Principles and Role in Psychopharmacology

A number of procedures have been developed for examining the pain-relieving (i.e., ► analgesic) properties of drugs in laboratory animals. Each of these procedures involves the presentation of a potentially painful (or ► nociceptive) stimulus, followed by the measurement of a clearly observable response. Typically, data are based on the time it takes the organism to respond to or withdraw from a nociceptive stimulus. Once baseline levels of responding in response to the nocicepetive stimulus are determined and considered reliable, a drug is administered, and response latencies are then redetermined in the presence of the drug. If the time it takes the organism to respond to the stimulus is longer following drug administration, and importantly, if this change is not because the animal is unable to make the response due to the sedative or motor effects of the drug, then the drug is said to produce antinociceptive effects.

Although this experimental paradigm appears relatively simple and straightforward, it becomes far more complex when one considers the multiple variables that influence the data that can be obtained with these procedures. For example, the type of nociceptive stimulus (e.g., thermal, mechanical, electrical, or chemical), its intensity, and the duration and outcome of its effect determine the drug-induced alterations in response latency, and therefore, these must be quantified very precisely. Similarly, the nature of the response itself - whether it involves an elementary reflex response or a more integrated escape or ► avoidance response - can influence the interpretation of a drug's effect. Antinociceptive tests also attempt to differentiate nonspecific response alterations from those that are specific to the nociceptive stimulus. This is particularly important in the assessment of drug-induced alterations in response to the nociceptive stimulus since a drug may produce motor effects that interfere with the ability of the organism to execute a particular response. A more extensive discussion of these issues can be found in a classic review by Beecher (1957) and a recent review by Le Bars et al. (2001). For the discussion of parallel issues related to pain assessment in humans, see Turk and Melzack (2001).

Although antinociceptive procedures vary in many ways, it is convenient to place them in one of two larger groups - those that involve acute pain and those that involve more persistent pain as might occur with tissue injury or nerve damage.

Models of Acute (or Phasic) Pain in Animals

In general, antinociceptive tests for examining acute pain involve the presentation of a brief thermal, mechanical, or electrical stimulus that is delivered to the skin, paws, or tail of an animal. One of the most common procedures in this category is the tail-flick procedure, originally developed by D'Amour and Smith (1941). In this procedure, a high intensity light is focused on an area close to the tip of the tail as shown for a mouse in Fig. 1, and the time (response latency) taken by the mouse to remove (i.e., flick) its tail from the light source is determined. The light intensity can be adjusted to yield baseline response latencies that are relatively short (i.e., between 3 and 5 s) or long (i.e., between 8 and 10 s).

Typically, the time taken by the mouse to remove its tail from the heat source is measured prior to the drug administration to determine baselines response latencies. After baseline latencies have been determined, drug administration takes place, and tail flick latencies are determined either once or multiple times over a set time period. However, caution has to be taken with repeated measurements, as the tail-flick response is prone to ► habituation. The tail flick response is easy to observe, and it is considered to involve simple spinal reflexes. It can be measured either with a stopwatch or through a system that uses photocells to determine when the tail has been removed from the light source. ► Opioid analgesics such as ► morphine are very active in this procedure, though their effects depend on the intensity of the thermal stimulus. For example, if the light intensity is very high and elicits short tail flick latencies, a higher dose of morphine is usually required to alter the tail flick latency than when the light is set at a lower intensity.

One very important characteristic of the tail flick procedure, and almost all other acute antinociceptive procedures, is the use of a cutoff time to prevent tissue damage. The cutoff time is defined as the maximal time

Antinociception Test Methods. Fig. 1. This figure provides a simple schematic of the tail-flick procedure, showing the position of the mouse's tail over a light source.

that an animal is exposed to the nociceptive stimulus. For example, if a high intensity light is used in the tail flick procedure and the animal (e.g., a mouse) does not remove its tail from the light source within 10 s (cutoff time), the mouse is removed from the apparatus by the experimenter. The cutoff time also influences certain aspects of the data analysis since it is one of the variables in the formula commonly used to quantify antinociceptive drug effects.

A drug's effect in the tail flick procedure is usually quantified by determining the difference between the response latency obtained under baseline conditions and response latency obtained after drug administration. This difference is then divided by the difference between the cutoff time and the baseline latency and a percent maximal effect (%MPE) is derived from these measures.

latency (s) following drug administration — latency (s) under baseline conditions

cutoff time(s) — latency (s) under baseline conditions

Since baseline latencies vary between animals, this formula accommodates individual differences in response to the nociceptive stimulus.

