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Enhancement of thermal hyperalgesia by α-adrenoceptors in capsaicin-treated skin
Journal of the Autonomic Nervous System 69 Ž1998. 96–102
Enhancement of thermal hyperalgesia by a-adrenoceptors in capsaicin-treated skin Peter D. Drummond
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Department of Psychology, Murdoch UniÕersity, 6150 Western Australia, Australia Received 23 July 1997; revised 15 November 1997; accepted 24 November 1997
Abstract This study aimed to investigate whether the endogenous release of noradrenaline would influence hyperalgesia to heat in skin sensitized by the topical application of 0.6% capsaicin. To release endogenous stores of noradrenaline, tyramine was introduced transcutaneously by iontophoresis into the volar aspect of the forearm of 19 healthy subjects. The heat pain threshold fell from 43.7 " 3.88C to 41.3 " 4.08C after the iontophoresis of tyramine in capsaicin-treated skin Ž P - 0.001., but did not change significantly after tyramine iontophoresis in untreated skin. The heat pain threshold decreased by 0.5 " 2.28C after the iontophoresis of saline, indicating that nonspecific factors did not fully account for the hyperalgesic effect of tyramine. Iontophoresis of the a-adrenergic antagonist, phenoxybenzamine, after the capsaicin treatment blocked the hyperalgesic effect of tyramine, suggesting that thermal hyperalgesia was mediated by a-adrenoceptors. However, iontophoresis of phenoxybenzamine before the capsaicin treatment was ineffective. These findings suggest that release of endogenous stores of noradrenaline increases sensitivity to heat in skin sensitized by capsaicin. In addition, neurogenic inflammation appears to increase access to the receptors that facilitate thermal hyperalgesia. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Noradrenaline; Pain; Capsaicin; Heat; Perineurium
1. Introduction Introducing noradrenaline into inflamed skin markedly increases sensitivity to heat ŽDrummond, 1995, 1996a, Drummond, in press., suggesting that an adrenergic excitation of sensitized cutaneous nociceptors can amplify heat pain sensations. Neurophysiological experiments have shown that noradrenaline further excites cutaneous nociceptors already discharging in response to inflammation. For example, Hu and Zhu Ž1989. reported that stimulating the sympathetic trunk or local arterial injection of noradrenaline increased the discharge of cutaneous nociceptors previously sensitized by local injection of an inflammatory soup containing serotonin, histamine, potassium and hydrogen ions. In rats chronically inflamed by adjuvant treatment, injection of noradrenaline and stimulation
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0165-1838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 8 3 8 Ž 9 8 . 0 0 0 1 7 - 4
of the lumbar sympathetic chain excited about 25% of C-fibre polymodal nociceptors in the saphenous nerve ŽSato et al., 1993, 1994.. In contrast, neither sympathetic stimulation nor injection of noradrenaline excited C-fibre polymodal nociceptors in non-inflamed skin ŽHu and Zhu, 1989; Sato et al., 1993, 1994.. Sympathetic vasoconstrictor fibres containing noradrenaline innervate arterioles deep in the dermis, whereas cutaneous nociceptors responsive to heat terminate in the superficial layers of the epidermis ŽDrummond et al., 1996a; Gibbins, 1992; Tillman et al., 1995.. In previous studies that demonstrated an adrenergic facilitation of hyperalgesia, noradrenaline was introduced transcutaneously by iontophoresis ŽDrummond, 1995, 1996a, Drummond, in press.. Unfortunately, this method does not simulate the endogenous release of noradrenaline because the concentration of noradrenaline would be highest in the epidermis, close to the iontophoresis capsule. Furthermore, local concentrations of noradrenaline were above the normal physiological range in previous studies, because the skin remained blanched for many minutes ŽDrummond, 1995,
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Drummond, in press.. Thus, whether the release of endogenous stores of noradrenaline increases sensitivity to heat is uncertain. To investigate this point in the present study, the sympathomimetic amine tyramine was used to release endogenous stores of noradrenaline. Tyramine itself does not excite adrenergic receptors, but displaces noradrenaline from vesicles into the cytosol of sympathetic nerves, and increases the loss of noradrenaline from the cytosol by a mechanism that does not involve nerve impulses or exocytosis ŽHoffman and Lefkowitz, 1996; Smith, 1973.. Since tyramine displaces endogenous stores of noradrenaline, the distribution and concentration of noradrenaline released into the skin should resemble levels achieved during neuronal discharge more closely than levels reached during the direct iontophoresis of noradrenaline. To investigate the influence of inflammation on adrenergic hyperalgesia, tyramine was iontophoresed into untreated skin and skin inflamed by the topical application of capsaicin, the pungent component of chili peppers. Injection or topical application of capsaicin provokes neurogenic inflammation mediated by neuropeptides released from sensory nerve terminals ŽWinter et al., 1995., and also directly sensitizes cutaneous nociceptors at the treated site to heat ŽCulp et al., 1989; LaMotte et al., 1992.. A second aim of the study was to determine whether pretreatment with an a-adrenergic antagonist would inhibit adrenergic hyperalgesia. Neurophysiological and behavioural experiments in animals suggest the presence of excitatory a-adrenoceptors on primary afferent neurons ŽOuseph and Levine, 1995; Sato and Perl, 1991; Sato et al., 1993, 1994.. Peripheral nerve fibres are usually separated from contact with the extracellular fluid by the perineurium, a membranous sheath that encloses nerve fascicles almost to the tip of free nerve endings, and which is continuous with the capsules of sensory ganglia and encapsulated end organs ŽOlsson, 1990.. Tight junctions between cells in the perineurium act as a barrier to the diffusion of large molecules from the extracellular fluid to the intrafascicular compartment. However, this barrier to diffusion weakens during inflammation, possibly because of disruption to tight intercellular junctions in the perineurium ŽAntonijevic et al., 1995. or because of vasodilatation and increased permeability of blood vessels that traverse the perineurium to supply the nerve fascicles ŽZochodne and Ho, 1993.. The perineurium normally separates opioid receptors on peripheral nerve trunks from the extracellular fluid ŽStein, 1995., and may also insulate a-adrenoceptors on the trunks of C-fibre afferents ŽShyu et al., 1989.. If so, inflammation could increase access to these a-adrenoceptors by adrenergic agonists and antagonists in the extracellular fluid. In the present study, the a-adrenergic antagonist, phenoxybenzamine, was introduced locally, either before or after capsaicin treatment. It was hypothesized that application of phenoxybenzamine after capsaicin treatment would inhibit adrenergic hyperal-
