Peripheral mechanisms of inflammatory pain with particular reference to TRPV1, burn injury and the actions of certain gaseous general anaesthetics
Creators: name: White, John P. M.
Publisher: name: Imperial College London
Year: 2011

description: 1. The literature relating to peripheral mechanisms of inflammatory pain is reviewed. 2. To investigate the role of the transient receptor potential vanilloid type 1 (TRPV1) ion channel in spinal nociceptive processing after burn injury, the candidate studied scalding-type burn injury-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the spinal dorsal horn of wild-type (WT) and TRPV1 knock-out (KO) mice. Activation of ERK1/2 in the spinal dorsal horn is a recognised marker for spinal nociceptive processing. At 5 minutes after severe scalding injury to WT mouse hind-paw, a substantial number of phosphorylated ERK1/2 (pERK1/2) immunopositive neurons were found in the ipsilateral dorsal horn. At 1 hour post-injury, the number of pERK1/2-labelled neurons remained substantially the same. However, at 3 hours post-injury, a further increase in the number of labelled neurons was found on the ipsilateral side, while a remarkable increase in the number of labelled neurons on the contralateral side resulted in there being no significant difference between the extent of the labelling on both sides. By 6 hours postinjury, the number of labelled neurons was reduced on both sides without there being significant difference between the two sides. A similar pattern of severe scalding injury-induced activation of ERK1/2 in spinal dorsal horn neurons over the same time-course was found in TRPV1 KO mice, except that the extent to which ERK1/2 was activated in the ipsilateral dorsal horn at 5 minutes post-injury was significantly greater in WT animals when compared to TRPV1 null animals. This difference in activation of ERK1/2 in spinal dorsal horn neurons was abolished within 1 hour after injury, demonstrating for the first time that TRPV1 is not essential for the maintenance of ongoing spinal nociceptive processing in inflammatory pain conditions in mouse resulting from at least certain types of severe burn injury. The facts that neuronal staining for activated ERK1/2 not only persisted, but also that the number of neurons expressing activated ERK1/2 increased with the passage of time are significant novel findings of this study which have important implications. After an acute noxious stimulus (provided by hind-paw injection of capsaicin), ERK1/2 activation reaches a peak within 2-5 min and then quickly subsides, such that it is barely detectable or absent by 2 hours after the stimulus (Ji et al., 1999). The findings of this study that activated ERK1/2 is: (i) still present in neurons at the 3 hour and 6 hour time-points post-burn injury; and (ii) that it is found in even more neurons at those later time-points than at the earlier time-points measured, strongly suggest that the ERK1/2 activation found at 3 hours and 6 hours post-injury, respectively, is not merely residual from the initial noxious heat stimulus, but rather that it has been caused by the ongoing inflammatory process itself. This is a key novel finding of this study. The data obtained contain evidence of a dissociation within the physiological process of the burn injury of these two distinct "noxious insults" comprising: first, the initial external heat stimulus of the burn injury, which causes direct damage to the tissue accompanied by heat-induced activation of heat-sensitive receptors which result in excitation of nociceptive primary afferents; and, second, the burn injury-induced massive inflammatory response whereby inflammatory mediators occasion an ongoing activation of their receptors on those nociceptors to maintain, and, indeed, increase, nociceptive signaling in those neurons. The further finding that, at 3 hours after scalding-type burn injury, in both TRPV1-null and wild-type mice, the contralateral side of the spinal cord now exhibited a substantial number of pERK1/2- immunopositive neurons remains intriguing, notwithstanding that the phenomenon of hyperalgesia developing in the hind-paw contralateral to a heat injury has long been noted (Coderre and Melzack, 1991; Polgar et al., 1998). One may speculate that this labelling is caused, or contributed to, by an action of descending fibres on these contralateral dorsal horn neurons since the concept of supraspinal pronociceptive modulation of pain is now well recognised (Lima et al., 2002; Tavares and Lima, 2007). In addition, or in the alternative, this labelling may result from cross-communication between opposite dorsal horn neurons (Coderre and Melzack, 1991; Shenker et al., 2003). In any event, the activation of ERK1/2 on both sides of the dorsal horn discloses a process in which spinal neurons are driving inflammatory nociceptive activity after the excitatory effect of peripheral inflammagens has abated in primary sensory afferents. 