The tail-withdrawal test (Janssen et al. 1963) is a modification of the tail-flick test. In this procedure, mice or rats are gently restrained, and their tail is placed into a water bath maintained at temperatures between 48° and 56° C. Some investigators have also used cold water as the painful stimulus, although this is less common. Latency to remove the tail from the water is measured, much in the same way as in the tail flick procedure, and data are usually described with the percent maximal effect formula described for the tail flick procedure.

The tail-withdrawal test has also been used in primates and the procedure parallels those used in rodents. In this procedure, a monkey is placed in a restraint chair and the lower portion of their tail is immersed in water maintained at a specific temperature (40, 50, or 55°C). The latency to remove the tail from the water is then determined. Typically, monkeys do not remove their tails from 40° C water; however, response latencies might average 10 s when the water is 50° C and 5 s or less when the water is 54°C (Dykstra and Woods 1986). If the monkey does not remove its tail from the water within the predetermined cutoff time (e.g., 20 s), the tail is removed by the experimenter and the latency assigned a value of 20 s (cutoff time). Investigators have shown that reliable data can be obtained with this procedure and tail withdrawal responses can be measured every 15-30 min for periods as long as 3 h.

The hot plate procedure is another acute antinocicep-tive test that is commonly used to assess drug effects. In this procedure, an animal (usually a rodent) is placed on a flat surface maintained at a set temperature (e.g., 52-56° C). The baseline nociceptive response to the hot plate is evaluated by recording the latency to lick or shuffle the hind paw(s), or jump from the hotplate surface. Following the determination of baseline response latencies, drug administration is initiated. Figure 2 illustrates data obtained with this procedure. Responses are measured using a stopwatch and a predetermined cutoff time is established to prevent tissue damage. If an animal does not exhibit a nociceptive response after this cutoff period, they are manually removed from the hot plate and assigned a response value equal to the cutoff time.

Figure 2 shows data obtained at two different hot plate temperatures following the administration of two different opioid analgesics, a high efficacy opioid, morphine, and a lower efficacy opioid, ► buprenorphine. The left portion of Fig. 2 shows data obtained when the hot plate temperature was 53° C. Under these conditions, both morphine and buprenorphine produced dose-dependent increases in the percent maximal possible effect (%MPE), with morphine producing 100% maximal effect at 10 mg/kg. The right portion of Fig. 2 shows data obtained when the hot plate temperature was 56° C. Under these conditions, a higher dose of morphine was required to produce 100% maximal effect (32 mg/kg), and buprenor-phine's effects were markedly attenuated at the higher temperature. (Adapted from Fischer et al. 2008.)

In contrast to the procedures described above, which usually involve assessment of an unlearned response following presentation of an acute stimulus, a few procedures, especially those conducted in primates, involve a period of training. In the titration procedure, monkeys are trained on an avoidance task in which a stimulus such as an electric shock is presented to the monkey's tail (or foot) in increasing intensities. If the monkey makes one or more responses on a lever, the shock is turned off (avoided) for a period of time and the intensity is reduced when the shock resumes again. The intensity at which each monkey maintains the shock is then used as a measure of sensitivity to the potentially painful stimulus. One advantage of this procedure is that the animal, rather than the experimenter determines the level at which the stimulus is maintained. Moreover, responding can be maintained for relatively long periods of time with this procedure, which provides a convenient way to examine onset and termination of a drug effect (Allen and Dykstra 2001).

Models of Persistent (Tonic) or Neuropathic Pain

Antinociceptive test methods that involve persistent or long duration pain typically administer an irritant, which

Antinociception Test Methods. Fig. 2. Antinociceptive effects of several doses of morphine or buprenorphine on a hot plate set either at 53°C (left) or 56°C (right).

produces inflammation, or they induce injury, often by ligation of a nerve such as the sciatic nerve. These procedures often examine multiple aspects of pain sensitivity, including heightened sensitivity to pain (► hyperalgesia) or ► allodynia, a condition in which stimuli that are not normally painful are perceived as painful.