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gesia more effectively than application of phenoxybenzamine before capsaicin treatment. 2. Method 2.1. Subjects The sample consisted of 6 male and 13 female university students aged between 17 and 39 years Žmean age 22.9 years.. Fifteen of the subjects returned 2–77 days later Žmean 26 days. for a second session. Each subject was paid A$10 per session for participating and provided informed consent for the procedures, which were approved by the Murdoch University Ethics committee. 2.2. Procedures The experiments were carried out in an airconditioned laboratory maintained at 22 " 18C. 2.2.1. Application of capsaicin Capsaicin powder ŽSigma, St. Louis, MI, USA. was dissolved in 50% ethanol in distilled water at a concentration of 0.02 M Ž0.6%.. The skin on the volar aspect of the left or right forearm near the elbow was cleaned with soap and water and an alcohol swab. The gauze pad of an elastic dressing Ž40 mm = 25 mm., containing 400 m l of the capsaicin solution, was then applied to the prepared skin. The dressing was covered with plastic tape to retard evaporation of the capsaicin solution. Thirty minutes later, the dressing was removed and the treated skin was washed with soap and water. 2.2.2. Iontophoresis of drugs Tyramine hydrochloride ŽSigma. and phenoxybenzamine hydrochloride ŽICN Pharmaceuticals, Costa Mesa, CA, USA. were prepared daily at 0.5 mM with distilled water from stock solutions at 10 mM. A capsule Žinternal diameter 0.8 cm. was attached to the site of application of capsaicin with an adhesive washer. The capsule was filled with one of the drug solutions or with 0.9% saline, and a weak direct current Ž50 m A. was passed through the solution to introduce positively-charged ions into the skin. The ground electrode was a silver plate measuring 3 cm = 5 cm, covered in electrode paste and attached to the volar aspect of the forearm near the wrist. 2.2.3. Heat pain thresholds The radiant heat from a halogen globe was focused through a 6-mm diameter aperture placed just above the skin. Skin temperature was monitored and servo-controlled with a thermocouple bead Ž0.8 mm in diameter. which indented the skin slightly in the centre of the aperture. To assess the heat pain threshold, skin temperature increased from arm temperature Žaround 328C. at 0.58C per second until the subject signalled pain by switching the lamp off or to a maximum of 498C. Skin temperature then returned passively to around 328C over the next 10–15 s. Before the experiment, subjects practised identifying the onset of
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P.D. Drummondr Journal of the Autonomic NerÕous System 69 (1998) 96–102
burning pain until they were able to do this consistently. The heat pain threshold was measured at six sites in the capsaicin-treated skin Žthree control sites and three sites of drug iontophoresis.. The heat pain threshold at each site was calculated as the average threshold from two or three temperature ramps. The interstimulus interval at each site averaged 4 min, and the entire sequence took 6–10 min. Phenoxybenzamine was iontophoresed Ž50 m A, 10 min. at a site in the capsaicin-treated skin, starting 15 min before the topical application of capsaicin or 5 to 10 min after the capsaicin had been washed from the skin. In 15 subjects who returned on another occasion, phenoxybenzamine was iontophoresed on the other forearm Žthe timing of the phenoxybenzamine iontophoresis, with respect to the capsaicin treatment, was counterbalanced across subjects and sessions.. Heat pain thresholds were first measured after spontaneous pain from the topically-applied capsaicin had subsided. When phenoxybenzamine was iontophoresed before capsaicin, thermal testing began 15 to 30 min after removal of the capsaicin-impregnated dressing Žmean interval 22 " 6 min.; however, spontaneous pain took longer to subside in some subjects when phenoxybenzamine was iontophoresed after the application of capsaicin Žmean interval before thermal testing 37 " 10 min, range 20–55 min.. Next, saline or tyramine was iontophoresed Ž50 m A, 4 min. into the capsaicin-treated skin, or tyramine was iontophoresed at the site pretreated with phenoxybenzamine Ž50 m A, 4 min.. Subjects were not told whether saline or tyramine had been iontophoresed at any particular site. However, mild pallor occasionally developed for a few minutes at the site of the tyramine iontophoresis. The order of the three iontophoreses was randomized across subjects and sessions, and the interval between iontophoreses averaged 24 " 5 min. Starting 5 min after each iontophoresis, heat pain thresholds were measured at each of the experimental and control sites. In 16 subjects, tyramine was iontophoresed Ž50 m A, 4 min. into the forearm at a site not previously treated with capsaicin. Five minutes after the iontophoresis, heat pain thresholds were measured at this site and at an adjacent site in the same arm.
3. Results Iontophoresis of tyramine into the untreated forearm did not influence the heat pain threshold. The heat pain threshold averaged 44.0 " 3.28C Žmean " S.D.. at this site compared with 44.6 " 2.68C at an adjacent control site Ždifference not statistically significant.. Sensitivity to heat increased after the application of capsaicin. In particular, the heat pain threshold averaged 42.4 " 3.98C at control sites in the capsaicin-treated skin during the first round of thermal testing, compared with 44.6 " 2.68C in the untreated skin of the same subjects w t Ž15. s 2.79, P - 0.05x. As shown in Fig. 1, the iontophoresis of tyramine influenced the heat pain threshold in capsaicin-treated skin wSite by Time interaction, F Ž1,18. s 30.6, P - 0.001x. Investigation of this interaction indicated that the heat pain threshold decreased after the tyramine iontophoresis Ž P 0.001. but did not change at control sites over the same time interval; in addition, the heat pain threshold was lower at the site of tyramine iontophoresis than at control sites after the iontophoresis Ž P - 0.01.. Variation in the intensity of hyperalgesia at the tyramine site was unrelated to variation in the time interval between the application of capsaicin and the iontophoresis of tyramine. After the iontophoresis of saline, the heat pain threshold decreased in relation to the upward drift in the heat pain threshold at the control sites wSite by Time interaction, F Ž1,18. s 5.57, P - 0.05x; however, the decrease in the heat pain threshold at the site of saline iontophoresis was not statistically significant ŽFig. 2.. Importantly, the decrease in the heat pain threshold was greater at the tyramine site than at the saline site wSite by Time interaction, F Ž1,18. s 6.08, P - 0.05x, indicating that the hyperalgesic effect of tyramine Žaveraging 2.5 " 2.48C. was greater
2.3. Statistical analyses Preliminary analyses indicated that hyperalgesia after the tyramine and saline iontophoreses at unblocked sites was similar in sessions 1 and 2 in the 15 subjects who participated in both sessions. Therefore, heat pain thresholds were averaged across sessions in these cases. The effect of each iontophoresis on heat pain thresholds was subsequently investigated in repeated measures analyses of variance with factors of Time Žbefore vs. after the iontophoresis. and Site Žthe pain threshold at the iontophoresis site vs. the pain threshold averaged across the control sites.. The source of significant interactions between site and time was investigated with paired t-tests.
Fig. 1. Effect of tyramine iontophoresis on the heat pain threshold in capsaicin-treated skin. The heat pain threshold decreased significantly after the iontophoresis of tyramine Ž) P - 0.001., and was lower at the site of tyramine iontophoresis than at the control sites Ž P - 0.01.. Bars in Figs. 1–5 represent standard errors.
P.D. Drummondr Journal of the Autonomic NerÕous System 69 (1998) 96–102
Fig. 2. Effect of saline iontophoresis on the heat pain threshold in capsaicin-treated skin. The heat pain threshold increased at control sites over the time period required for iontophoresis Ž) P - 0.05., but did not change significantly from baseline after the saline iontophoresis.
than the hyperalgesic effect of saline Žaveraging 0.5 " 2.28C.. During the first round of thermal testing, the heat pain threshold was 1.1 " 1.68C lower at the site of phenoxybenzamine iontophoresis than at control sites in the capsaicintreated skin, regardless of whether phenoxybenzamine was iontophoresed before or after the application of capsaicin wdifference between the phenoxybenzamine and control sites, F Ž1,14. s 5.20, P - 0.05x. However, the hyperalgesic effect of phenoxybenzamine had largely dissipated before tyramine was iontophoresed Ždifference in heat pain thresholds between the phenoxybenzamine and control sites not significant, see Figs. 3 and 4.. Phenoxybenzamine iontophoresed after the topical application of capsaicin blocked the hyperalgesic effect of tyramine ŽFig. 3., whereas phenoxybenzamine iontophoresed before the topi-
Fig. 3. Effect of phenoxybenzamine pretreatment after capsaicin treatment on the hyperalgesic response to tyramine. Administration of phenoxybenzamine after capsaicin treatment blocked the hyperalgesic effect of tyramine completely.
