 mg/ml for intraperitoneal use) to achieve a better control of the dose. The recommended method of anesthetizing healthy animals with intravenous pentobarbital is to inject rapidly one-third to one-half the total calculated dose and then slowly infuse more to the desired depth of anesthesia. Ill animals are at an increased risk of effects of anesthesia and require less anesthetic. The oxybarbiturates (including pentobarbital) transfer across the blood-brain barrier slowly. Thus, an initial rapid injection is recommended to produce a high initial plasma concentration to avoid the excitement that can occur during the induction of anesthesia with pentobarbital. Because veins are often not readily accessible in laboratory rodents, the most common route of administration of pentobarbital is intraperitoneal. Slower induction, longer recovery, more variable anesthesia, and a greater mortality rate can be

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Recognition and Alleviation of Pain and Distress in Laboratory Animals TABLE 5-3 Doses of Barbiturates in Various Species Species Drug Dose, mg/kg (Routea) References Dog and cat Pentobarbital 25–30 (iv) Warren, 1983   Thiopental 8–30 (iv) Warren, 1983   Thiamylal 6–25 (iv) Warren, 1983 Guinea pig Pentobarbital 15–30 (iv) Hughes et al., 1975   Thiopental 20 (iv) Hughes et al., 1975 Hamster Pentobarbital 50–90 (ip) Hughes, 1981   Thiopental 20 (iv) Vanderlip and Gilroy, 1981 Mouse Pentobarbital 40–90 (ip) Hughes, 1981; Clifford, 1984   Thiopental 25–50 (iv) Hughes, 1981; Clifford, 1984   Thiamylal 25–50 (iv) Harkness and Wagner, 1983; Clifford, 1984 Rabbit Pentobarbital 40 (ip) Vanderlip and Gilroy, 1981   Pentobarbital 20–40 (iv) Hughes, 1981   Thiopental 15–50 (iv) Sedgewick, 1980; Hughes, 1981   Thiamylal 15–30 (iv) Sedgewick, 1980; Vanderlip and Gilroy, 1981 Rat Pentobarbital 25–45 (ip) Hughes, 1981; Clifford, 1984   Thiopental 20–25 (iv) Sedgewick, 1980; Clifford, 1984   Thiamylal 20–50 (iv) Sedgewick, 1980; Clifford, 1984 a iv = intravenous; ip = intraperitoneal. expected with intraperitoneal administration. Excitement can occur during the induction period because of the slow absorption from the peritoneal cavity. Variability in dose and sleeping time has been reported, especially in rats and mice. A dose range of 30–60 mg/kg has been reported for rodents (Wixson et al., 1987a), but doses as high as 90 mg/kg have also been given (Hughes, 1981). The short-acting pentobarbital can be used in smaller species of laboratory and companion animals for 2–3 hours of general anesthesia. Sex, age, diet, type of bedding, and strain have been suggested as reasons for dose variability (Harkness and Wagner, 1983; Clifford, 1984). Therefore, a specific dose might have to be established for a given colony of rodents. Variability has also been noted in rabbits. DISSOCIATIVE ANESTHETICS-CYCLOHEXAMINES Common Example Ketamine hydrochloride and tiletamine-zolazepam combination (Telazol®).

