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Involvement of the sympathetic postganglionic neuron in capsaicin-induced secondary hyperalgesia in the rat
Neuroscience Vol. 65, No. 1, pp. 283-291, 1995
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Pergamon
0306-4522(94)00474-9
Elsevier ScienceLtd Copyright © 1995IBRO Printed in Great Britain. All rights reserved 0306-4522/95 $9.50+ 0.00
I N V O L V E M E N T OF THE S Y M P A T H E T I C P O S T G A N G L I O N I C N E U R O N IN C A P S A I C I N - I N D U C E D S E C O N D A R Y H Y P E R A L G E S I A IN THE RAT E. K I N N M A N * and J. D. L E V I N E t Departments of Anatomy/Medicine/Oral Surgery, Box 0452A, University of California, San Francisco, CA 94143, U.S.A. Abstract--The involvement of the sympathetic postganglionic neuron in secondary hyperalgesia was evaluated using a model of secondary hyperalgesia induced by a small intradermal injection of capsaicin in the rat, a procedure known to produce mechanical hyperalgesia/allodynia in humans. Capsaicin injection into the glabrous skin of the hind paw led to increased sensitivity to mechanical stimulation with von Frey filaments at the injection site (i.e. primary hyperalgesia) as well as in an area of the hind paw remote from the site of injection (i.e. secondary hyperalgesia). Surgical removal of the sympathetic postganglionic neurons innervating the hind paw plantar skin before the capsaicin injection prevented secondary hyperalgesia. However, decentralization of the sympathetic postganglionic neurons subserving the hind paw did not effect secondary hyperalgesia. Phentolamine, an a-adrenergic receptor antagonist, as well as prazosin, an ~-adrenergic receptor antagonist, given systemically, both blocked the development of secondary hyperalgesia. Yohimbine, an ct2-adrenergic receptor antagonist, was without effect. Prazosin also blocked the development of secondary hyperalgesia when given intradermally at the site of capsaicin injection, Activation of C-fibres with capsaicin induces secondary hyperalgesia, which is sympathetic postganglionic neuron-dependent. This sensory-sympathetic interaction is, however, independent of preganglionic sympathetic outflow and seems to be mediated by an cq-adrenergic mechanism. Sensory-sympathetic interaction appears to take place in the area of capsacin-induced C-fibre nociceptor activation.
Prolonged pain after trauma is accompanied by increased sensitivity towards stimuli. In the area of tissue injury, increases sensitivity towards both thermal and mechanical stimuli exists, termed primary hyperalgesia. 4,14'27'31'43 Thermal primary hyperalgesia is thought to be due to sensitization of primary afferents (for references see Refs 6 and 55), whereas the pathophysiology of mechanical hyperalgesia is less clear but seems to involve both peripheral 9'23'26'31'35'37 and central mechanisms.1°~14'21'24'26'36'52'56'61The increased mechanical sensitivity surrounding the area of tissue injury (secondary hyperalgesia 14'31) is proposed to involve input from Aft-fibre afferents from the area of secondary hyperalgesia onto wide dynamic range (WDR) neurons that have been sensitized by C-fibre activity arising from the site of tissue injury. 21,24,26,56 Neuropathy patients, who report pain and sensory abnormalities in an area outside the innervation territory of the injured peripheral nerve, may also demonstrate a sympathetically dependent
*On leave from: Departments of Neurology/Rehabilitation Medicine, Karolinska Hospital, S-171 76 Stockholm, Sweden. tTo whom correspondence should be addressed. Abbreviations: SPGN, sympathetic postganglionic neuron; WDR, wide dynamic range.
component to their pain, 2,5'~'57'58 and it has recently been shown that partial nerve injury-induced increased mechanical sensitivity in rats is dependent upon activity in sympathetic postganglionic neurons (SPGNs) 18'2°'38'5°'64 ( K i n n m a n and Levine, unpublished observations). A contribution of the S P G N to secondary hyperalgesia has, however, not been evaluated. Capsaicin applied to the h u m a n skin gives rise to burning pain as well as mechanical and thermal hyperalgesia at the site of capsaicin application, and mechanical hyperalgesia/allodynia outside the area of flare induced by capsaicin (i.e. secondary hyperalgesia) (see e.g. Ref. 21). These sensory symptoms of the capsaicin model in m a n resemble those of some neuropathy patients (see e.g. Ref. 32). Ongoing burning pain in the capsaicin model has been associated with C-fibre activity, and the secondary hyperalgesia has been shown to be mediated by Aft-fibre afferents.21,24,26,56 In the present study the possibility that secondary hyperalgesia is sympathetically maintained has been evaluated. Capsaicin was injected into the glabrous skin of the rat hind paw to evoke secondary hyperalgesia and to allow analysis of its dependence on the presence of the S P G N and the site of a sensorysympathetic interaction, as well as to investigate an ct-adrenergic receptor involvement. A role of the
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S P G N , involving an cq-adrenergic m e c h a n i s m , is p r o p o s e d in this acute m o d e l o f s e c o n d a r y h y p e r algesia. A s e n s o r y - s y m p a t h e t i c i n t e r a c t i o n a p p e a r s to take place at t h e site o f capsaicin injection. In a d d i t i o n , this s y m p a t h e t i c a l l y m a i n t a i n e d h y p e r algesia is i n d e p e n d e n t o f p r e g a n g l i o n i c s y m p a t h e t i c outflow, s u g g e s t i n g a novel m e c h a n i s m by w h i c h the S P G N c a n regulate afferent fibre i n p u t to the C N S , a n d c o n t r i b u t e to n o c i c e p t i o n .