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Fig. 4. Effect of phenoxybenzamine pretreatment before capsaicin treatment on the hyperalgesic response to tyramine. Administration of phenoxybenzamine before capsaicin treatment did not block the hyperalgesic effect of tyramine; the heat pain threshold decreased significantly after the tyramine iontophoresis Ž) P - 0.05. and was lower at this site than at the control sites after the iontophoresis Ž P - 0.05..
cal application of capsaicin did not ŽFig. 4.. In particular, when phenoxybenzamine was iontophoresed after the topical application of capsaicin, the interaction between Site and Time was not significant w F Ž1,16. s 0.04x, indicating that the iontophoresis of tyramine did not induce hyperalgesia at the phenoxybenzamine-pretreated site ŽFig. 3.. In contrast, thermal hyperalgesia increased after the iontophoresis of tyramine when phenoxybenzamine was administered before capsaicin wSite by Time interaction
Fig. 5. Summary of findings. Hyperalgesia was calculated as the difference in heat pain thresholds from before to after the iontophoresis of saline or tyramine. The iontophoresis of tyramine induced greater hyperalgesia than the iontophoresis of saline in capsaicin-treated skin. Adrenergic blockade after capsaicin treatment blocked the hyperalgesic effect of tyramine, whereas adrenergic blockade before capsaicin treatment did not.
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F Ž1,16. s 12.66, P - 0.01, see Fig. 4x. Investigation of this interaction indicated that the heat pain threshold decreased after the tyramine iontophoresis at the phenoxybenzamine-pretreated site Ž P - 0.05. and was lower at this site than at control sites after the tyramine iontophoresis Ž P - 0.05.. The interval between removal of the capsaicin-impregnated dressing and thermal testing at the phenoxybenzamine-tyramine site averaged 67 " 22 min when phenoxybenzamine was iontophoresed before capsaicin, and 74 " 20 min when phenoxybenzamine was iontophoresed after capsaicin Ždifference not significant.. There was no relationship between the heat pain threshold at the tyramine site before the iontophoresis and the change in threshold after the iontophoresis, irrespective of whether the site had been pretreated with phenoxybenzamine before or after the capsaicin treatment. The intensity of thermal hyperalgesia after the tyramine iontophoresis did not differ between blocked and unblocked sites when phenoxybenzamine was administered before the capsaicin treatment ŽFig. 5..
4. Discussion Previous studies in our laboratory demonstrated that thermal hyperalgesia increased in capsaicin-treated skin at the site of noradrenaline iontophoresis ŽDrummond, 1995, 1996a, Drummond, in press.. The present findings extend upon those observations by demonstrating that release of endogenous stores of noradrenaline increased thermal hyperalgesia in capsaicin-treated skin but not in untreated skin. In addition, the findings suggest that thermal hyperalgesia induced by tyramine iontophoresis was mediated by a-adrenoceptors, and was inhibited by a-adrenergic blockade after, but not before, capsaicin treatment. 4.1. Thermal hyperalgesia Sensitivity to heat was greater after the iontophoresis of tyramine than after the iontophoresis of saline in capsaicin-treated skin. Since tyramine displaces noradrenaline from the synaptic vesicles and cytosol of sympathetic nerve terminals ŽHoffman and Lefkowitz, 1996; Smith, 1973., the findings suggest that sympathetic fibres terminating in the skin contain enough noradrenaline to excite cutaneous nociceptors already sensitized by capsaicin treatment. The hyperalgesic effect of tyramine in capsaicintreated skin was blocked by pretreatment with phenoxybenzamine. Phenoxybenzamine irreversibly blocks aadrenoceptors, but at high concentrations will also inhibit responses to serotonin, acetylcholine and histamine ŽHoffman and Lefkowitz, 1996.. The pharmacological effects of tyramine are abolished by chronic postganglionic sympathetic denervation or by pretreatment with cocaine or reserpine ŽHoffman and Lefkowitz, 1996., indicating that tyramine targets adrenergic stores in preference to stores of
serotonin or histamine. In a previous study, iontophoretic pretreatment with the competitive a-adrenergic antagonist phentolamine Ž50 m A, 10 min. inhibited hyperalgesia to noradrenaline in skin that had been sensitized by the topical application of capsaicin ŽDrummond, in press.. In the present study, the same iontophoretic dose of phenoxybenzamine blocked the hyperalgesic effect of tyramine. Taken together, the findings suggest that hyperalgesia to tyramine was mediated by a-adrenoceptors. If endogenously-released noradrenaline facilitates thermal hyperalgesia in inflamed skin, then the iontophoresis of an a-adrenergic antagonist such as phenoxybenzamine should inhibit hyperalgesia; however, sensitivity to heat increased after the iontophoresis of phenoxybenzamine in the present study. The most likely reason for this finding is that nonspecific effects of iontophoresis contributed to thermal hyperalgesia. In previous studies, sensitivity to heat increased after a 10–min, 50 m A iontophoresis of saline in the capsaicin-treated skin of the forearm ŽDrummond, in press., and blood flow increased for several minutes when saline was iontophoresed in the forehead ŽDrummond, 1996b.. A 1-min iontophoresis of saline at 50 m A current strength had no detectable effect on cutaneous blood flow or sensitivity to heat in capsaicin-treated skin ŽDrummond, 1995., but sensitivity to heat increased slightly after a 4-min iontophoresis of saline in the present study. During transcutaneous iontophoresis, the electrical current induces hyperaemia in proportion to its strength and duration ŽGrossman et al., 1995., presumably because of neurogenic inflammation. The inflammatory response might increase sensitization of cutaneous nociceptors to heat. Phenoxybenzamine might also have a more direct influence on thermal hyperalgesia. If levels of noradrenaline in the extracellular fluid were too low to excite cutaneous nociceptors, phenoxybenzamine would not inhibit the adrenergic component of hyperalgesia. However, phenoxybenzamine itself enhances the neuronal release of noradrenaline via a 2-blockade, and inhibits the reuptake of noradrenaline ŽHoffman and Lefkowitz, 1996.; thus, an increase in extracellular levels of noradrenaline may have facilitated the discharge of cutaneous nociceptors Žassuming, of course, that phenoxybenzamine did not also block the mechanism responsible for adrenergic hyperalgesia.. Further research is needed to determine whether an augmented release of noradrenaline in response to natural stimuli such as cold or mental stress unmasks an inhibitory effect of phenoxybenzamine. Capsaicin increases blood flow inside nerve fascicles by releasing substance P and calcitonin gene-related peptide from capsaicin-sensitive afferents ŽZochodne and Ho, 1993.; these peptides liberate histamine and other inflammatory mediators from mast cells and increase vascular permeability. These effects of capsaicin might weaken the perineurial diffusion barrier and permit noradrenaline and other inflammatory mediators to access nerve fibres in the dermis. In support of this view, tyramine increased thermal