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Recognition and Alleviation of Pain and Distress in Laboratory Animals or slightly higher doses, are necessary. Good agreement was found in the transposition of relative drug potencies from monkeys to humans (Malis, 1973). Meperidine can be combined with a tranquilizer to produce sedation in cats and other animals in which excitement can occur (Clifford, 1957). The dose of meperidine in nonrodents, with or without a tranquilizer, should not exceed 10 mg/kg by the intramuscular route. The lack of marked sedation after the use of meperidine or other opioids in cats and other animals that lack the classic response to opioids should not mislead clinicians into administering general anesthetics and other depressants later at normal rates and dosages. Meperidine also has been used in rats, rabbits, guinea pigs, and mice (Hughes, 1981). The doses in rodents are variable and generally higher than in nonrodents. The durations of analgesic action of meperidine in rats at 5, 10, and 20 mg/kg were 2, 45, and 72 minutes, respectively. At the time of disappearance of analgesia, the plasma concentrations were similar (200–220 ng/ml) after the three different doses (Dahlstrom et al., 1979). In horses and ponies, meperidine has been used for pain, especially after gastrointestinal and orthopedic surgery. The suggested dose is 2.2 mg/kg administered intramuscularly; it should not be given intravenously. The analgesic effect of oxymorphone in dogs and cats has long been recognized (Palminteri, 1963). Oxymorphone has been shown to have several important advantages over morphine and meperidine in dogs (Copland et al., 1987). For example, it does not promote histamine release and associated vasodilation and bronchoconstriction; as a preanesthetic, it is less likely than morphine to provoke emesis. A study is under way to develop a canine pain scaling approach to assess several postsurgical canine behaviors with and without the administration of oxymorphone (Hansen et al., 1990). Behaviors are being correlated with plasma concentrations of stress hormones, physiologic signs, type of surgery, and amount of tissue trauma. The goal is to identify behaviors most likely associated with pain and stress, so that an objective basis can be established for diagnosing pain and optimizing analgesic therapy. Agonist-antagonists: Analgesic and behavioral effects of butorphanol (0.1–0.8 mg/kv intravenously and subcutaneously) and nalbuphine and pentazocine (each at 0.75–3 mg/kg intraveneously) have been compared in cats (Sawyer and Rech, 1987). Visceral analgesia was produced by all drugs over dose ranges given. However, somatic analgesia could be achieved with butorphanol at 0.8 mg/kg intravenously. Neither sedation nor behavioral effects were observed with any of the drugs over the dose ranges stated, except with higher doses of pentazocine. Sawyer and Rech (1987) recommended doses of butorphanol of 0.1–0.2 mg/kg intravenously or 0.4 mg/kg subcutaneously. In dogs, dose-dependent sedation occurred over a dose range of 0.1–0.4 mg/kg intravenously (Trim, 1983). Buprenorphine, approved for use in the United States in 1989, has been recommended as an analgesic to alleviate moderate to severe pain in several laboratory animal species (Flecknell, 1987). Table 5-8 lists doses and recom-

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Recognition and Alleviation of Pain and Distress in Laboratory Animals TABLE 5-8 Doses of Buprenorphine in Various Species for Management of Moderate to Severe Pain Species Dose, mg/kg (Routea) Dose Interval, hours Reference Cat 0.005–0.01 (iv, sc) 12 Clifford, 1957; Flecknell, 1987 Dog 0.01–0.02 (iv, sc) 8–12 Harvey and Walberg, 1987; Flecknell, 1987   0.01–0.02 (iv, im, sc) 12   Guinea pig 0.05 (sc) 8–12 Flecknell, 1987 Mouse 2.5 (ip) 6–8 Harvey and Walberg, 1987; Flecknell, 1987   2.0 (sc) 12   Pig 0.1 (im) 12 Flecknell, 1987 Primate 0.01 (iv, im) 8–12 Harvey and Walberg, 1987; Flecknell, 1987   0.01 (iv, im) 12   Rabbit 0.02–0.05 (iv, sc) 8–12 Harvey and Walberg, 1987; Flecknell, 1987 Rat 0.02–0.08 (sc, ip) 8–12 Harvey and Walberg, 1987; Flecknell, 1987   0.1–0.5 (iv, sc) 8–12   Sheep and goat 0.005 (im) 4–6 Flecknell, 1987 a im = intramuscular; iv = intravenous; ip = intraperitoneal; sc = subcutaneous. mended dose intervals to maintain the analgesic effect in several species. The actual dose interval used in an animal should depend on the analgesic needs of the animal. Intrathecal and epidural administration of opioids and local anesthetics for postoperative analgesia (Gregg, 1989) and for intractable sacral and perineal pain (Finley, 1990) is gaining widespread use in human medicine. In dogs, the epidural administration of morphine has successfully provided prolonged postsurgical analgesia (Valverde et al., 1989). Its major advantage is the long duration of analgesia (10–23 hours) after an epidural dose of 0.1 mg/kg of body weight. (See also Dodman et al., 1992.) Antagonists: Naloxone (0.04 mg/kg intravenously) is as effective in animals as in humans in reversing the analgesic, sedative, cardiovascular, and respiratory effect of opioid agonists. NONSTEROIDAL ANTI-INFLAMMATORY DRUGS Common Examples Salicylates (aspirin), pyrazolones (phenylbutazone [Butazolidin®], oxyphenbutazone and dipyrone), anthranilic acids (mefenamic acid and meclofenamic acid), nicotinic acid derivatives (flunixin [Banamine®]), phenylpropionic acids

