Characterization of variables defining hindpaw withdrawal latency evoked by radiant thermal stimuli

Abstract

We have examined the stability and sources of variation within the nociceptive model of rat hind paw withdrawal from an under-glass radiant stimulus (Hargreaves et al., 1988) using a system where stimulus intensity and floor temperature can be controlled and reproducibly changed. The current study demonstrates that: (i) increased stimulus intensity with a fixed surface temperature is associated with a monotonic decrease in mean response latency and its variance; (ii) for a fixed stimulus intensity, the mean paw withdrawal latency and variance increased as the glass floor temperature is lowered from 30°C to room temperature (25°C). Using subcutaneously-implanted thermocouples and a 30°C glass surface, the subcutaneous paw temperature observed at an interval corresponding to the time at which the animal displayed a paw withdrawal did not differ across multiple heating rates (41–42.5°C). This finding is in agreement with human studies of pain thresholds and C-fiber activity. These studies emphasize the importance of maintaining a fixed surface temperature to reduce experimental variability and the utility of this apparatus across multiple stimulus intensities to define agonist efficacy.

Introduction

The acute application of a sufficiently intense thermal stimulus to the body surface of the unanesthetized animal will evoke an organized escape response targeted at removing the stimulated site from the heat source. The adequate stimulus is believed to reflect the activation of small high threshold thermoreceptors and polymodal C-fibers, with the frequency of firing being proportional to stimulus intensity (Beitel et al., 1977; Konietzny, 1984). The response to the afferent input may be organized at the spinal level (e.g. a nociceptive reflex in the Sherringtonian sense) or the supraspinal level, reflecting a complex coordinated behavioral adjustment to the stimulus environment.

Behavioral models in the rodent have been developed which are believed to reflect these underlying physiological substrates (Yaksh, 1997). For assessment of spinally mediated nociceptive reflexes, the rat tail displays a brisk withdrawal consequent to being placed in a heated water bath (Janssen et al., 1963) or the application of a radiant stimulus (D'Amour and Smith, 1941). For supraspinally organized nociceptive reflexes, a common paradigm has been the withdrawal or licking of the hind paw or the jumping response evoked by placing the animal on a hot surface (Woolfe and MacDonald, 1944). The use of models based on spinal versus supraspinal organization is predicated on specific questions being addressed. However, it is certain that an essential element of nociception is the supraspinal processing of the input generated by the noxious stimulus and emphasizes the importance of assessing the supraspinal component of the afferent-evoked response.

An important variant on the paw withdrawal model is the application of a radiant thermal stimulus to the plantar surface of the hindpaw of a rodent standing on a glass surface (Hargreaves et al., 1988). This model has gained favor as an analgesic drug bioassay for several technical reasons: (i) it permits independent testing of either hind paw; (ii) testing can be undertaken with minimum handling (i.e. the animal can be placed in the chamber before the stimulus is initiated); (iii) the end point (paw withdrawal) can be automatically detected in un-anesthetized, un-restrained animals; and (iv) stimulus intensity is easily and reproducibly changed. Utilization of this model as a drug bioassay merits consideration of model properties that contribute to response variability and sensitivity. Reduced variability permits smaller groups to be employed to define statistical differences, but if reduced variability also significantly reduces sensitivity, model utility in defining analgesic drug action will be lost. We believe that two variables merit specific consideration: stimulus intensity and glass surface temperature.

With respect to intensity, increasing thermal stimulus intensity in a variety of models will result in a reduced response latency (Carstens et al., 1979; Carstens and Wilson, 1993; Dirig and Yaksh, 1996). We sought to determine the relationship between stimulus intensity, mean response latency, and group variation. With the radiant stimulus model, increasing lamp current (amperage) results in an increased peak temperature as well an increased rate of rise. One question addressed in the current study was whether the temperature associated with the withdrawal response was independent of the rate of rise.

With respect to glass temperature, the glass surface upon which the rodent is placed acts as a heat sink or source, depending upon the relative temperature gradient (Hirata et al., 1990). Glass temperature can thus affect paw skin temperature. Changes in basal glass temperature could potentially alter withdrawal latency. This has been demonstrated in the tail flick test where decreases in basal tail skin temperature increased tail flick latencies (Berge et al., 1988). Accordingly, failure to control glass temperature could lead to sources of random variation. The current study thus incorporates the radiant thermal stimulus described above to examine the relationship between thermal intensity, glass surface temperature, tissue temperature, and withdrawal latency.