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hyperalgesia in capsaicin-treated skin but did not induce thermal hyperalgesia in untreated skin. Furthermore, phenoxybenzamine inhibited adrenergic hyperalgesia when administered after the capsaicin treatment, but was ineffective when administered before the capsaicin treatment. Thus, the adrenoceptors that facilitate thermal hyperalgesia apparently were more accessible after the capsaicin treatment than beforehand. For unknown reasons, the capsaicin treatment induced greater sensitization to heat in experimental and control sites when phenoxybenzamine was administered after the capsaicin application than beforehand. However, it is unlikely that the intensity of capsaicin-induced sensitization modified the inhibitory effect of phenoxybenzamine on adrenergic hyperalgesia, because phenoxybenzamine applied after the capsaicin treatment inhibited the tyramine effect both in very sensitive and less sensitive skin. The findings suggest that the inflammatory effect of capsaicin influenced adrenergic hyperalgesia and its modulation by adrenergic blockade, whereas the sensitizing effect of capsaicin did not. However, further investigation is required to determine whether inflammation without nociceptor sensitization influences adrenergic hyperalgesia. The lack of a hyperalgesic effect of tyramine in untreated skin is consistent with a substantial body of literature, demonstrating that noradrenaline usually does not increase nociceptor discharge ŽJanig and Koltzenburg, ¨ 1992.. However, Drummond Ž1996a. reported that thermal hyperalgesia developed in untreated skin after the iontophoresis of noradrenaline, and Meyer and Raja Ž1996. confirmed that thermal hyperalgesia developed after an intradermal injection of noradrenaline in untreated skin. The development of thermal hyperalgesia could depend on the epidermal concentration of noradrenaline; in particular, an inflammatory reaction to high concentrations of noradrenaline might allow noradrenaline to sensitize cutaneous nociceptors to heat. The concentration of noradrenaline around terminal projections of cutaneous nociceptors in the epidermis almost certainly was far higher during direct iontophoresis than during the tyramine-provoked release of noradrenaline, because noradrenaline released from perivascular sympathetic nerves in the dermis would have to diffuse a considerable distance to activate cutaneous nociceptors in the epidermis; furthermore, we observed that blanching was more intense and persisted far longer after the direct iontophoresis of noradrenaline than after the tyramine iontophoresis. Westerman et al. Ž1992. reported that transcutaneous iontophoresis of the adrenergic agonist, phenylephrine, induced neurogenic inflammation; thus, vasoactive peptides released from cutaneous nociceptors by adrenergic agonists might interact with the adrenergic agonists to sensitize nociceptors to heat. 4.2. Mechanisms of adrenergic hyperalgesia The vasoconstrictive effect of noradrenaline might trap inflammatory mediators or prevent heat transfer. To inves-
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tigate this possibility, blood flow through the capsaicintreated site on the forearm was blocked by inflating a cuff on the upper arm to above-systolic blood pressure ŽDrummond, 1996a.. Thermal hyperalgesia increased during arterial occlusion; nevertheless, sensitivity to heat was greater at the site of noradrenaline iontophoresis than elsewhere in the capsaicin-treated skin, indicating that some mechanisms, in addition to vasoconstriction, contributed to adrenergic hyperalgesia. In addition, arterial occlusion did not affect nociceptor discharge to noradrenaline in the inflamed skin of rats ŽHu and Zhu, 1989.. Noradrenaline might release inflammatory mediators from injured or inflamed tissue. For example, activation of prejunctional a 2-adrenoceptors apparently releases prostaglandins E 2 and I 2 from sympathetic postganglionic neurons ŽGonzales et al., 1991.. In addition, a-adrenoceptor activation increases the secretion of nerve growth factor, a potent nociceptive peptide ŽLewin and Mendell, 1993; Rueff et al., 1996., from cultured vascular smooth muscle ŽTuttle et al., 1993.. Alternatively, noradrenaline might act directly on excitatory a-adrenoceptors on primary afferent neurons. At present, direct evidence of excitatory adrenoceptors on primary afferent neurons is lacking; however, a 1-adrenoceptors were identified recently by autoradiography in epidermal tissue ŽDrummond et al., 1996b.. In addition, neurophysiological and behavioural experiments are consistent with the presence of a-adrenoceptors on primary afferent neurons. For example, Ouseph and Levine Ž1995. reported that mechanical hyperalgesia, produced by intradermal injection of prostaglandin E 2 with the phosphodiesterase inhibitor, rolipram, was antagonized by phentolamine and the a 1-antagonist, prazosin. Since prostaglandins sensitize primary afferent nociceptors directly, the findings support the presence of a-adrenoceptors on these neurons. Similarly, Kinnman and Levine Ž1995. reported that prazosin blocked the development of hyperalgesia to punctate stimulation around the site of an intradermal capsaicin injection in rats. Sato et al. Ž1994. found that the administration of an a 2-antagonist blocked adrenergic excitation of cutaneous nociceptors in adjuvant-inflamed rats 4 to 6 weeks after chemical sympathectomy, ruling out a prejunctional sympathetic mechanism. Sato et al. postulated that a 2-adrenoceptors on Cfibre polymodal nociceptors mediated adrenergic excitation of these nociceptors during inflammation. Even in the absence of inflammation, cutaneous nociceptors develop an adrenergic excitability after sympathectomy, a finding which is of relevance to the mechanism of postsympathectomy pain ŽBossut et al., 1996.. 4.3. Clinical implications Adrenergic mechanisms appear to contribute to chronic pain syndromes that sometimes develop after nerve or tissue injury ŽChoi and Rowbotham, 1997; Torebjork ¨ et al.,
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1995.. The present findings suggest that cutaneous nociceptors quickly develop an abnormal excitability to noradrenaline during inflammation, possibly because inflammation enhances access to a-adrenoceptors on primary afferent neurons. Since a-adrenergic blockade during neurogenic inflammation seems to prevent primary adrenergic hyperalgesia Žpresent study. and inhibits the development of central sensitization ŽKinnman et al., 1997., the therapeutic response to a-adrenergic blockade, starting immediately after nerve or soft-tissue injury, should be investigated.
Acknowledgements This study was supported by the National Health and Medical Research Council of Australia. I gratefully acknowledge the technical assistance of Ms Nadene Friday.