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Recognition and Alleviation of Pain and Distress in Laboratory Animals (ibuprofen [Motrin®] and fenoprofen [Nalfon®]), naphthylpropionic acids (naproxen [Naprosyn®]), indoles (indomethacin [Indocin®]), and p-aminophenols (acetaminophen [Tylenol®]). Clinical Use The nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used in the treatment of myositis, arthritis, and other surgical and nonsurgical acute and chronic inflammatory conditions (Table 5-9). The NSAIDs are the preferred drugs for the treatment of acute and chronic inflammatory conditions, because they do not interfere with the secretion of glucocorticoids from the adrenal gland, as do the steroid drugs. Pharmacologic Effects The analgesic, antipyretic, and anti-inflammatory actions of NSAIDs are attributable mainly to inhibition of prostaglandin synthesis in the peripheral nervous system and CNS (e.g., antipyresis by inhibition of prostaglandin synthesis in the thermoregulatory center). Acetaminophen, however, is considered to have poor anti-inflammatory activity, because it is a weak prostaglandin synthetase inhibitor in vitro. The pain produced by the inflammatory process is mediated through endogenous eicosanoids and other substances (see Chapter 2). Many of the products of eicosanoid metabolism are responsible for the classic signs of the inflammatory process: redness, pain, and edema. Eicosanoids, released as a consequence of injury, produce vasodilation, increase in capillary permeability, edema, and leukocyte migration, which are associated with the pain produced by inflammation. The eicosanoids are not stored in cells, and their synthesis is initiated by the enzymatic release of fatty acids from cellular phospholipids (Higgins, 1985; Moore, 1985). The release of arachidonic acid from membrane phospholipids is the first event in the synthesis of the eicosanoids. Cyclo-oxygenase catalyzes the initial formation of prostaglandin from arachidonic acid. NSAIDs, with the possible exception of acetaminophen, are potent inhibitors of cyclo-oxygenase (Gilman et al., 1990). The eicosanoids are synthesized by all cells except red blood cells and have a major effect on cellular functions. For example, the release of the eicosanoids or their blockage has an effect on the microcirculation, producing vasodilation or vasoconstriction. NSAIDs interfere with or modify the effects of many drugs that depend on the release of eicosanoids. The postoperative use of NSAIDs to minimize inflammation associated with surgical trauma is encouraged. But they have a broad effect on the arachidonic acid cascade, so they can modify the actions of many drugs and normal physiologic activity that might be under study. Because they do not produce obvious behavioral changes, as do many other drugs used for the relief of pain and distress, these

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Recognition and Alleviation of Pain and Distress in Laboratory Animals TABLE 5-9 Doses of Nonsteroidal Anti-inflammatory Drugs Species Drug Dose, mg/kg (Routea) Dose Interval, hours References Cat Aspirin 10 (po) 48 Jenkins, 1987 Dog Aspirin 10 (po) 6–8 Booth and McDonald, 1982   Ibuprofen 10 (po) 24–48 Jenkins, 1987   Flunixin 1.1 (iv, im)   Hardie et al., 1985   Acetaminophen 15 (po) 8 Jenkins, 1987   Phenylbutazone 15 (po) 8 Booth and McDonald, 1982 Horse Aspirin 25 (po) 12 Jenkins, 1987   Flunixin 1.1 (iv, im, po)   Houdeshell and Hennessey, 1977; Soma et al., 1988b   Phenylbutazone 4.4 (iv, po) 12–24 Piperno et al., 1968 Mice Aspirin 120–300 (po)   Jenkins, 1987   Ibuprofen 7.5 (po)   Jenkins, 1987   Acetaminophen 300 (ip)   Jenkins, 1987 Primate Aspirin 20 (po) 6–8 Flecknell, 1987 Rabbit Aspirin 500 (po)   CCAC, 1980 Rat Aspirin 100 (po) 4 Flecknell, 1987   Ibuprofen 10–30 (po)   Jenkins, 1987   Acetaminophen 110–300 (po)   Jenkins, 1987   Phenylbutazone 30–100 (po)   Kruckenberg, 1979   Phenylbutazone 7.5–15 (sc)   Kruckenberg, 1979 Ruminant Aspirin 50–100 (po) 12 Jenkins, 1987   Phenylbutazone 6 (iv, im, po)   Eberhardson et al., 1979 a iv = intravenous; im = intramuscular; ip = intraperitoneal; sc = subcutaneous; po = oral. interactions might not be considered. Drugs other than NSAIDs (for example, corticosteroids) can also modify the release of arachidonic acid and can interfere with experimental studies. NSAIDs share many properties; all are effective analgesics, antipyretics, and (except for acetaminophen) potent anti-inflammatory agents. They also share the tendency to produce adverse effects on the gastrointestinal, renal, and, to a lesser extent, hepatic and hematopoietic systems. The tendency to induce toxic manifestations differs markedly among species and NSAIDs. Most NSAIDs are weak