EXPERIMENTAL PROCEDURES
Animals Thirty-five male Sprague-Dawley rats (250-300g; Bantin and Kingman, Fremont, U.S.A.) were used in this study. The animals were housed in groups of two to three under a 12h light/dark cycle throughout the experiments. Food and water were available ad libitum. Experiments were carried out under the approval o f the Institutional Animal Care Committee of the University o f California, San Francisco. Rats were allowed to habituate in the local animal care facility for at least one week prior to surgery. The animals were anaesthetized with sodium pentobarbital (50mg/kg, i.p.) prior to surgeries. Behavioural testing Behavioural testing was done before capsaicin/vehicle injections in the skin and again 20 min after the injections. Mechanical sensitivity of the plantar skin of the hind paw was tested by application of calibrated von Frey filaments. Six different nylon yon Frey filaments (A. Ainsworth, London, U.K.) were used (5.89, 9.81, 27.0, 74.4, 124 and 205mN). The animals were placed on a metal mesh floor and covered by a transparent plastic box (8 x 8 x 18cm). Each yon Frey filament was tested in the following way: 19 a yon Frey filament was inserted through the mesh and applied to the heel of the foot (see Fig. 1) until the filament just bent. The distance between the capsaic!n injection to the site o f testing was 1.5-2 cm. A trial consisted of eight repetitive yon Frey filament applications (at a frequency o f approximately l/s). Paw withdrawal any time during the application of the five stimuli was considered a response. Five trials each were performed, on the injected and control sides, with about 3min between trials. The six yon Frey filaments were applied in increasing magnitude o f force. The behavioural testing after the capsaicin/vehicle injection was ended with yon Frey filament applications as above at the site of capsaicin injection with the strongest filament (205 raN). The foot withdrawal response frequency (i.e. no. o f trials producing a paw withdrawals/5 x 100) was recorded for each yon Frey filament. Capsaicin and vehicle injections Capsaicin (30#g in a volume of 6/tg; dissolved in 5: 5 : 90, v: v: v of Tween-80~thanol-saline) (Sigma, St Louis, U.S.A.) was injected intradermally in an area just proximal to the anterior two foot pads on the left hind paw (Fig. 1). The corresponding area on the right hind paw was injected with vehicle (6#1, 5:5:90, v : v : v of Tween-80~thanol-saline), except in local prazosin treatment (see below), where capsaicin was also injected on the contralateral side. Experimental groups Normal rats (n = 6). To determine if increased sensitivity towards mechanical stimulation with von Frey filaments would take place in an area remote from that o f the capsaicin injection (i.e. at a site of secondary hyperalgesia),
Fig. 1. Schematic drawing of experimental paradigm to study capsaicin secondary hyperalgesia (left hind paw). Filled black circle represents the area of capsaicin injection. Shaded area corresponds to area o f flare induced by capsaicin injection, von Frey filaments were applied onto the cross-hatched area (area of secondary hyperalgesia) and onto the flare area around the injection site (area o f primary hyperalgesia).
rats were injected with capsaicin/vehicle and behavioural testing was done as described above. Surgical sympathectomy at the L 2 - L 4 level (n = 5). To determine if the SPGN is involved in the development of secondary hyperalgesia, a bilateral sympathectomy at the L2-L4 paravertebral ganglion level was performed. The sympathetic chain was exposed by an extraperitoneal approach from the left side. The L2-L4 level (nomenclature used as described in Ref. 3) of the sympathetic chain was visualized. The L2-L4 ganglia and sympathetic trunk were then resected bilaterally causing at least an 85% sympathetic denervation of the plantar hind p a w ) Capsaicin/vehicle injection and behavioural testing were done one week after the sympathectomy. Decentralization o f the sympathetic postganglionic neuron (n = 6). An involvement of activity in preganglionic sympathetic neurons was examined by decentralizing the sympathetic neuron prior to the capsaicin injection. The sympathetic chain was visualized and the sympathetic trunk above the left L2 ganglia as well as the white rami of the left L2 and L3 (when present) paravertebral ganglia were cut, causing a decentralization of postganglionic neurons projecting to the tibial nerve innervating the tested plantar foot. 3 Capsaicin/vehicle injections were performed one week after the decentralization. c~-adrenoreceptor antagonist treatments. To determine if an ~-adrenoreceptor was involved in the development of the increased mechanical sensitivity systemic injections o f adrenergic receptor antagonists were given. Phentolamine (n = 6; 1 mg/kg, i.p.; 2,2°,44'46 Regetine ~ , Ciba, Basle, Switzerland), an ~-adrenergic receptor antagonist, was given 10rain before the baseline readings. Four hours later, capsaicin/vehicle injections were given and 10rain before behavioural testing phentolamine (1 mg/kg, i.p.) was
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Sympathetic neurons and secondary hyperalgesia again injected since the drug is known to have a short half life. Prazosin (n = 6; 2 mg/kg, i.p.; t,47'$4 Sigma, St Louis, U.S.A.) a relatively specific ~t-adrenergic receptor antagonist. Yohimbine (n = 6; 2 mg/kg, i.p.; 1s'16'47Sigma, St Louis, U.S.A.) a relatively specific ~t2-adrenergic receptor antagonist was given 10 min before the baseline readings and then capsaicin/vehicle injections were given. Local prazosin treatment. To determine the site of sensory-sympathetic interaction, prazosin (which blocked secondary hyperalgesia when given systemically) was administrated intradermally (1 pg in 5 #1 volume, 3° dissolved in distilled water) in the capsaicin injection area (n = 6) or in the test area for secondary hyperalgesia (n = 6). Prazosin was injected in the left hind paw and vehicle (5 #1 distilled water) in the corresponding area on the right side, 10 min before the baseline readings, and capsaicin was then injected bilaterally as a control for systemic effects of the prazosin injection.