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bird, L.E., Molinoff, P.B., Ruddon, R.W., Gilman, A.G. ŽEds.., Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 9th edn., McGraw-Hill, New York, pp. 199–248. Hu, S., Zhu, J., 1989. Sympathetic facilitation of sustained discharges of polymodal nociceptors. Pain 38, 85–90. Janig, W., Koltzenburg, M., 1992. Possible ways of sympathetic–afferent ¨ interactions. In: Janig, W., Schmidt, R.F. ŽEds.., Reflex Sympathetic ¨ Dystrophy: Pathophysiological Mechanisms and Clinical Implications, VCH, New York, pp. 213–243. Kinnman, E., Levine, J.D., 1995. Involvement of the sympathetic postganglionic neuron in capsaicin-induced secondary hyperalgesia in the rat. Neuroscience 65, 283–291. Kinnman, E., Nygards, E.B., Hansson, P., 1997. Peripheral a-adrenor˚ eceptors are involved in the development of capsaicin-induced ongoing and stimulus-evoked pain in humans. Pain 69, 79–85. LaMotte, R.H., Lundberg, L.E.R., Torebjork, ¨ H.E., 1992. Pain, hyperalgesia and activity in nociceptive C units in humans after intradermal injection of capsaicin. J. Physiol. 448, 749–764. Lewin, G.R., Mendell, L.M., 1993. Nerve growth factor and nociception. TINS 16, 353–359. Meyer, R.A., Raja, S.N., 1996. Intradermal norepinephrine produces a dose-dependent hyperalgesia to heat in humans, Abstracts of the 8th World Congress on Pain, IASP Press, Seattle, p. 398. Olsson, Y., 1990. Microenvironment of the peripheral nervous system under normal and pathological conditions. Crit. Rev. Neurobiol. 5, 265–311. Ouseph, A.K., Levine, J.D., 1995. a 1 -Adrenoceptor-mediated sympathetically dependent mechanical hyperalgesia in the rat. Eur. J. Pharmacol. 273, 107–112. Rueff, A., Dawson, A.J.L.R., Mendell, L.M., 1996. Characteristics of nerve growth factor-induced hyperalgesia in rats: dependence on enhanced bradykinin-1 receptor activity but not neurokinin-1 receptor activation. Pain 66, 359–372. Sato, J., Perl, E.R., 1991. Adrenergic excitation of cutaneous pain receptors induced by peripheral nerve injury. Science 251, 1608–1610. Sato, J., Suzuki, S., Iseki, T., Kumazawa, T., 1993. Adrenergic excitation of cutaneous nociceptors in chronically inflamed rats. Neurosci. Lett. 164, 225–228. Sato, J., Suzuki, S., Tamura, R., Kumazawa, T., 1994. Norepinephrine excitation of cutaneous nociceptors in adjuvant-induced inflamed rats does not depend on sympathetic neurons. Neurosci. Lett. 177, 135– 138. Shyu, B.C., Olausson, B., Andersson, S.A., 1989. Sympathetic and noradrenaline effects on C-fibre transmission: single-unit analysis. Acta Physiol. Scand. 137, 85–91. Smith, A.D., 1973. Mechanisms involved in the release of noradrenaline from sympathetic nerves. Br. Med. Bull. 29, 123–129. Stein, C., 1995. The control of pain in peripheral tissue by opioids. New Engl. J. Med. 332, 1685–1690. Tillman, D.B., Treede, R.D., Meyer, R.A., Campbell, J.N., 1995. Response of C-fibre nociceptors in the anaesthetized monkey to heat stimuli: estimates of receptor depth and threshold. J. Physiol. 485, 753–765. Tuttle, J.B., Etheridge, R., Creedon, D.J., 1993. Receptor-mediated stimulation and inhibition of nerve growth factor secretion by vascular smooth muscle. Exp. Cell. Res. 208, 350–361. Torebjork, ¨ E., Wahren, L.K., Wallin, G., Hallin, R., Koltzenburg, M., 1995. Noradrenaline-evoked pain in neuralgia. Pain 63, 11–20. Westerman, R.A., Pano, I., Rabavilas, A., Nunn, A., Hahn, A., Roberts, R.G.D., Burry, H., 1992. Reflex sympathetic dystrophy: altered axon reflex and autonomic responses. Clin. Exp. Neurol. 29, 210–233. Winter, J., Bevan, S., Campbell, E.A., 1995. Capsaicin and pain mechanisms. Br. J. Anaesth. 75, 157–168. Zochodne, D.W., Ho, L.T., 1993. Evidence that capsaicin hyperaemia of rat sciatic vasa nervorum is local, opiate-sensitive and involves mast cells. J. Physiol. 468, 325–333.
Enhancement of thermal hyperalgesia by a-adrenoceptors in capsaicin-treated skin Peter D. Drummond
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Department of Psychology, Murdoch UniÕersity, 6150 Western Australia, Australia Received 23 July 1997; revised 15 November 1997; accepted 24 November 1997
Abstract This study aimed to investigate whether the endogenous release of noradrenaline would influence hyperalgesia to heat in skin sensitized by the topical application of 0.6% capsaicin. To release endogenous stores of noradrenaline, tyramine was introduced transcutaneously by iontophoresis into the volar aspect of the forearm of 19 healthy subjects. The heat pain threshold fell from 43.7 " 3.88C to 41.3 " 4.08C after the iontophoresis of tyramine in capsaicin-treated skin Ž P - 0.001., but did not change significantly after tyramine iontophoresis in untreated skin. The heat pain threshold decreased by 0.5 " 2.28C after the iontophoresis of saline, indicating that nonspecific factors did not fully account for the hyperalgesic effect of tyramine. Iontophoresis of the a-adrenergic antagonist, phenoxybenzamine, after the capsaicin treatment blocked the hyperalgesic effect of tyramine, suggesting that thermal hyperalgesia was mediated by a-adrenoceptors. However, iontophoresis of phenoxybenzamine before the capsaicin treatment was ineffective. These findings suggest that release of endogenous stores of noradrenaline increases sensitivity to heat in skin sensitized by capsaicin. In addition, neurogenic inflammation appears to increase access to the receptors that facilitate thermal hyperalgesia. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Noradrenaline; Pain; Capsaicin; Heat; Perineurium
1. Introduction Introducing noradrenaline into inflamed skin markedly increases sensitivity to heat ŽDrummond, 1995, 1996a, Drummond, in press., suggesting that an adrenergic excitation of sensitized cutaneous nociceptors can amplify heat pain sensations. Neurophysiological experiments have shown that noradrenaline further excites cutaneous nociceptors already discharging in response to inflammation. For example, Hu and Zhu Ž1989. reported that stimulating the sympathetic trunk or local arterial injection of noradrenaline increased the discharge of cutaneous nociceptors previously sensitized by local injection of an inflammatory soup containing serotonin, histamine, potassium and hydrogen ions. In rats chronically inflamed by adjuvant treatment, injection of noradrenaline and stimulation
) Corresponding author. Tel.: q61 8 93602415; fax: q61 8 93606492; e-mail: [email protected]
0165-1838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 8 3 8 Ž 9 8 . 0 0 0 1 7 - 4
of the lumbar sympathetic chain excited about 25% of C-fibre polymodal nociceptors in the saphenous nerve ŽSato et al., 1993, 1994.. In contrast, neither sympathetic stimulation nor injection of noradrenaline excited C-fibre polymodal nociceptors in non-inflamed skin ŽHu and Zhu, 1989; Sato et al., 1993, 1994.. Sympathetic vasoconstrictor fibres containing noradrenaline innervate arterioles deep in the dermis, whereas cutaneous nociceptors responsive to heat terminate in the superficial layers of the epidermis ŽDrummond et al., 1996a; Gibbins, 1992; Tillman et al., 1995.. In previous studies that demonstrated an adrenergic facilitation of hyperalgesia, noradrenaline was introduced transcutaneously by iontophoresis ŽDrummond, 1995, 1996a, Drummond, in press.. Unfortunately, this method does not simulate the endogenous release of noradrenaline because the concentration of noradrenaline would be highest in the epidermis, close to the iontophoresis capsule. Furthermore, local concentrations of noradrenaline were above the normal physiological range in previous studies, because the skin remained blanched for many minutes ŽDrummond, 1995,
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Drummond, in press.. Thus, whether the release of endogenous stores of noradrenaline increases sensitivity to heat is uncertain. To investigate this point in the present study, the sympathomimetic amine tyramine was used to release endogenous stores of noradrenaline. Tyramine itself does not excite adrenergic receptors, but displaces noradrenaline from vesicles into the cytosol of sympathetic nerves, and increases the loss of noradrenaline from the cytosol by a mechanism that does not involve nerve impulses or exocytosis ŽHoffman and Lefkowitz, 1996; Smith, 1973.. Since tyramine displaces endogenous stores of noradrenaline, the distribution and concentration of noradrenaline released into the skin should resemble levels achieved during neuronal discharge more closely than levels reached during the direct iontophoresis of noradrenaline. To investigate the influence of inflammation on adrenergic hyperalgesia, tyramine was iontophoresed into untreated skin and skin inflamed by the topical application of capsaicin, the pungent component of chili peppers. Injection or topical application of capsaicin provokes neurogenic inflammation mediated by neuropeptides released from sensory nerve terminals ŽWinter et al., 1995., and also directly sensitizes cutaneous nociceptors at the treated site to heat ŽCulp et al., 1989; LaMotte et al., 1992.. A second aim of the study was to determine whether pretreatment