The abdominal constriction (or writhing) test is one example of a persistent pain model. In the abdominal constriction test, an irritant is injected directly into the peritoneal cavity of a mouse, resulting in a characteristic writhing response. Although a number of compounds have been used in this assay (acetic acid, acetylcholine, bradykinin, hypertonic saline, phenylquinone, etc.), a solution of 0.6% acetic acid is one of the most commonly used. The onset of inflammation following acetic acid occurs about 5 min after the injection and lasts for approximately 30 min, but has been reported to last for longer periods, depending on the concentration of acetic acid. In order for this assay to produce consistent data, the writhing response must be clearly defined. For example, writhes are often defined as "lengthwise stretches of the torso with a concomitant concave arching of the back'' often in combination with a reduction in motor activity and some loss of coordination. Scoring is done by experimenter observations, usually of videotaped experimental sessions. One of the advantages of this procedure is the fact that it is sensitive to weak analgesics such as aspirin and other ► nonsteroidal anti-inflammatory drugs (NSAIDs); however, the lack of specificity in response to drugs without analgesic activity (false positives) sometimes makes interpretation more difficult.

The Formalin test (Dubuisson and Dennis 1977) is one of the most commonly used tests for examining persistent pain. With this test, responses are measured following a subcutaneous injection of a formalin solution into the hind paw. Two spontaneous responses are typically measured with the formalin test, an acute, early phase response and a tonic, inflammatory phase response that occurs somewhat later. These responses are usually scored on a four-level scale, from normal posture to licking, nibbling, and/or shaking of the injected paw. Given the two types of responses elicited by the formalin injection, it is possible to examine both acute pain and tonic pain in the same animal, in response to a single injection of formalin.

Carrageenan, a substance derived from Irish sea moss, is another compound that has been used in persistent pain models as it produces inflammation and consequent hy-persensitivity in rodents. In a typical procedure, a 2% suspension of carrageenan is injected subcutaneously into one of the hind paws of a mouse; the opposite hind paw is not injected. This two-paw protocol in which comparisons are made between an inflamed paw and healthy paw was first introduced by Randall and Selitto

(1957) and is now the standard protocol for tests of this nature. Several hours after the carrageenan injection, mice are tested for pain sensitivity and the Hargreaves' test is often used for this assessment. To conduct the Hargreaves' test, mice are placed in individual, transparent chambers with a glass floor. After adaptation to the chamber, a high-intensity beam such as a projector bulb is directed at the plantar surface of the mouse's hind paw. Time to withdraw the paw is then measured.

The von Frey test of mechanical sensitivity provides another way to examine pain sensitivity in animals that have been treated with irritants or have experienced nerve damage. In this procedure, animals are placed in a transparent box which rests on a metal mesh floor. Von Frey monofilaments (or hairs) are then applied to the foot with a single, steady application. Since each monofilament has a different bending force, experimenters can determine the monofilament that elicits foot withdrawal 50% of the time under baseline conditions, and then following drug administration.

Another procedure that has been used to examine pain sensitivity as well as hyperalgesia and/or allodynia is capsaicin, the pungent ingredient in hot chili peppers. Exposure to capsaicin produces inflammation and allo-dynia, which is defined as pain that is produced by a stimulus that is not normally painful. A modification of the primate tail withdrawal procedure, using warm water rather than hot water, has been used to examine allodynia following exposure to capsaicin. The monkey is restrained and the distal part of its tail is immersed in water maintained at a comfortable temperature such as 46° C. At this temperature, monkeys usually leave their tails in the water for at least 20 s, which is used as the cutoff time. After baseline latencies are determined, capsaicin is injected above the tip of the tail. Following administration of capsaicin, allodynia develops as revealed by a decrease in tail-withdrawal latencies from 20 s to approximately 2 s. Local administration of an opioid analgesic has been shown to inhibit the allodynia induced by capsaicin under these conditions (Ko et al. 1998).

Advantages and Limitations of Antinociceptive Tests

One clear advantage of most of the antinociceptive procedures described here is that they are relatively easy to perform in animals. As a group, they require very little training time and produce reliable, repeatable, and easily quantified measures of nociception across a range of stimuli. The acute antinociceptive procedures, such as the tail flick and hot plate, are predictive of the effects of morphine-like opioid analgesics, but do not reveal activity for a number of other drugs, including the nonsteroidal anti-inflammatory drugs (NSAIDS). In contract, persistent pain procedures such as the abdominal constriction test, and formalin test, are predictive of the antinoci-ceptive effects of nonsteroidal anti-inflammatory drugs (NSAIDS) as well as morphine-like analgesics. The fact that these procedures can predict the analgesic activity of drugs in humans, certainly verifies their usefulness as animal models. Beyond being predictive of analgesia in humans, investigations using these procedures have also advanced understanding of the multiple variables that influence the processing of nociceptive stimuli as well as the alteration in nociception following administration of different classes of drugs.

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