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Statistical analyses
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Data are given as mean __+S.E.M. A two-way repeated measures ANOVA was performed to compare response frequency values before and after capsaicin/vehicle injection. A Student's t-test was performed, when appropriate, to compare different experimental groups. P < 0.05 was considered statistically significant,
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R E S U L T S
Effect o f capsaicin injection in normal rats The normal rats never responded to stimulation with the three weakest yon Frey filaments (5.89, 9.81 and 27.0mN) and the response frequency to the stronger yon Frey filaments (74.4, 124 and 2 0 5 m N ) was only 0 - 2 5 % , when tested prior to capsaicin injection. After a capsaicin injection the rats responded in 35~50% of trials for all individual von Frey filament. Capsaicin injection produced a flare response on the plantar surface of the paw in all rats not extending beyond the two most proximal foot pads, allowing a distance of approximately 1 cm between the proximal border o f the flare and the test area for secondary hyperalgesia on the heel (see Fig. 1). The rats showed spontaneous licking and guarding of the capsaicin-injected paw. Capsaicin injection significantly increased the response frequency for all von Frey filaments used when applied to the heel, as well as over the injection site (all P =0.005, A N O V A ; Fig. 2). Von Frey filament stimulation often led to licking and shaking of the hindpaw on the capsaicin-injected side, throughout the course of behavioural testing, lasting approximately 1 h, not seen on the vehicle-injected side nor before injections. On the vehicle-injected side, no significant changes in response frequencies took place when von Frey filaments were applied on the heel, although the injection led to a slight increase in response frequency (P < 0.05, A N O V A ; Fig. 2) when the injection area was tested. Thus, intradermal capsaicin injection in rats leads to increased sensitivity to von Frey filament stimulation for forces in both the A/3- and A~-/C-fibre range (see Ref. 28) in an area remote from that of capsaicin injection, henceforth called the area of secondary hyperalgesia, in addition to increased mechanical NSC
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Fig. 2. Mechanical sensitivity was tested in the experimental groups before (precap) and after (postcap) 30 #g of capsaicin was injected into the left and vehicle into the right hind paw. von Frey filaments, with increasing forces (A/~-to A6-/C-fibre range~8), were applied onto the heel [5.89 ( I ) , 9.81 (O), 27.0 (A), 74.4 (0), 124 ([]) and 205 mN ((3)]. The strongest von Frey filament was also applied on the injection area [205 mN (~r)]. A significant increase in mechanical sensitivity occurred for all von Frey filaments tested when applied onto the heel of the capsaicin-injected hind paw (P < 0.005 for all yon Frey filaments, two-way repeated measures ANOVA). A significant increase in mechanical sensitivity also occurred when the strongest yon Frey filament was applied over the injection area on the capsaicin-injected hind paw (P <0.0001, two-way repeated measures ANOVA). No significant increase in mechanical sensitivity was observed when yon Frey filaments were applied onto the heel on the vehicle-injected side, although a small increase (P < 0.05, two-way repeated measures ANOVA) was seen in response to the strongest yon Frey filament over the injection site.
sensitivity over the area of injection (primary hyperalgesia).
Effect o f capsaicin injection in surgically sympathectomized rats In sympathectomized rats the capsaicin injection only produced increased mechanical sensitivity for one von Frey filament in the area of secondary hyperalgesia (5.89 mN; P < 0.05, A N O V A ; Fig. 3). It did, however, significantly increase mechanical sensitivity in the area of primary hyperalgesia (P < 0.001, A N O V A ; Fig. 3), although significantly smaller in magnitude compared to primary hyper-
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Effect of capsaicin injection in phentolamine-treated rats Capsaicin injection after p h e n t o l a m i n e t r e a t m e n t led to secondary hyperalgesia for only one von Frey filament (9.81 mN, P < 0.05, A N O V A ; Fig. 5). Primary hyperalgesia was still seen ( P < 0.01, A N O V A ; Fig. 5) on the capsaicin-injected side, a l t h o u g h significantly decreased from that seen in n o r m a l rats ( P < 0 . 0 0 5 , t-test). N o significant changes took place on the vehicle-injected side. A n c~-adrenergic m e c h a n i s m would thus seem to be involved in the d e v e l o p m e n t o f secondary and to a lesser degree in p r i m a r y hyperalgesia.
Effect of capsaicin injection in prazosin-treated rats
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Prazosin t r e a t m e n t h a d a preventive effect similar to t h a t produced by p h e n t o l a m i n e , since secondary hyperalgesia only developed for one von Frey filament (9.81raN, P < 0 . 0 5 , A N O V A ; Fig. 6). N o significant effect was seen on primary hyper-
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Fig. 3. Sympathetic postganglionic neuron-sympathectomized rats. Significant increase in mechanical sensitivity when von Frey filaments were applied onto the heel occurred for only one yon Frey filament tested on the capsaicin-injected hind paw (5.89 mN, P < 0.05, two-way repeated measures ANOVA). A significant increase in mechanical sensitivity was observed when the strongest von Frey filament was applied over the injection area on the capsaicin-injected hind paw (P <0.001, two-way repeated measures ANOVA). No significant increase in mechanical sensitivity occurred when von Frey filaments were applied onto the heel of the capsaicin-injected hind paw, or when applied over the injection site on the vehicle-injected side (two-way repeated measures ANOVA). In this figure and in Figs 4-7, injections were done and mechanical testing was performed as described in the legend to Fig. 2.
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algesia in n o r m a l rats ( P < 0.05, t-test). N o significant changes took place on the vehicle-injected side. These results suggest a role of the S P G N in the d e v e l o p m e n t o f secondary hyperalgesia, a n d to a lesser degree in p r i m a r y hyperalgesia.