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Recognition and Alleviation of Pain and Distress in Laboratory Animals acids, are highly bound to serum proteins, and are biotransformed extensively by hepatic mixed-function oxidases. Differences in rates of drug biotransformation might underlie much of the variation among species in the elimination kinetics of NSAIDs (Mazue et al., 1982)—for example, variation in the plasma half-life of aspirin (horse, 1 hour; dog, 8 hours; cat, 38 hours) and phenylbutazone (horse, 3–6 hours; cattle, 35–72 hours) (Jenkins, 1987). Such differences should be taken into account in the establishment of dose schedules to provide effective drug concentrations in the body during therapy and to prevent drug toxicity. Dose Recommendations (Table 5-9) Before NSAIDs are used, references should be consulted for more information on length of treatment and reactions specific to the animals in question. For example, acetaminophen should not be used in cats, because of deficiencies in detoxifying mechanisms and an inherent sensitivity of feline red blood cells to oxidative damage. NSAIDS constitute a potentially valuable group of drugs for producing analgesia in laboratory animals. Dose recommendations are available for treating individual animals of a given species, but information on administration in drinking water (e.g., dose, stability, and palatability) is lacking and would be especially valuable for treating large numbers of animals, such as rodents, simultaneously with a minimum of handling. SPECIAL ANESTHETIC CONSIDERATIONS α-Chloralose α-Chloralose (40–80 mg/kg intravenously) is useful for providing prolonged anesthesia for nonsurvival experiments (6–10 hours) with minimal cardiovascular and respiratory depression (Van Citters et al., 1964). However, the depth of analgesia is usually inadequate for surgical procedures (Flecknell, 1987; Holzgrefe et al., 1987), and its use for this purpose requires clear justification. When combined with adjunctive drugs in nonsurvival procedures, chloralose can be used for preparative surgery and later for maintenance (Holzgrefe et al., 1987). Its onset of action is slow (15 minutes after intravenous administration), so a short-acting barbiturate usually is given first to induce anesthesia. But chloralose can be administered to animals already deeply sedated with opioids, tranquilizers, or cyclohexamines. Urethane Urethane, like chloralose, produces prolonged anesthesia with minimal cardiovascular and respiratory depression. Unlike chloralose, it produces analgesia

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Recognition and Alleviation of Pain and Distress in Laboratory Animals sufficient for surgical procedures. Urethane has been reported to be both mutagenic and carcinogenic, so it should be handled as a mild carcinogen, and animals anesthetized with it should not be allowed to recover (Flecknell, 1987). Use of Skeletal Muscle Relaxants Muscle relaxants do not provide relief from pain. They are used to paralyze skeletal muscles while an animal is fully anesthetized. If a procedure will cause no pain and the animal is properly ventilated, general anesthesia may be discontinued under carefully controlled conditions for specific neurophysiologic studies (Van Sluyters and Oberdorfer, 1991). Such use of muscle relaxants requires prior approval of an animal care and use committee, because acute stress is believed to be a consequence of paralysis in a conscious state. Table 5-10 lists does of the neuromuscular blocking agent pancuronium, which is commonly used in experimental animals. Klein (1987) has compiled an extensive review of neuromuscular blocking agents and their use in dogs, cats, pigs, horses, sheep, and calves. This TABLE 5-10 Doses of Pancuronium in Domestic Species Species Dose, mg/kg Duration, minutes Anesthetic Reference Calf 43 ± 9a 43 ± 19a Halothane-O2 Hildebrand and Howitt, 1984 Sheep 5.0 ± 0.61 21 ± 2.5 Halothane-O2 Klein et al., 1985; Cass et al., 1980   0.15/min steady state     Horse 82 ± 7.3 20–35 Halothane-O2 Klein et al., 1983 Pony 125 ± 20a 16 Halothane-O2 Manley et al., 1983 Pig 50   Thiopental- N2O-O2, or ketamine Denny and Lucke, 1977   10     Lumb and Jones, 1984 Dog 22 ± 3 108 ± 10 Halothane Booij et al., 1980 Cat 20 15 ± 2a Halothane-α-chloralose Hughes and Chapple, 1976   22 14 ± 2 Pentobarbital Durant et al., 1980   34 8.8 ± 2.3 α-Chloralose-pentobarbital Durant et al., 1979 a Mean ± standard deviation. SOURCE: Modified from Klein, 1987.