A reassessment of stress-induced ‘‘analgesia’’ in the rat using an unbiased method Pascal Carrive a,⇑ , Maxim Churyukanov b , Daniel Le Bars c a School of Medical Sciences, University of New South Wales, NSW 2052, Australia b Department of Neurology, Moscow Medical Academy, Moscow 119021, Russia c Team ‘‘Pain’’, INSERM UMRS 975, CNRS UMR 7225, Faculté de Médecine UPMC, Université Pierre et Marie Curie, Paris, France Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article. article info Article history: Received 29 July 2010 Received in revised form 2 December 2010 Accepted 13 December 2010 Keywords: Tail-flick Conditioned fear Infrared thermography Cutaneous vasoconstriction Noxious heat Pain abstract An increased tail-flick latency to noxious heat during or after stress in the rodent is usually interpreted as a stress-induced reduction in pain sensitivity and often described as a form of stress-induced ‘‘analgesia.’’ However, this measure is an indirect and flawed measure of the change in nociceptive threshold to nox- ious heat. A major confound of the latency measure is the initial temperature of the tail, which can drop down to room temperature during stress, the consequence of a marked sympathetically mediated vaso- constriction in the skin of the extremities. We addressed this issue with tail-flick tests during contextual fear using infrared thermography to monitor temperature changes and a CO 2 laser to deliver the heat stimulus. The experiment revealed a 4.2°C increase of the nociceptive threshold, confirming a true antin- ociceptive effect. However, its contribution to the increased withdrawal latency was less than two-thirds (63.2%). Nearly one-third (32.2%) was due to the drop in tail temperature (4.4°C), which also slowed con- duction along sensory fibers (2.2%, included in the 32.2%). The remaining 4.6% was due to an increase in decisional/motor latency. This new unbiased method establishes beyond doubt that a conditioned stress response is associated with true antinociception to noxious heat. It also confirms that stress-induced changes in skin temperature can be a major confound in tail-flick tests. The present study shows, for the first time, the exact contribution of these two components of the tail-flick latency for a stress response. Ó 2010 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. 1. Introduction Stress-induced analgesia is a well-described phenomenon that can be observed and reproduced in laboratory animals [2,11,12,41,43,46,59]. The common view is that this form of re- duced reactivity to pain is due to activation of descending inhibi- tory pain pathways leading to an increase in nociceptive threshold, that is, an increase of the threshold at which the noci- ceptive stimulus becomes painful and triggers a withdrawal re- sponse. However, the actual nociceptive threshold and its changes during stress in the conscious animal have in fact never been measured. While a number of nociceptive tests have been used to study the effect of stress on pain, the most common ones are thermal tests such as the tail-flick, in which the degree of presumed analgesia is evaluated from changes in the withdrawal reaction time (or latency) [39]. This is a convenient behavioral measure; however, it is an integrated measure that depends only partly on the nocicep- tive threshold [5]. As has been pointed out already, one critical variable that can affect the withdrawal reaction time is the initial temperature of the skin where the stimulus is applied [5,20,30,38]. The confounding effect of the initial temperature is a major con- cern when testing thermal nociception during stress because stress can lead to large variations in skin temperature. It is well known that centrally evoked defense reactions or naturally evoked stress responses are associated with sympathetically mediated vasocon- strictions in the skin that lead to cooling of cutaneous territories, especially in the extremities [1,3,8,9,19,22,47]. This is best ob- served with infrared thermography, as we recently reported, in fear-conditioned rats [56]. In this study, which was conducted at room temperature, marked reductions in skin temperature (4– 6°C) were observed in the tail and paws. Such a drop in skin tem- perature is not trivial. It could significantly increase the with- drawal reaction time of a tail-flick independently of any change in nociceptive threshold. At present, there is no way to determine the contributions of these 2 factors. Methods to minimize or con- trol for the confounding effect of the initial temperature have been proposed, however, they are tedious and not always amenable to some forms of stress, such as conditioned fear, where the animals must be freely moving and preferably untethered [54]. The aim of this study was to reassess the stress-induced ‘‘anal- gesia’’ evoked by conditioned fear to context (also known as condi- 0304-3959/$36.00 Ó 2010 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2010.12.019 ⇑ Corresponding author. Address: School of Medical Sciences, University of New South Wales, NSW 2052, Australia. Tel.: +61 293852467. E-mail address: p.carrive@unsw.edu.au (P. Carrive). www.elsevier.com/locate/pain PAIN Ò 152 (2011) 676–686