Effect of eapsaicin injection in rats with decentralized sympathetic postganglionic neurons Capsaicin injection still gave rise to secondary hyperalgesia for all v o n Frey filaments ( P < 0.005, ANOVA; Fig. 4) a n d p r i m a r y hyperalgesia ( P < 0.0005, A N O V A ; Fig. 4) after transection of the preganglionic efferents. N o significant changes took place on the vehicle-injected side. Thus, preganglionic sympathetic activity does n o t seem to be necessary for the d e v e l o p m e n t o f either secondary or p r i m a r y hyperalgesia.
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Fig. 4. Decentralization sympathectomized rats. Significant increase in mechanical sensitivity when von Frey filaments were applied onto the heel occurred for all von Frey filaments tested on the capsaicin-injected hind paw (P < 0.005, two-way repeated measures ANOVA). A significant increase in mechanical sensitivity was observed when the strongest von Frey filament was applied over the injection area on the capsaicin-injected hind paw (P <0.0005, two-way repeated measures ANOVA). No significant increase in mechanical sensitivity occurred when von Frey filaments were applied onto the heel, or when applied over the injection site on the vehicle-injected side (two-way repeated measures ANOVA).
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(t-test), when prazosin was injected in the area subsequently injected with capsaicin. Increased mechanical sensitivity was seen for all v o n Frey filaments in the secondary hyperalgesia area a n d over the capsaicin-injected area (all P < 0.0005, A N O V A ; Fig. 8) o n the contralateral vehicle-injected side. These results suggest t h a t the site of s e n s o r y sympathetic interaction is the capsaicin-injected skin. Capsaicin injection into b o t h h i n d paws led to significant increase in mechanical sensitivity, as in n o r m a l rats, w h e n prazosin was injected into the area of secondary hyperalgesia in one h i n d paw a n d vehicle into the c o r r e s p o n d i n g area o n the contralateral paw (data not shown). Increased mechanical sensitivity developed bilaterally, b o t h in the area o f p r i m a r y hyperalgesia a n d in the area of secondary hyperalgesia (all P < 0.0001, A N O V A ; data not shown). Thus, the site of s e n s o r y - s y m p a t h e t i c interaction is n o t the area of secondary hyperalgesia.
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Fig. 5. Phentolamine-treated rats. Significant increase in mechanical sensitivity when von Frey filaments were applied onto the heel occurred for only one von Frey filament on the capsaicin-injected hind paw (P < 0.05, two-way repeated measures (ANOVA). A significant increase in mechanical sensitivity was observed when the strongest von Frey filament was applied over the injection area on the capsaicin-injected hind paw (P < 0.01, two-way repeated measures ANOVA). Significant increase in mechanical sensitivity when von Frey filaments were applied onto the heel occurred for only one yon Frey filament on the vehicle-injected side (27.0 mN, P < 0.05, two-way repeated measures ANOVA). No significant increase in mechanical sensitivity took place when von Frey filaments were applied over the injection site on the vehicle-injected side (two-way repeated measures ANOVA).
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algesia (Fig. 6). This indicates a n i n v o l v e m e n t of ~ - a d r e n e r g i c receptors in secondary hyperalgesia, b u t not in p r i m a r y hyperalgesia.
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Effect of capsaicin injection in yohimbine-treated rats R a t s treated with y o h i m b i n e developed secondary a n d p r i m a r y hyperalgesia ( b o t h P < 0.005, A N O V A ; Fig. 7) as in n o r m a l rats. T h u s ~2-adrenergic receptors do n o t a p p e a r to be involved in the d e v e l o p m e n t of either secondary or p r i m a r y hyperalgesia.
Effect of capsaicin in rats treated with prazosin in the law Significantly increased mechanical sensitivity was observed for only one v o n Frey filament (9.81 m N , P < 0.05, A N O V A ; Fig. 8), a n d p r i m a r y hyperalgesia was present in the area of the capsaicin injection ( P < 0 . 0 0 0 1 , A N O V A ; Fig. 8) a n d n o t significantly different f r o m t h a t in n o r m a l rats
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Fig. 6. Prazosin-treated rats. Significant increase in mechanical sensitivity when von Frey filaments were applied onto the heel occurred for only one yon Frey filament tested on the capsaicin-injected hind paw (9.81 mN, P < 0.05, twoway repeated measures ANOVA). A significant increase in mechanical sensitivity was observed when the strongest von Frey filament was applied over the injection area on the capsaicin-injected hind paw (P < 0.0001, two-way repeated measures ANOVA). No significant increase in mechanical sensitivity occurred when von Frey filaments were applied onto the heel, or when they were applied over the injection site on the vehicle-injected side (two-way repeated measures ANOVA).
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Is secondary hyperalgesia dependent upon afferent C-fibre input? Capsaicin is a selective activator o f C-fibre m e c h a n o - h e a t nociceptor fibres in rats ~7'53 a n d h u m a n s , 22'25 a n d produces pain a n d hyperalgesia/ allodynia in h u m a n s (e.g. Ref. 21). Secondary hyperalgesia developing after capsaicin injection in h u m a n s has been suggested to be produced by capsaicinevoked C-fibre activity, leading to central sensitization. 2124'26'56 F u r t h e r m o r e , blocking painful foci in some n e u r o p a t h y patients 13 can abolish touch-evoked allodynia, which could c o r r e s p o n d to the secondary hyperalgesia in the present capsaicin injection model, suggesting t h a t ongoing C-fibre input is also necessary for central sensitization in the clinical situation. The present experiments provide confirmation for the suggestion t h a t ongoing C-fibre activity leads to central sensitization 1°'12'36'48'49'5L59'62'63 and
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precap postcap Fig. 7. Yohimbine-treated rats. A significant increase in mechanical sensitivity occurred for all yon Frey filaments tested when applied onto the heel on the capsaicin-injected hind paw (P < 0.005 for all yon Frey filaments, two-way repeated measures ANOVA). A significant increase in mechanical sensitivity was also observed when the strongest von Frey filament was applied over the injection area on the capsaicin-injected hind paw (P < 0.0001, two-way repeated measures ANOVA). No significant increase in mechanical sensitivity occurred when von Frey filaments were applied onto the heel on the vehicle-injected side, although an increase (P < 0.0005, two-way repeated measures ANOVA) was seen over the injection site.
