Force of limb withdrawals elicited by graded noxious heat compared with other behavioral measures of carrageenan-induced hyperalgesia and allodynia

Abstract

We investigated if carrageenan-induced thermal hyperalgesia and mechanical allodynia are associated with a reduction in threshold and–or enhancement of suprathreshold nocifensive responses, using a method to measure the force of a hind limb withdrawal reflex elicited by graded noxious heat stimuli (36–52°C, 5 s) delivered by Peltier thermode tethered to the ventral hind paw of conscious rats. Withdrawal reflexes were recorded 2.5 h after intraplantar injection of carrageenan (1 or 0.1%) or saline vehicle in sessions >2 weeks apart: baseline reflexes were assessed the day before. Withdrawal reflex force increased linearly from 42–52°C. Carrageenan 1% significantly enhanced withdrawal reflexes at 40–46°C, reducing the slope and threshold of the stimulus-response function. This was associated with significant reductions in thermal paw withdrawal latency (Hargreaves test: by 50%), mechanical withdrawal threshold (by 82%) and weight bearing on the injected side (by 81%) measured with independent force plates. Smaller reductions in thermal paw withdrawal latency and mechanical withdrawal threshold, and smaller enhancement of withdrawal reflex force, were observed following 0.1% carrageenan. Intraplantar saline was ineffective. This method allows assessment of hyperalgesic changes in stimulus-response coding over a broad range of noxious stimulus intensities.

Introduction

A variety of behavioral tests are used to assess pain and hyperalgesia in animals. Many tests measure the latency of reflexive withdrawal of a limb away from a noxious stimulus. Two sensitive and widely used reflex tests are the tail flick reflex assay and the thermal paw withdrawal test (Hargreaves et al., 1988). While the measured endpoint in these tests is a motor response, there is considerable evidence that nociceptive reflexes reasonably reflect pain sensation in humans and animals (Carstens, 1997). Other frequently used animal pain assays include the hot-plate and the formalin tests. The latter test scores behavioral responses (limb guarding, flinching, postural changes) following intracutaneous injection of dilute formalin, and is thought to reflect persistent pain associated with inflammation. To assess mechanical allodynia, variants of the Randall–Selitto method (Randall and Selitto, 1957) have been used to determine the threshold at which mechanical stimuli elicit limb withdrawal responses.

A major drawback of most currently employed animal pain tests is that they measure response latency, which provides information relevant to the pain threshold (Ness and Gebhart, 1986) but does not assess the magnitude of suprathreshold responses. Several recent reviews have emphasized the importance of pain assessment methods to measure responses elicited by stimuli across the noxious range from threshold to near-tolerance limits, thus allowing one to determine the effects of analgesic and hyperalgesic manipulations on pain threshold and suprathreshold processing (Dubner, 1985, Dubner, 1989, Watkins, 1989, Carstens, 1993). There are relatively few methods which allow this. Monkeys (Cooper and Vierck, 1986) and rats (Douglass, 1993, Vierck et al., 1995) have been operantly conditioned to press a bar to terminate noxious electrical or thermal stimuli, and the speed and force of bar presses were shown to increase as a function of stimulus intensity. We have recently developed methods to measure the magnitude of a hind limb withdrawal reflex (measuring limb flexor EMG) and tail flick reflex (measuring the force vector of tail movements in orthogonal planes) elicited by graded noxious heat stimuli in conscious rats (Carstens and Ansley, 1993, Carstens and Wilson, 1993, Carstens and Douglass, 1995). Noxious heat was chosen because it selectively excites cutaneous nociceptors but not warm receptors and is thus a more nociceptive-specific stimulus compared to electrical shock which non-selectively activates all fiber types. In both limb withdrawal and tail flick models, response magnitude increased as a function of stimulus temperature from a threshold near 40°C, and responses were depressed by administration of morphine and by electrical stimulation at analgesic brainstem sites. These results indicate that such models are useful in assessing the effects of analgesic manipulations across a range of noxious stimulus intensities.

Several animal models of persistent pain have been introduced recently (Bennett and Xie, 1988, Selzer et al., 1990, Kim and Chung, 1992) that require sensitive methods to assess enhanced nociceptive responses. In the present study we wished to determine if our model measuring the magnitude of limb withdrawals is useful in assessing hyperalgesia. A drawback of our previous studies is that the measured endpoint (integrated EMG magnitude) provides a relative but not absolute measure of an animal's response. However, it would be valuable to have an absolute measure of response magnitude because this would allow direct comparisons across animals and treatment groups. For this reason, we have presently devised a method to directly record the force of a hind limb withdrawal reflex elicited by graded noxious thermal stimulation of the hind paw in conscious rats. We have tested if our measure of limb withdrawal magnitude is useful in assessing the hyperalgesia that develops following intraplantar injection of carrageenan. We have additionally assessed thermal hyperalgesia and mechanical allodynia using established methods, and have also adapted a recent novel method to measure weight bearing by the hind limbs (Schott et al., 1994) before and after intraplantar injection of carrageenan into one paw. The sensitivity of our new method was assessed by comparing effects of carrageenan on limb withdrawal reflex force with carrageenan's effects in each of the other behavioral assays. An abstract of this work has appeared (Eisele et al., 1997).