3. Xenon provides effective analgesia in several pain states at sub-anaesthetic doses. The aim of this second study was to examine whether xenon may mediate its analgesic effect, in part, through reducing the activity of TRPV1, a receptor known to be involved in certain inflammatory pain conditions. The candidate studied the effect of xenon on capsaicin-evoked cobalt uptake in rat cultured primary sensory neurons and in human TRPV1 (hTRPV1)-expressing human embryonic kidney 293 (HEK293) cells. He also examined xenon's effect on the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the rat spinal dorsal horn evoked by hind-paw injection of capsaicin. Xenon (75%) reduced the number of primary sensory neurons responding to the TRPV1 agonist, capsaicin (100 nM-1 μM) by ~25% to ~50%. Xenon reduced the number of heterologously-expressed hTRPV1 activated by 300 nM capsaicin by ~50%. Xenon (80%) reduced by ~40% the number of phosphorylated ERK1/2-expressing neurons in rat spinal dorsal horn resulting from hind-paw capsaicin injection. The key original finding is that xenon substantially reduces the activity of TRPV1 in response to noxious stimulation by the specific TRPV1 agonist, capsaicin, suggesting a possible role for xenon as an adjunct analgesic where hTRPV1 is an active contributor to the excitation of primary afferents which initiates the pain sensation. Next, to investigate whether the xenon-induced inhibition of TRPV1 in rat DRG neurons reduces nociceptive processing, the effect of xenon in reducing the release of calcitonin gene-related peptide (CGRP) from those neurons was examined. Exposure to xenon failed to effect a reduction of capsaicin-evoked CGRP release from cultured primary sensory neurons when stimulated by capsaicin. This latter novel finding suggests that xenon acts on several molecular targets on nociceptive primary sensory neurons, and that xenon’s action on one, or more, of those targets serves to offset the inhibitory, proanalgesic, effect of xenon on TRPV1. It is concluded that xenon may not produce any analgesic effect through peripheral nociceptors. 4. Clinically relevant concentrations of isoflurane or sevoflurane sensitize TRPV1 to several of its activators, including capsaicin. It has, moreover, been suggested these volatile general anaesthetics may augment nociceptive signalling arising from surgical procedures and thereby contribute to postoperative pain. To investigate this suggestion, the candidate studied intraplantar capsaicin injection-induced phosphorylation of extracellular signal-regulated kinase 1/2 (pERK1/2) in spinal dorsal horn neurons (which is a recognised marker of spinal nociceptive processing) in rat during isoflurane or sevoflurane anaesthesia after 60 min under anaesthesia. Control animals were anaesthetised with pentobarbital (which of itself does not activate ERK1/2 in spinal dorsal horn neurons). Unilateral intraplantar capsaicin injection in control animals evoked pERK1/2 in a group of neurons in lamina I and lamina II of the ipsilateral spinal dorsal horn in a somatotopically appropriate area. In contrast, both anaesthetic gases (given for 60 min and without subsequent capsaicin injection) induced ERK1/2 activation in a different group of mainly lamina I neurons bilaterally. The total number of spinal dorsal horn neurons labelled on the ipsilateral side following capsaicin injection into the isoflurane-, or sevoflurane-, anaesthetised animals was significantly less than that produced by capsaicin alone. Further, capsaicin injection into isoflurane-, or sevoflurane-, anaesthetised animals reduced pERK1/2 induced by the gases alone on both sides. These key original findings are inconsistent with the suggestion that isoflurane-, or sevoflurane-, induced sensitisation of TRPV1 by capsaicin, or other agonist, is translated into induction of spinal nociceptive processing and consequential pain sensation.