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DISCUSSION Local injection of capsaicin into the g l a b r o u s skin o f the h i n d paw o f the rat was s h o w n to increse mechanical sensitivity b o t h at the site o f injection (i.e. p r i m a r y hyperalgesia) a n d at a site distant from the site of injection (i.e. secondary hyperalgesia). Increased mechanical sensitivity can be detected by applying repetitive mechanical stimulation with yon Frey filaments (weakest forces giving rise to A/~-fibre activity a n d the strongest forces giving rise to A f a n d C-fibre activity as well 28) o n t o a n area o f skin remote to the site o f capsaicin injection a n d for t h e strongest force also in the area o f flare immediately a r o u n d the site o f capsaicin injection (Fig. 1). Since capsaicin induces mechanical hyperalgesia/allodynia in h u m a n s , H'21'26'52the b e h a v i o u r a l response to mechanical s t i m u l a t i o n is suggested to be painful also in rats.
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Fig. 8. Prazosin injected in the paw. Significant increase in mechanical sensitivity when yon Frey filaments were applied onto the heel occurred for only one von Frey filament tested when prazosin was injected into the capsaicin injection area (9.81 mN, P < 0.05, two-way repeated measures ANOVA). A significant increase in mechanical sensitivity was observed when the strongest von Frey filament was appfied over the capsaicin injection area on the prazosin-injected hind paw (P < 0.0005, two-way repeated measures ANOVA). Significant increase in mechanical sensitivity after capsaicin injection was observed for all von Frey filaments tested on the vehicle-injected side both over the capsaicin/vehicle injection area and the secondary hyperalgesia area (all P < 0.0005, two-way repeated measures ANOVA). Mechanical testing was performed as described in the legend to Fig. 2.
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secondary hyperalgesia, since a capsaicin injection was also sufficient to produce secondary hyperalgesia in the rat.
Can the sympathetic postganglionic neuron modulate secondary hyperalgesia? Recent investigations have shown that a partial nerve injury ls'2°,3s'aT,5°'64and local treatment with the irritant chloroform 3° leads to sympathetically dependent, evoked and spontaneous pain. Furthermore, symptoms in some neuropathy patients are alleviated by sympathectomy. 2'4'~'57'5s Thus, neuropathic pain conditions could, in part, share a common pathophysiology with the present capsaicin model in giving rise to an interaction between sensory and sympathetic fibres, which in turn modulates the afferent activity leading to central sensitization. Sympathectomy completely prevented secondary hyperalgesia in the present study, indicating that the SPGN is necessary for secondary hyperalgesia to develop. An ~:adrenergic mechanism is suggested for capsaicininduced secondary hyperalgesia, since phentolamine and prazosin, but not yohimbine, blocked secondary hyperalgesia. Since sympathetic stimulation can activate nociceptors in traumatized skin or after nerve injury, in cats and rabbits, 4s'47's~ it is postulated that sympathetic terminals, in capsaicin-injected rat skin, act on C-fibre nociceptor afferents to cause central sensitization. The site of sensory-sympathetic interaction for capsaicin-induced secondary hyperalgesia is conceivably the capsaicin injection area, since prazosin also blocked secondary hyperalgesia when injected locally at this site. Alternatively, the sensory-sympathetic interaction could take place via an existing anatomical coupling between sympathetic postganglionic axons and sensory neurons in dorsal root ganglia. 8,34 This possibility is less likely as these sensorysympathetic couplings seem to be very rare in normal rats and take days to develop following nerve injury. The effect of the SPGN on sensory afferents, independent of preganglionic efferent activity, is possibly produced by changes in SPGN varicosities by a local change in milieu initiated by effects on primary sensory afferents (for reference see Ref. 29). Alternatively, a reflex input to the SPGN could be due to direct primary afferent input onto SPGNs. 42'65
Is the secondary hyperalgesia mediated by Aft-fibres? The increased mechanical sensitivity in the area of secondary hyperalgesia was similar for all yon Frey filaments used, whether they provided forces only in the Aft- or also in the AbC-fibre range, 2~ suggesting that Aft-fibres carry the signal leading to secondary hyperalgesia. This is consistent with the proposed mechanism for secondary hyperalgesia whereby ongoing C-fibre activity sensitizes W D R neurons allowing normally innocuous Aft-fibre stimulation to evoke behavioural responses indicative of dysaesthesia/pain. 21's6 If secondary hyperalgesia
Fig. 9. Schematic drawing of proposed mechanisms involved in capsaicin-induced secondary hyperalgesia. Capsaicinevoked sensory nerve activity from the capsaicin-injected area (1) sensitizes WDR neurons in the dorsal horn of the spinal cord (2). The development of secondary hyperalgesia after capsaicin injection is dependent upon the presence of SPGN terminals in the vicinity of the injected area (1) acting directly or indirectly on sensory afferents via an aq-adrenergic receptor. The effect of the SPGN is independent of preganglionic sympathetic outflow. Secondary hyperalgesia evoked by repetitive yon Frey filament stimulation is mediated by uninjured Aft-fibres converging onto sensitized WDR neurons (3).