with an a-adrenergic antagonist would inhibit adrenergic hyperalgesia. Neurophysiological and behavioural experiments in animals suggest the presence of excitatory a-adrenoceptors on primary afferent neurons ŽOuseph and Levine, 1995; Sato and Perl, 1991; Sato et al., 1993, 1994.. Peripheral nerve fibres are usually separated from contact with the extracellular fluid by the perineurium, a membranous sheath that encloses nerve fascicles almost to the tip of free nerve endings, and which is continuous with the capsules of sensory ganglia and encapsulated end organs ŽOlsson, 1990.. Tight junctions between cells in the perineurium act as a barrier to the diffusion of large molecules from the extracellular fluid to the intrafascicular compartment. However, this barrier to diffusion weakens during inflammation, possibly because of disruption to tight intercellular junctions in the perineurium ŽAntonijevic et al., 1995. or because of vasodilatation and increased permeability of blood vessels that traverse the perineurium to supply the nerve fascicles ŽZochodne and Ho, 1993.. The perineurium normally separates opioid receptors on peripheral nerve trunks from the extracellular fluid ŽStein, 1995., and may also insulate a-adrenoceptors on the trunks of C-fibre afferents ŽShyu et al., 1989.. If so, inflammation could increase access to these a-adrenoceptors by adrenergic agonists and antagonists in the extracellular fluid. In the present study, the a-adrenergic antagonist, phenoxybenzamine, was introduced locally, either before or after capsaicin treatment. It was hypothesized that application of phenoxybenzamine after capsaicin treatment would inhibit adrenergic hyperal-
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gesia more effectively than application of phenoxybenzamine before capsaicin treatment. 2. Method 2.1. Subjects The sample consisted of 6 male and 13 female university students aged between 17 and 39 years Žmean age 22.9 years.. Fifteen of the subjects returned 2–77 days later Žmean 26 days. for a second session. Each subject was paid A$10 per session for participating and provided informed consent for the procedures, which were approved by the Murdoch University Ethics committee. 2.2. Procedures The experiments were carried out in an airconditioned laboratory maintained at 22 " 18C. 2.2.1. Application of capsaicin Capsaicin powder ŽSigma, St. Louis, MI, USA. was dissolved in 50% ethanol in distilled water at a concentration of 0.02 M Ž0.6%.. The skin on the volar aspect of the left or right forearm near the elbow was cleaned with soap and water and an alcohol swab. The gauze pad of an elastic dressing Ž40 mm = 25 mm., containing 400 m l of the capsaicin solution, was then applied to the prepared skin. The dressing was covered with plastic tape to retard evaporation of the capsaicin solution. Thirty minutes later, the dressing was removed and the treated skin was washed with soap and water. 2.2.2. Iontophoresis of drugs Tyramine hydrochloride ŽSigma. and phenoxybenzamine hydrochloride ŽICN Pharmaceuticals, Costa Mesa, CA, USA. were prepared daily at 0.5 mM with distilled water from stock solutions at 10 mM. A capsule Žinternal diameter 0.8 cm. was attached to the site of application of capsaicin with an adhesive washer. The capsule was filled with one of the drug solutions or with 0.9% saline, and a weak direct current Ž50 m A. was passed through the solution to introduce positively-charged ions into the skin. The ground electrode was a silver plate measuring 3 cm = 5 cm, covered in electrode paste and attached to the volar aspect of the forearm near the wrist. 2.2.3. Heat pain thresholds The radiant heat from a halogen globe was focused through a 6-mm diameter aperture placed just above the skin. Skin temperature was monitored and servo-controlled with a thermocouple bead Ž0.8 mm in diameter. which indented the skin slightly in the centre of the aperture. To assess the heat pain threshold, skin temperature increased from arm temperature Žaround 328C. at 0.58C per second until the subject signalled pain by switching the lamp off or to a maximum of 498C. Skin temperature then returned passively to around 328C over the next 10–15 s. Before the experiment, subjects practised identifying the onset of
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burning pain until they were able to do this consistently. The heat pain threshold was measured at six sites in the capsaicin-treated skin Žthree control sites and three sites of drug iontophoresis.. The heat pain threshold at each site was calculated as the average threshold from two or three temperature ramps. The interstimulus interval at each site averaged 4 min, and the entire sequence took 6–10 min. Phenoxybenzamine was iontophoresed Ž50 m A, 10 min. at a site in the capsaicin-treated skin, starting 15 min before the topical application of capsaicin or 5 to 10 min after the capsaicin had been washed from the skin. In 15 subjects who returned on another occasion, phenoxybenzamine was iontophoresed on the other forearm Žthe timing of the phenoxybenzamine iontophoresis, with respect to the capsaicin treatment, was counterbalanced across subjects and sessions.. Heat pain thresholds were first measured after spontaneous pain from the topically-applied capsaicin had subsided. When phenoxybenzamine was iontophoresed before capsaicin, thermal testing began 15 to 30 min after removal of the capsaicin-impregnated dressing Žmean interval 22 " 6 min.; however, spontaneous pain took longer to subside in some subjects when phenoxybenzamine was iontophoresed after the application of capsaicin Žmean interval before thermal testing 37 " 10 min, range 20–55 min.. Next, saline or tyramine was iontophoresed Ž50 m A, 4 min. into the capsaicin-treated skin, or tyramine was iontophoresed at the site pretreated with phenoxybenzamine Ž50 m A, 4 min.. Subjects were not told whether saline or tyramine had been iontophoresed at any particular site. However, mild pallor occasionally developed for a few minutes at the site of the tyramine iontophoresis. The order of the three iontophoreses was randomized across subjects and sessions, and the interval between iontophoreses averaged 24 " 5 min. Starting 5 min after each iontophoresis, heat pain thresholds were measured at each of the experimental and control sites. In 16 subjects, tyramine was iontophoresed Ž50 m A, 4 min. into the forearm at a site not previously treated with capsaicin. Five minutes after the iontophoresis, heat pain thresholds were measured at this site and at an adjacent site in the same arm.
3. Results Iontophoresis of tyramine into the untreated forearm did not influence the heat pain threshold. The heat pain threshold averaged 44.0 " 3.28C Žmean " S.D.. at this site compared with 44.6 " 2.68C at an adjacent control site Ždifference not statistically significant.. Sensitivity to heat increased after the application of capsaicin. In particular, the heat pain threshold averaged 42.4 " 3.98C at control sites in the capsaicin-treated skin during the first round of thermal testing, compared with 44.6 " 2.68C in the untreated skin of the same subjects w t Ž15. s 2.79, P - 0.05x. As shown in Fig. 1, the iontophoresis of tyramine influenced the heat pain threshold in capsaicin-treated skin wSite by Time interaction, F Ž1,18. s 30.6, P - 0.001x. Investigation of this interaction indicated that the heat pain threshold decreased after the tyramine iontophoresis Ž P 0.001. but did not change at control sites over the same time interval; in addition, the heat pain threshold was lower at the site of tyramine iontophoresis than at control sites after the iontophoresis Ž P - 0.01.. Variation in the intensity of hyperalgesia at the tyramine site was unrelated to variation in the time interval between the application of capsaicin and the iontophoresis of tyramine. After the iontophoresis of saline, the heat pain threshold decreased in relation to the upward drift in the heat pain threshold at the control sites wSite by Time interaction, F Ž1,18. s 5.57, P - 0.05x; however, the decrease in the heat pain threshold at the site of saline iontophoresis was not statistically significant ŽFig. 2.. Importantly, the decrease in the heat pain threshold was greater at the tyramine site than at the saline site wSite by Time interaction, F Ž1,18. s 6.08, P - 0.05x, indicating that the hyperalgesic effect of tyramine Žaveraging 2.5 " 2.48C. was greater
2.3. Statistical analyses Preliminary analyses indicated that hyperalgesia after the tyramine and saline iontophoreses at unblocked sites was similar in sessions 1 and 2 in the 15 subjects who participated in both sessions. Therefore, heat pain thresholds were averaged across sessions in these cases. The effect of each iontophoresis on heat pain thresholds was subsequently investigated in repeated measures analyses of variance with factors of Time Žbefore vs. after the iontophoresis. and Site Žthe pain threshold at the iontophoresis site vs. the pain threshold averaged across the control sites.. The source of significant interactions between site and time was investigated with paired t-tests.
