Unilateral hot plate test: a simple and sensitive method for detecting central and peripheral hyperalgesia in mice

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Abstract

The application of thermal noxious stimuli forms the basis of some widely used tests to detect either hyperalgesic or analgesic reactions. In the classical hot plate test, mice react by licking their paws and/or jumping. However, tests relying on the unilateral application of thermal radiant heat to the plantar side of the hindpaw, have become popular in recent years since unilateral changes in nociceptive sensitivity can be detected. Based on the aforementioned tests, we developed a testing procedure in mice, the unilateral hot plate (UHP): the plantar side of one hindpaw is placed on a hot plate surface and, thus, the withdrawal latency of each paw can be measured separately. The effectiveness of several analgesic and hyperalgesic drugs measured by the UHP was compared with that measured by a method based on the application of radiant heat (RH) stimuli. In the UHP method, morphine (1–10 mg/kg) increases latencies, while spinal NMDA (0.001–1 ng) or PGE2 (30–300 ng), intraplantar carrageenan (2–4%) or PGE2 (30–300 ng) decrease latencies. In all cases, the UHP method detected changes in pain reactivity at lower doses than the RH test. The sensitivity and usefulness of the UHP test for performing pain studies in mice is described.

Introduction

Pain behavioral studies in the laboratory measure, in many instances, animal nocifensive reactions, generally in mice or rats. The testing methods used in these studies are a critical variable for the obtainment and interpretation of data. In fact, it is well known that the methods used are especially directed towards detecting different modalities of pain through variation of the type of stimulus, its duration, intensity or localization (Franklin and Abbott, 1989). Furthermore, the nociceptive testing method can even determine the outcome with opposing alterations in pain reactivity (i.e. hypo and hyperalgesia) when testing a single treatment by different methods (Autier et al., 2000).

Among the various analgesic tests using thermal nociceptive stimulation, the most popular are the tail-flick (D'Amour and Smith, 1941) and the hot plate test (Woolfe and McDonald, 1944). In the hot plate test, animals are exposed only once to the heat stimulus, resulting in minimal tissue injury. The assay may be performed without any previous habituation and offers good reliability and reproducibility (Cochin, 1968). Subsequently, other methods based on the reaction of the paws to thermal nociceptive stimuli, were developed. Among these, the method described by Hargreaves et al. (1998) is very popular, particularly for testing responses in rats, and less frequently in mice (Mogil et al., 1999, Ramer et al., 1998). In this method, a thermal radiant stimulus is applied to the plantar side of one hindpaw and the latency for paw withdrawal is measured. Although animals must be adapted to the apparatus before testing (Rupniak et al., 1997, Sammons et al., 2000), it has proved to be a useful and sensitive method for detecting both hyperalgesic and analgesic responses.

In the hot plate test, in addition to analgesic responses, hyperalgesic responses can also be detected, usually by summarizing several withdrawal behaviors (licking feet, jumping or rapidly stamping paws) which are computed as a whole (Nishihara et al., 1995, Richardson et al., 1997, Richardson et al., 1998). However, one of the clearest advantages offered by tests based on radiant heat in comparison to the other thermal tests described so far, is to enable independent measures in each paw and, thus, allow their comparison in the same animal. This latter fact is especially applicable to the detection of local peripheral effects of drugs on pain reactivity.

Thus, aiming to take advantage of the properties exhibited by the hot plate test and those of the radiant heat method, we attempted to study the reaction of mice tested by a modified hot plate test in which the latency to withdrawal of only one hindpaw was measured at each time without any previous training period.

Section snippets

Animals

Swiss male mice, 40–50 days old from the Animalario of the Universidad de Oviedo (Reg. 33044 13A) exposed to a light–dark cycle of 12 h and with free access to water and food, were used. The experiments were performed between 10:00 and 15.00 h and animals were tested only once. Experiments were conducted according to ethical guidelines (Zimmermann, 1983) and approved by the Comisión de Ensayos Clı́nicos y Bioética del Principado de Asturias (Spain).

Drug administration

Morphine hydrochloride was intraperitoneally

Study of the analgesic effect of intraperitoneal morphine on withdrawal latencies measured by RH and UHP methods

The study of the analgesic effect of systemic morphine was used as a first approach to assess the sensitivity of the UHP test for detecting changes in nociceptive reactivity. By either testing method (RH or UHP), the repeated application of the thermal stimuli at different times did not modify the withdrawal latencies, as can be seen by the reactivity of the control groups shown in Fig. 1. As can also be seen in this figure, the administration of doses up to 1 mg/kg of morphine at the times

Discussion

In this work, we measured the nociceptive reactivity to thermal stimuli in mice using a testing method based on the classical hot plate test: the UHP. In the UHP test, only one hindpaw (on its plantar side) is exposed at each time to the heat stimulus. We performed a comparative study of the responses obtained with several analgesic and hyperalgesic drugs in this UHP method, and those obtained in a similar test which uses a focal radiant heat (RH) nociceptive stimulus. Globally, our results

Acknowledgements

This work was supported by grants from the Universidad de Oviedo (NP-01-519-2) and Fondo de Investigación Sanitaria (FIS 01/0261).

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