was C-fibre mediated, it would be expected to give rise to a larger increase in mechanical response frequency as the force was increased into the C-fibre range. Thus, this study is consistent with a contribution of Aft-fibres to secondary hyperalgesia. This conclusion is also consistent with experiments showing that Aft-fibres signal allodynia in neuropathy patients, 7'13'33'39~2'57,6°, since the symptoms of both mechanical allodynia in neuropathy patients as well as secondary mechanical hyperalgesia after a capsaicin injection in humans, 21'56are thought to be carried by Aft-fibres. CONCLUSIONS
The present study shows that activation of C-fibre nociceptors by capsaicin is sufficient to produce secondary hyperalgesia to develop in the rat, corroborating recent findings on secondary hyperalgesia in humans. Furthermore, the results indicate that "activity" in the SPGN independent of preganglionic input to the SPGN and acting via an Ctl-adrenergic receptor in the injected skin, is needed for secondary hyperalgesia to develop. It is hypothesized that an interaction can take place between SPGN terminals and sensory afferents in the area of intense C-fibre nociceptor activation, enabling the amount of primary afferent activity to be regulated, and thus also regulating the amount of central sensitization (primarily W D R neurons 52) that takes place (Fig. 9).
Acknowledgements--Erik Kinnman's fellowship has been funded by the Reflex Sympathetic Dystrophy Syndrome Association. Supported by NIH grant NS21445 and the Peninsula Community Foundation.
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E. Kinnman and J. D. Levine REFERENCES
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~
Pergamon
0306-4522(94)00474-9
Elsevier ScienceLtd Copyright © 1995IBRO Printed in Great Britain. All rights reserved 0306-4522/95 $9.50+ 0.00
I N V O L V E M E N T OF THE S Y M P A T H E T I C P O S T G A N G L I O N I C N E U R O N IN C A P S A I C I N - I N D U C E D S E C O N D A R Y H Y P E R A L G E S I A IN THE RAT E. K I N N M A N * and J. D. L E V I N E t Departments of Anatomy/Medicine/Oral Surgery, Box 0452A, University of California, San Francisco, CA 94143, U.S.A. Abstract--The involvement of the sympathetic postganglionic neuron in secondary hyperalgesia was evaluated using a model of secondary hyperalgesia induced by a small intradermal injection of capsaicin in the rat, a procedure known to produce mechanical hyperalgesia/allodynia in humans. Capsaicin injection into the glabrous skin of the hind paw led to increased sensitivity to mechanical stimulation with von Frey filaments at the injection site (i.e. primary hyperalgesia) as well as in an area of the hind paw remote from the site of injection (i.e. secondary hyperalgesia). Surgical removal of the sympathetic postganglionic neurons innervating the hind paw plantar skin before the capsaicin injection prevented secondary hyperalgesia. However, decentralization of the sympathetic postganglionic neurons subserving the hind paw did not effect secondary hyperalgesia. Phentolamine, an a-adrenergic receptor antagonist, as well as prazosin, an ~-adrenergic receptor antagonist, given systemically, both blocked the development of secondary hyperalgesia. Yohimbine, an ct2-adrenergic receptor antagonist, was without effect. Prazosin also blocked the development of secondary hyperalgesia when given intradermally at the site of capsaicin injection, Activation of C-fibres with capsaicin induces secondary hyperalgesia, which is sympathetic postganglionic neuron-dependent. This sensory-sympathetic interaction is, however, independent of preganglionic sympathetic outflow and seems to be mediated by an cq-adrenergic mechanism. Sensory-sympathetic interaction appears to take place in the area of capsacin-induced C-fibre nociceptor activation.
Prolonged pain after trauma is accompanied by increased sensitivity towards stimuli. In the area of tissue injury, increases sensitivity towards both thermal and mechanical stimuli exists, termed primary hyperalgesia. 4,14'27'31'43 Thermal primary hyperalgesia is thought to be due to sensitization of primary afferents (for references see Refs 6 and 55), whereas the pathophysiology of mechanical hyperalgesia is less clear but seems to involve both peripheral 9'23'26'31'35'37 and central mechanisms.1°~14'21'24'26'36'52'56'61The increased mechanical sensitivity surrounding the area of tissue injury (secondary hyperalgesia 14'31) is proposed to involve input from Aft-fibre afferents from the area of secondary hyperalgesia onto wide dynamic range (WDR) neurons that have been sensitized by C-fibre activity arising from the site of tissue injury. 21,24,26,56 Neuropathy patients, who report pain and sensory abnormalities in an area outside the innervation territory of the injured peripheral nerve, may also demonstrate a sympathetically dependent
*On leave from: Departments of Neurology/Rehabilitation Medicine, Karolinska Hospital, S-171 76 Stockholm, Sweden. tTo whom correspondence should be addressed. Abbreviations: SPGN, sympathetic postganglionic neuron; WDR, wide dynamic range.