Fig. 1. Effect of tyramine iontophoresis on the heat pain threshold in capsaicin-treated skin. The heat pain threshold decreased significantly after the iontophoresis of tyramine Ž) P - 0.001., and was lower at the site of tyramine iontophoresis than at the control sites Ž P - 0.01.. Bars in Figs. 1–5 represent standard errors.
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Fig. 2. Effect of saline iontophoresis on the heat pain threshold in capsaicin-treated skin. The heat pain threshold increased at control sites over the time period required for iontophoresis Ž) P - 0.05., but did not change significantly from baseline after the saline iontophoresis.
than the hyperalgesic effect of saline Žaveraging 0.5 " 2.28C.. During the first round of thermal testing, the heat pain threshold was 1.1 " 1.68C lower at the site of phenoxybenzamine iontophoresis than at control sites in the capsaicintreated skin, regardless of whether phenoxybenzamine was iontophoresed before or after the application of capsaicin wdifference between the phenoxybenzamine and control sites, F Ž1,14. s 5.20, P - 0.05x. However, the hyperalgesic effect of phenoxybenzamine had largely dissipated before tyramine was iontophoresed Ždifference in heat pain thresholds between the phenoxybenzamine and control sites not significant, see Figs. 3 and 4.. Phenoxybenzamine iontophoresed after the topical application of capsaicin blocked the hyperalgesic effect of tyramine ŽFig. 3., whereas phenoxybenzamine iontophoresed before the topi-
Fig. 3. Effect of phenoxybenzamine pretreatment after capsaicin treatment on the hyperalgesic response to tyramine. Administration of phenoxybenzamine after capsaicin treatment blocked the hyperalgesic effect of tyramine completely.
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Fig. 4. Effect of phenoxybenzamine pretreatment before capsaicin treatment on the hyperalgesic response to tyramine. Administration of phenoxybenzamine before capsaicin treatment did not block the hyperalgesic effect of tyramine; the heat pain threshold decreased significantly after the tyramine iontophoresis Ž) P - 0.05. and was lower at this site than at the control sites after the iontophoresis Ž P - 0.05..
cal application of capsaicin did not ŽFig. 4.. In particular, when phenoxybenzamine was iontophoresed after the topical application of capsaicin, the interaction between Site and Time was not significant w F Ž1,16. s 0.04x, indicating that the iontophoresis of tyramine did not induce hyperalgesia at the phenoxybenzamine-pretreated site ŽFig. 3.. In contrast, thermal hyperalgesia increased after the iontophoresis of tyramine when phenoxybenzamine was administered before capsaicin wSite by Time interaction
Fig. 5. Summary of findings. Hyperalgesia was calculated as the difference in heat pain thresholds from before to after the iontophoresis of saline or tyramine. The iontophoresis of tyramine induced greater hyperalgesia than the iontophoresis of saline in capsaicin-treated skin. Adrenergic blockade after capsaicin treatment blocked the hyperalgesic effect of tyramine, whereas adrenergic blockade before capsaicin treatment did not.
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F Ž1,16. s 12.66, P - 0.01, see Fig. 4x. Investigation of this interaction indicated that the heat pain threshold decreased after the tyramine iontophoresis at the phenoxybenzamine-pretreated site Ž P - 0.05. and was lower at this site than at control sites after the tyramine iontophoresis Ž P - 0.05.. The interval between removal of the capsaicin-impregnated dressing and thermal testing at the phenoxybenzamine-tyramine site averaged 67 " 22 min when phenoxybenzamine was iontophoresed before capsaicin, and 74 " 20 min when phenoxybenzamine was iontophoresed after capsaicin Ždifference not significant.. There was no relationship between the heat pain threshold at the tyramine site before the iontophoresis and the change in threshold after the iontophoresis, irrespective of whether the site had been pretreated with phenoxybenzamine before or after the capsaicin treatment. The intensity of thermal hyperalgesia after the tyramine iontophoresis did not differ between blocked and unblocked sites when phenoxybenzamine was administered before the capsaicin treatment ŽFig. 5..
4. Discussion Previous studies in our laboratory demonstrated that thermal hyperalgesia increased in capsaicin-treated skin at the site of noradrenaline iontophoresis ŽDrummond, 1995, 1996a, Drummond, in press.. The present findings extend upon those observations by demonstrating that release of endogenous stores of noradrenaline increased thermal hyperalgesia in capsaicin-treated skin but not in untreated skin. In addition, the findings suggest that thermal hyperalgesia induced by tyramine iontophoresis was mediated by a-adrenoceptors, and was inhibited by a-adrenergic blockade after, but not before, capsaicin treatment. 4.1. Thermal hyperalgesia Sensitivity to heat was greater after the iontophoresis of tyramine than after the iontophoresis of saline in capsaicin-treated skin. Since tyramine displaces noradrenaline from the synaptic vesicles and cytosol of sympathetic nerve terminals ŽHoffman and Lefkowitz, 1996; Smith, 1973., the findings suggest that sympathetic fibres terminating in the skin contain enough noradrenaline to excite cutaneous nociceptors already sensitized by capsaicin treatment. The hyperalgesic effect of tyramine in capsaicintreated skin was blocked by pretreatment with phenoxybenzamine. Phenoxybenzamine irreversibly blocks aadrenoceptors, but at high concentrations will also inhibit responses to serotonin, acetylcholine and histamine ŽHoffman and Lefkowitz, 1996.. The pharmacological effects of tyramine are abolished by chronic postganglionic sympathetic denervation or by pretreatment with cocaine or reserpine ŽHoffman and Lefkowitz, 1996., indicating that tyramine targets adrenergic stores in preference to stores of
serotonin or histamine. In a previous study, iontophoretic pretreatment with the competitive a-adrenergic antagonist phentolamine Ž50 m A, 10 min. inhibited hyperalgesia to noradrenaline in skin that had been sensitized by the topical application of capsaicin ŽDrummond, in press.. In the present study, the same iontophoretic dose of phenoxybenzamine blocked the hyperalgesic effect of tyramine. Taken together, the findings suggest that hyperalgesia to tyramine was mediated by a-adrenoceptors. If endogenously-released noradrenaline facilitates thermal hyperalgesia in inflamed skin, then the iontophoresis of an a-adrenergic antagonist such as phenoxybenzamine should inhibit hyperalgesia; however, sensitivity to heat increased after the iontophoresis of phenoxybenzamine in the present study. The most likely reason for this finding is that nonspecific effects of iontophoresis contributed to thermal hyperalgesia. In previous studies, sensitivity to heat increased after a 10–min, 50 m A iontophoresis of saline in the capsaicin-treated skin of the forearm ŽDrummond, in press., and blood flow increased for several minutes when saline was iontophoresed in the forehead ŽDrummond, 1996b.. A 1-min iontophoresis of saline at 50 m A current strength had no detectable effect on cutaneous blood flow or sensitivity to heat in capsaicin-treated skin ŽDrummond, 1995., but sensitivity to heat increased slightly after a 4-min iontophoresis of saline in the present study. During transcutaneous iontophoresis, the electrical current induces hyperaemia in proportion to its strength and duration ŽGrossman et al., 1995., presumably because of neurogenic inflammation. The inflammatory response might increase sensitization of cutaneous nociceptors to heat. Phenoxybenzamine might also have a more direct influence on thermal hyperalgesia. If levels of noradrenaline in the extracellular fluid were too low to excite cutaneous nociceptors, phenoxybenzamine would not inhibit the adrenergic component of hyperalgesia. However, phenoxybenzamine itself enhances the neuronal release of noradrenaline via a 2-blockade, and inhibits the reuptake of noradrenaline ŽHoffman and Lefkowitz, 1996.; thus, an increase in extracellular levels of noradrenaline may have facilitated the discharge of cutaneous nociceptors Žassuming, of course, that phenoxybenzamine did not also block the mechanism responsible for adrenergic hyperalgesia.. Further research is needed to determine whether an augmented release of noradrenaline in response to natural stimuli such as cold or mental stress unmasks an inhibitory effect of phenoxybenzamine. Capsaicin increases blood flow inside nerve fascicles by releasing substance P and calcitonin gene-related peptide from capsaicin-sensitive afferents ŽZochodne and Ho, 1993.; these peptides liberate histamine and other inflammatory mediators from mast cells and increase vascular permeability. These effects of capsaicin might weaken the perineurial diffusion barrier and permit noradrenaline and other inflammatory mediators to access nerve fibres in the dermis. In support of this view, tyramine increased thermal