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals reference contains much useful information and it should be consulted by individuals considering the use of neuromuscular blocking agents in anesthetized animals. The maintenance of a constant depth of anesthesia or general knowledge of the depth of anesthesia is especially important when neuromuscular blocking agents are used with light anesthesia. When a neuromuscular blocking agent is necessary, the general anesthesia to be used in a specific study should be administered to a nonparalyzed animal to an appropriate depth in a test situation before the neuromuscular agent is used, to determine the adequacy of the general anesthesia. Many signs of anesthesia are eliminated when muscle paralysis occurs, and delivery of an adequate depth of anesthesia is essential. Because of an animal's inability to respond to stimuli when paralyzed, it is difficult to evaluate whether the animal is anesthetized or can feel pain. Observable signs that might indicate pain under paralysis include autonomic nervous system changes such as lacrimation and salivation, sudden changes in heart rate and arterial blood pressure, and changes in pupil size. Any attempt to breathe out of synchrony with a ventilator might indicate a response to pain, lack of adequate anesthesia, inadequate ventilation (as reflected in increased arterial CO2 concentration), or hypoxia. Those signs, either singly or in combination, can provide valuable information about an animal's condition, but they are not infallibly linked to the animal's state and should be used cautiously and validated separately for each experimental situation. The following recommendations, adapted from Movshon (1988), might be helpful. Necessary surgical procedures should be performed under general surgical anesthesia and before the induction of paralysis. If such procedures might have painful consequences during the later paralysis, local anesthesia should be used in a manner shown by the experimenter to be effective over comparable periods after surgery in alert, freely moving animals of the same species. Endotracheal intubation and other preparatory procedures should take place under general anesthesia, as should the induction of paralysis. Fixation of an animal in any restraining device requires suitable implanted devices previously fitted under surgical anesthesia. Those are known to be well tolerated and to be neither painful nor stressful in awake, unparalyzed animals. Local anesthesia of pressure points is recommended. (Pressure points are parts of the body that become painful owing to continued application of pressure.) Other devices attached to the animal should be placed to provide maximal comfort. The pharynx and larynx should be treated with a topical anesthetic. The tracheal cannula should be coated with a lubricant that contains a local anesthetic agent and must be immobilized so that it cannot be moved inadvertently when general anesthesia has subsided. Intravenous catheters likewise must be immobilized. The animal should be placed in one of its natural resting positions. Minor adjustments in posture from time to time might be helpful in preventing pooling of blood in large veins.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals Salivation should be controlled with a suitable drug, or some other innocuous means should be used to prevent the accumulation of secretion in the throat. The duration of paralysis must be dictated by the time over which the physiologic condition of the animal, and hence its comfort and well-being, can readily be maintained. For most species that will not exceed about 4–6 hours. Some form of intravenous supplementation might be needed, as will cannulation of the bladder in some species (such as tree shrews) if emptying does not occur spontaneously. If an animal is to recover from the paralysis and re-establish its respiration, it should happen while the animal is under general anesthesia. The skilled use of chemical antidotes can ease the process of recovery so that anesthesia can be brief and light. No procedure should be undertaken in an unanesthetized paralyzed animal until it has been shown—when the animal is fully alert, unparalyzed, and capable of expressing its reactions—that an identical procedure elicits no sign of discomfort or distress. That specifically includes, but is not limited to, insertion or manipulation of recording or other devices; electric, chemical, or other stimulation; and the measurement of optical reflexes. In an otherwise comfortable situation, the principal source of stress that occurs in paralyzed humans (and therefore, presumably, animals) is the sense of respiratory distress that accompanies an increase in arterial PaCO2. Therefore, end-tidal CO2 should be continuously monitored with a reliable, calibrated instrument and its concentration should be kept substantially below the 4.7% at which respiratory distress can begin. It should be