component to their pain, 2,5'~'57'58 and it has recently been shown that partial nerve injury-induced increased mechanical sensitivity in rats is dependent upon activity in sympathetic postganglionic neurons (SPGNs) 18'2°'38'5°'64 ( K i n n m a n and Levine, unpublished observations). A contribution of the S P G N to secondary hyperalgesia has, however, not been evaluated. Capsaicin applied to the h u m a n skin gives rise to burning pain as well as mechanical and thermal hyperalgesia at the site of capsaicin application, and mechanical hyperalgesia/allodynia outside the area of flare induced by capsaicin (i.e. secondary hyperalgesia) (see e.g. Ref. 21). These sensory symptoms of the capsaicin model in m a n resemble those of some neuropathy patients (see e.g. Ref. 32). Ongoing burning pain in the capsaicin model has been associated with C-fibre activity, and the secondary hyperalgesia has been shown to be mediated by Aft-fibre afferents.21,24,26,56 In the present study the possibility that secondary hyperalgesia is sympathetically maintained has been evaluated. Capsaicin was injected into the glabrous skin of the rat hind paw to evoke secondary hyperalgesia and to allow analysis of its dependence on the presence of the S P G N and the site of a sensorysympathetic interaction, as well as to investigate an ct-adrenergic receptor involvement. A role of the
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S P G N , involving an cq-adrenergic m e c h a n i s m , is p r o p o s e d in this acute m o d e l o f s e c o n d a r y h y p e r algesia. A s e n s o r y - s y m p a t h e t i c i n t e r a c t i o n a p p e a r s to take place at t h e site o f capsaicin injection. In a d d i t i o n , this s y m p a t h e t i c a l l y m a i n t a i n e d h y p e r algesia is i n d e p e n d e n t o f p r e g a n g l i o n i c s y m p a t h e t i c outflow, s u g g e s t i n g a novel m e c h a n i s m by w h i c h the S P G N c a n regulate afferent fibre i n p u t to the C N S , a n d c o n t r i b u t e to n o c i c e p t i o n .
EXPERIMENTAL PROCEDURES
Animals Thirty-five male Sprague-Dawley rats (250-300g; Bantin and Kingman, Fremont, U.S.A.) were used in this study. The animals were housed in groups of two to three under a 12h light/dark cycle throughout the experiments. Food and water were available ad libitum. Experiments were carried out under the approval o f the Institutional Animal Care Committee of the University o f California, San Francisco. Rats were allowed to habituate in the local animal care facility for at least one week prior to surgery. The animals were anaesthetized with sodium pentobarbital (50mg/kg, i.p.) prior to surgeries. Behavioural testing Behavioural testing was done before capsaicin/vehicle injections in the skin and again 20 min after the injections. Mechanical sensitivity of the plantar skin of the hind paw was tested by application of calibrated von Frey filaments. Six different nylon yon Frey filaments (A. Ainsworth, London, U.K.) were used (5.89, 9.81, 27.0, 74.4, 124 and 205mN). The animals were placed on a metal mesh floor and covered by a transparent plastic box (8 x 8 x 18cm). Each yon Frey filament was tested in the following way: 19 a yon Frey filament was inserted through the mesh and applied to the heel of the foot (see Fig. 1) until the filament just bent. The distance between the capsaic!n injection to the site o f testing was 1.5-2 cm. A trial consisted of eight repetitive yon Frey filament applications (at a frequency o f approximately l/s). Paw withdrawal any time during the application of the five stimuli was considered a response. Five trials each were performed, on the injected and control sides, with about 3min between trials. The six yon Frey filaments were applied in increasing magnitude o f force. The behavioural testing after the capsaicin/vehicle injection was ended with yon Frey filament applications as above at the site of capsaicin injection with the strongest filament (205 raN). The foot withdrawal response frequency (i.e. no. o f trials producing a paw withdrawals/5 x 100) was recorded for each yon Frey filament. Capsaicin and vehicle injections Capsaicin (30#g in a volume of 6/tg; dissolved in 5: 5 : 90, v: v: v of Tween-80~thanol-saline) (Sigma, St Louis, U.S.A.) was injected intradermally in an area just proximal to the anterior two foot pads on the left hind paw (Fig. 1). The corresponding area on the right hind paw was injected with vehicle (6#1, 5:5:90, v : v : v of Tween-80~thanol-saline), except in local prazosin treatment (see below), where capsaicin was also injected on the contralateral side. Experimental groups Normal rats (n = 6). To determine if increased sensitivity towards mechanical stimulation with von Frey filaments would take place in an area remote from that o f the capsaicin injection (i.e. at a site of secondary hyperalgesia),
Fig. 1. Schematic drawing of experimental paradigm to study capsaicin secondary hyperalgesia (left hind paw). Filled black circle represents the area of capsaicin injection. Shaded area corresponds to area o f flare induced by capsaicin injection, von Frey filaments were applied onto the cross-hatched area (area of secondary hyperalgesia) and onto the flare area around the injection site (area o f primary hyperalgesia).
rats were injected with capsaicin/vehicle and behavioural testing was done as described above. Surgical sympathectomy at the L 2 - L 4 level (n = 5). To determine if the SPGN is involved in the development of secondary hyperalgesia, a bilateral sympathectomy at the L2-L4 paravertebral ganglion level was performed. The sympathetic chain was exposed by an extraperitoneal approach from the left side. The L2-L4 level (nomenclature used as described in Ref. 3) of the sympathetic chain was visualized. The L2-L4 ganglia and sympathetic trunk were then resected bilaterally causing at least an 85% sympathetic denervation of the plantar hind p a w ) Capsaicin/vehicle injection and behavioural testing were done one week after the sympathectomy. Decentralization o f the sympathetic postganglionic neuron (n = 6). An involvement of activity in preganglionic sympathetic neurons was examined by decentralizing the sympathetic neuron prior to the capsaicin injection. The sympathetic chain was visualized and the sympathetic trunk above the left L2 ganglia as well as the white rami of the left L2 and L3 (when present) paravertebral ganglia were cut, causing a decentralization of postganglionic neurons projecting to the tibial nerve innervating the tested plantar foot. 3 Capsaicin/vehicle injections were performed one week after the decentralization. c~-adrenoreceptor antagonist treatments. To determine if an ~-adrenoreceptor was involved in the development of the increased mechanical sensitivity systemic injections o f adrenergic receptor antagonists were given. Phentolamine (n = 6; 1 mg/kg, i.p.; 2,2°,44'46 Regetine ~ , Ciba, Basle, Switzerland), an ~-adrenergic receptor antagonist, was given 10rain before the baseline readings. Four hours later, capsaicin/vehicle injections were given and 10rain before behavioural testing phentolamine (1 mg/kg, i.p.) was
285
Sympathetic neurons and secondary hyperalgesia again injected since the drug is known to have a short half life. Prazosin (n = 6; 2 mg/kg, i.p.; t,47'$4 Sigma, St Louis, U.S.A.) a relatively specific ~t-adrenergic receptor antagonist. Yohimbine (n = 6; 2 mg/kg, i.p.; 1s'16'47Sigma, St Louis, U.S.A.) a relatively specific ~t2-adrenergic receptor antagonist was given 10 min before the baseline readings and then capsaicin/vehicle injections were given. Local prazosin treatment. To determine the site of sensory-sympathetic interaction, prazosin (which blocked secondary hyperalgesia when given systemically) was administrated intradermally (1 pg in 5 #1 volume, 3° dissolved in distilled water) in the capsaicin injection area (n = 6) or in the test area for secondary hyperalgesia (n = 6). Prazosin was injected in the left hind paw and vehicle (5 #1 distilled water) in the corresponding area on the right side, 10 min before the baseline readings, and capsaicin was then injected bilaterally as a control for systemic effects of the prazosin injection.