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hyperalgesia in capsaicin-treated skin but did not induce thermal hyperalgesia in untreated skin. Furthermore, phenoxybenzamine inhibited adrenergic hyperalgesia when administered after the capsaicin treatment, but was ineffective when administered before the capsaicin treatment. Thus, the adrenoceptors that facilitate thermal hyperalgesia apparently were more accessible after the capsaicin treatment than beforehand. For unknown reasons, the capsaicin treatment induced greater sensitization to heat in experimental and control sites when phenoxybenzamine was administered after the capsaicin application than beforehand. However, it is unlikely that the intensity of capsaicin-induced sensitization modified the inhibitory effect of phenoxybenzamine on adrenergic hyperalgesia, because phenoxybenzamine applied after the capsaicin treatment inhibited the tyramine effect both in very sensitive and less sensitive skin. The findings suggest that the inflammatory effect of capsaicin influenced adrenergic hyperalgesia and its modulation by adrenergic blockade, whereas the sensitizing effect of capsaicin did not. However, further investigation is required to determine whether inflammation without nociceptor sensitization influences adrenergic hyperalgesia. The lack of a hyperalgesic effect of tyramine in untreated skin is consistent with a substantial body of literature, demonstrating that noradrenaline usually does not increase nociceptor discharge ŽJanig and Koltzenburg, ¨ 1992.. However, Drummond Ž1996a. reported that thermal hyperalgesia developed in untreated skin after the iontophoresis of noradrenaline, and Meyer and Raja Ž1996. confirmed that thermal hyperalgesia developed after an intradermal injection of noradrenaline in untreated skin. The development of thermal hyperalgesia could depend on the epidermal concentration of noradrenaline; in particular, an inflammatory reaction to high concentrations of noradrenaline might allow noradrenaline to sensitize cutaneous nociceptors to heat. The concentration of noradrenaline around terminal projections of cutaneous nociceptors in the epidermis almost certainly was far higher during direct iontophoresis than during the tyramine-provoked release of noradrenaline, because noradrenaline released from perivascular sympathetic nerves in the dermis would have to diffuse a considerable distance to activate cutaneous nociceptors in the epidermis; furthermore, we observed that blanching was more intense and persisted far longer after the direct iontophoresis of noradrenaline than after the tyramine iontophoresis. Westerman et al. Ž1992. reported that transcutaneous iontophoresis of the adrenergic agonist, phenylephrine, induced neurogenic inflammation; thus, vasoactive peptides released from cutaneous nociceptors by adrenergic agonists might interact with the adrenergic agonists to sensitize nociceptors to heat. 4.2. Mechanisms of adrenergic hyperalgesia The vasoconstrictive effect of noradrenaline might trap inflammatory mediators or prevent heat transfer. To inves-
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tigate this possibility, blood flow through the capsaicintreated site on the forearm was blocked by inflating a cuff on the upper arm to above-systolic blood pressure ŽDrummond, 1996a.. Thermal hyperalgesia increased during arterial occlusion; nevertheless, sensitivity to heat was greater at the site of noradrenaline iontophoresis than elsewhere in the capsaicin-treated skin, indicating that some mechanisms, in addition to vasoconstriction, contributed to adrenergic hyperalgesia. In addition, arterial occlusion did not affect nociceptor discharge to noradrenaline in the inflamed skin of rats ŽHu and Zhu, 1989.. Noradrenaline might release inflammatory mediators from injured or inflamed tissue. For example, activation of prejunctional a 2-adrenoceptors apparently releases prostaglandins E 2 and I 2 from sympathetic postganglionic neurons ŽGonzales et al., 1991.. In addition, a-adrenoceptor activation increases the secretion of nerve growth factor, a potent nociceptive peptide ŽLewin and Mendell, 1993; Rueff et al., 1996., from cultured vascular smooth muscle ŽTuttle et al., 1993.. Alternatively, noradrenaline might act directly on excitatory a-adrenoceptors on primary afferent neurons. At present, direct evidence of excitatory adrenoceptors on primary afferent neurons is lacking; however, a 1-adrenoceptors were identified recently by autoradiography in epidermal tissue ŽDrummond et al., 1996b.. In addition, neurophysiological and behavioural experiments are consistent with the presence of a-adrenoceptors on primary afferent neurons. For example, Ouseph and Levine Ž1995. reported that mechanical hyperalgesia, produced by intradermal injection of prostaglandin E 2 with the phosphodiesterase inhibitor, rolipram, was antagonized by phentolamine and the a 1-antagonist, prazosin. Since prostaglandins sensitize primary afferent nociceptors directly, the findings support the presence of a-adrenoceptors on these neurons. Similarly, Kinnman and Levine Ž1995. reported that prazosin blocked the development of hyperalgesia to punctate stimulation around the site of an intradermal capsaicin injection in rats. Sato et al. Ž1994. found that the administration of an a 2-antagonist blocked adrenergic excitation of cutaneous nociceptors in adjuvant-inflamed rats 4 to 6 weeks after chemical sympathectomy, ruling out a prejunctional sympathetic mechanism. Sato et al. postulated that a 2-adrenoceptors on Cfibre polymodal nociceptors mediated adrenergic excitation of these nociceptors during inflammation. Even in the absence of inflammation, cutaneous nociceptors develop an adrenergic excitability after sympathectomy, a finding which is of relevance to the mechanism of postsympathectomy pain ŽBossut et al., 1996.. 4.3. Clinical implications Adrenergic mechanisms appear to contribute to chronic pain syndromes that sometimes develop after nerve or tissue injury ŽChoi and Rowbotham, 1997; Torebjork ¨ et al.,
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P.D. Drummondr Journal of the Autonomic NerÕous System 69 (1998) 96–102
1995.. The present findings suggest that cutaneous nociceptors quickly develop an abnormal excitability to noradrenaline during inflammation, possibly because inflammation enhances access to a-adrenoceptors on primary afferent neurons. Since a-adrenergic blockade during neurogenic inflammation seems to prevent primary adrenergic hyperalgesia Žpresent study. and inhibits the development of central sensitization ŽKinnman et al., 1997., the therapeutic response to a-adrenergic blockade, starting immediately after nerve or soft-tissue injury, should be investigated.
Acknowledgements This study was supported by the National Health and Medical Research Council of Australia. I gratefully acknowledge the technical assistance of Ms Nadene Friday.
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