Peripheral mechanisms of inflammatory pain with particular reference to TRPV1, burn injury and the actions of certain gaseous general anaesthetics
Author(s)
White, John P. M.
Type
Thesis
Abstract
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.
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.
Date Issued
2011
Date Awarded
2011-08
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Nagy, Istvan
Creator
White, John P. M.
Publisher Department
Medicine: Department of Anaesthesia
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)