understood that CO2 monitors designed for use in humans typically do not accurately measure end-tidal CO2 in smaller animals, because excessive gas mixing occurs in the sampling apparatus. Also, relatively short periods of artificial ventilation are often accompanied by blood-chemical and pulmonary-function changes that alter the normal relationship between end-tidal CO2 and arterial PaCO2. Regular direct measurement of arterial blood gases is an indispensable component of monitoring, and monitoring with a respiratory-gas analyzer alone is inadequate. The heart rate should be monitored. Preferably, a rate-meter sounds an alarm if heart rate is above or below its natural resting range. Indeed, once an immobilized animal has recovered from general anesthesia, the heart rate might provide the experimenter with a ready index of the animal's state; an increase due to a relatively innocuous stimulus provides some assurance that the animal is alert and is not experiencing a severe stress. Body temperature should be monitored and strictly maintained within the limits normal for resting animals of the species in question. Body temperature will fall gradually because of immobilization, and there should be provision for keeping the animal warm. But devices used for that purpose should be incapable of overheating to a point that causes dangerously high temperatures or uncomfortable heating of the skin.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals Provision should be made for the rapid and effective delivery of an anesthetic agent, in case an indication of pain or distress occurs and its cause cannot be immediately identified and rectified. If an animal is to be repeatedly subjected to experiments requiring the use of muscle relaxants, signs of specific aversion to the experimental setting should be taken as evidence that the precautions being used are not adequate, and the experiments should be discontinued until appropriate procedural changes are made. Control of Pain in Nonmammalian Species Pain and nociception have been studied extensively in mammals, but there are few reports on the presence or absence of pain perception in other animals. Several investigators have attempted to determine that insects can perceive pain, but have concluded that they probably do not (Wigglesworth, 1980; Eisemann et al., 1984; Fiorito, 1986). A working party at the Institute of Medical Ethics in London has examined the evidence that cephalopods might perceive pain (Smith and Morton, 1988). Cephalopods can be trained with negative reinforcers; they learn to escape and avoid noxious stimuli, and repeated noxious stimulation appears to have long-term effects on behavior. But there is very little evidence that would permit a conclusion that cephalopods perceive pain. Attempts of cold-blooded vertebrates and invertebrates to escape or avoid aversive stimuli are not sufficient to conclude that these animals experience the affective and sensory qualities of painful stimuli as do warm-blooded vertebrates, but they do indicate that at least some degree of stress can be accompanied by a negative motivational state, and they should not be ignored. In the absence of contrary information, consideration should be given to alleviating and preventing potential pain and stress in cold-blooded vertebrates and invertebrates, as for warm-blooded vertebrates (Eisemann et al., 1984). Fish have neuropeptides in their central nervous systems, such as substance P and enkephalin, that are similar to those in mammalian nociceptive systems. They respond to injury or irritants by withdrawing, but their responses to repeated stimuli are small or absent, and fish with severe wounds appear to behave normally. Therefore, it is difficult to describe the responses of fish to noxious stimuli as unequivocal signs of pain. Despite the lack of direct evidence that fish perceive pain as do other vertebrates (Medway, 1980; Arena and Richardson, 1990), it seems prudent at present to try to minimize pain in fish as in warm-blooded vertebrates. Neonatal and Fetal Surgery The traditional view that the human neonate and fetus are not capable of perceiving pain has recently been seriously questioned (Anand et al., 1987a). A review of the literature, including reports of animal experiments, showed that premature human fetuses and neonates indeed show physiologic and probably

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Recognition and Alleviation of Pain and Distress in Laboratory Animals psychologic evidence of severe stress when subjected to surgical procedures that result in marked nociceptive activity. The authors concluded that ''current knowledge suggests that humane considerations should apply as forcefully to the case of neonates and young, non-verbal infants as they do to children and adults in similar painful and stressful situations." Drug regimens that are safe for and tolerated by adult animals and humans can produce toxicity or even death in neonates or fetuses. Protective mechanisms—including drug-biotransforming enzyme systems, the blood-brain barrier, and renal excretory mechanisms—are undeveloped in fetal and neonatal animals (Stoelting, 1987). We underscore the published recommendation that "the choice of methods to be used in neonates is another area in which the flexible cooperation of principal investigators and veterinarians is particularly important" (Van Sluyters and Oberdorfer, 1991). Some general recommendations can be made: Consider the use of a local anesthetic when surgery is to be performed on a fetus. Consider the use of an inhaled anesthetic for neonates whenever possible, because biotransformation is not required for its elimination, and the depth of anesthesia can be readily controlled. Analgesia can be provided by using fentanyl in preterm human babies (Anand et al., 1987b). NONPHARMACOLOGIC CONTROL OF PAIN HYPOTHERMIA One analgesic technique that is applicable to altricious neonates that have not yet developed effective thermoregulatory mechanisms is hypothermia, which has a wide margin of safety and appears to be effective when surgery is necessary. It is also useful for restraint and as an adjunct to general anesthesia in cold-blooded animals (CCAC, 1980; Phifer and Terry, 1986; Arena and Richardson, 1990). TONIC IMMOBILITY Physical restraint can produce severe stress in some animal species (Gärtner et al., 1980; Pare and Glavin, 1986). Such stress is usually accompanied by functional CNS changes and hormonal responses. In some highly susceptible species—such as reptiles, birds, guinea pigs, and rabbits—restraint can result, usually after an initial period of struggling, in immobility that persists without continued restraint. The most common terms for the phenomenon are immobility reflex, animal hypnosis, and tonic immobility. Tonic immobility (TI) is probably the least ambiguous, because it is more of a behavioral description. Other terms—such as playing possum, mesmerism, and dead feint—have been used. The reflexive behavior is considered to be a mechanism of defense against predators, in that it renders the animal less sensitive to pain. Those intending to use TI should consult

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Recognition and Alleviation of Pain and Distress in Laboratory Animals Klemn (1976) and Porro and Carli (1988) for rationale, technique, and species differences. TI abolishes voluntary motor activity. Spinal reflexes are suppressed, but not abolished. Muscle tone varies with species and with the induction procedure. In rabbits, perhaps the most susceptible species, fine muscle tremors can occur initially or be induced by stimulation of the patellar tendon reflex. Rabbits' eyes remain open and fixed, but the corneal reflex is still present. Initially, the heart rate of immobilized animals can increase; at later stages, the rate tends to decrease. Fully immobilized rabbits exhibit pronounced catalepsy with reduced muscle tone. In susceptible species, TI is relatively easy to achieve. A rabbit, for example, is grasped from behind around the neck at the base of the skull by one hand. With the other hand under the rump (and preventing kicking by the hind legs), the animal is turned over onto its back with one quick smooth motion while gentle traction is maintained on the neck. Gentle neck traction might stimulate receptors in the carotid body to play a role in the induction and maintenance of immobility. Rubbing the animal's abdomen and talking in a soft monotone are said to facilitate the reflex. An animal immobilized can commonly be left lying on its back for a short period without additional restraint. The reflex can last from a few seconds to several minutes; the period depends on the species, the animal, previous experience, and the time that the animal is held in the supine position. Gentle traction on its neck will continue maintenance of TI and its associated analgesia. Loud noises or sudden poking or probing of the animal will cause it to arouse. TI is useful in performing physical examinations, deep abdominal palpation, and ophthalmologic examinations. It is readily reversible by returning the animal to its normal posture. After immobility is terminated, some degree of analgesia and disorientation can persist for a period that depends on the duration and depth of TI. ACUPUNCTURE The use of acupuncture is limited primarily to the treatment of specific chronic, painful disorders (Klide, 1989; Klide and Martin, 1989). It has been used successfully in the management of chronic pain in horses and dogs. It is not curative, and it has to be repeated periodically to allow the animal mobility and comfort. Acupuncture has been successful in modifying acute pain, but has no lingering effect on removal of the stimulation. It has been used for surgical analgesia under limited circumstances.