30 ~tg capsaicin 100-
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Data are given as mean __+S.E.M. A two-way repeated measures ANOVA was performed to compare response frequency values before and after capsaicin/vehicle injection. A Student's t-test was performed, when appropriate, to compare different experimental groups. P < 0.05 was considered statistically significant,
~ 100~
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Effect o f capsaicin injection in normal rats The normal rats never responded to stimulation with the three weakest yon Frey filaments (5.89, 9.81 and 27.0mN) and the response frequency to the stronger yon Frey filaments (74.4, 124 and 2 0 5 m N ) was only 0 - 2 5 % , when tested prior to capsaicin injection. After a capsaicin injection the rats responded in 35~50% of trials for all individual von Frey filament. Capsaicin injection produced a flare response on the plantar surface of the paw in all rats not extending beyond the two most proximal foot pads, allowing a distance of approximately 1 cm between the proximal border o f the flare and the test area for secondary hyperalgesia on the heel (see Fig. 1). The rats showed spontaneous licking and guarding of the capsaicin-injected paw. Capsaicin injection significantly increased the response frequency for all von Frey filaments used when applied to the heel, as well as over the injection site (all P =0.005, A N O V A ; Fig. 2). Von Frey filament stimulation often led to licking and shaking of the hindpaw on the capsaicin-injected side, throughout the course of behavioural testing, lasting approximately 1 h, not seen on the vehicle-injected side nor before injections. On the vehicle-injected side, no significant changes in response frequencies took place when von Frey filaments were applied on the heel, although the injection led to a slight increase in response frequency (P < 0.05, A N O V A ; Fig. 2) when the injection area was tested. Thus, intradermal capsaicin injection in rats leads to increased sensitivity to von Frey filament stimulation for forces in both the A/3- and A~-/C-fibre range (see Ref. 28) in an area remote from that of capsaicin injection, henceforth called the area of secondary hyperalgesia, in addition to increased mechanical NSC
65/I--J
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0
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postcap
Fig. 2. Mechanical sensitivity was tested in the experimental groups before (precap) and after (postcap) 30 #g of capsaicin was injected into the left and vehicle into the right hind paw. von Frey filaments, with increasing forces (A/~-to A6-/C-fibre range~8), were applied onto the heel [5.89 ( I ) , 9.81 (O), 27.0 (A), 74.4 (0), 124 ([]) and 205 mN ((3)]. The strongest von Frey filament was also applied on the injection area [205 mN (~r)]. A significant increase in mechanical sensitivity occurred for all von Frey filaments tested when applied onto the heel of the capsaicin-injected hind paw (P < 0.005 for all yon Frey filaments, two-way repeated measures ANOVA). A significant increase in mechanical sensitivity also occurred when the strongest yon Frey filament was applied over the injection area on the capsaicin-injected hind paw (P <0.0001, two-way repeated measures ANOVA). No significant increase in mechanical sensitivity was observed when yon Frey filaments were applied onto the heel on the vehicle-injected side, although a small increase (P < 0.05, two-way repeated measures ANOVA) was seen in response to the strongest yon Frey filament over the injection site.
sensitivity over the area of injection (primary hyperalgesia).
Effect o f capsaicin injection in surgically sympathectomized rats In sympathectomized rats the capsaicin injection only produced increased mechanical sensitivity for one von Frey filament in the area of secondary hyperalgesia (5.89 mN; P < 0.05, A N O V A ; Fig. 3). It did, however, significantly increase mechanical sensitivity in the area of primary hyperalgesia (P < 0.001, A N O V A ; Fig. 3), although significantly smaller in magnitude compared to primary hyper-
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Effect of capsaicin injection in phentolamine-treated rats Capsaicin injection after p h e n t o l a m i n e t r e a t m e n t led to secondary hyperalgesia for only one von Frey filament (9.81 mN, P < 0.05, A N O V A ; Fig. 5). Primary hyperalgesia was still seen ( P < 0.01, A N O V A ; Fig. 5) on the capsaicin-injected side, a l t h o u g h significantly decreased from that seen in n o r m a l rats ( P < 0 . 0 0 5 , t-test). N o significant changes took place on the vehicle-injected side. A n c~-adrenergic m e c h a n i s m would thus seem to be involved in the d e v e l o p m e n t o f secondary and to a lesser degree in p r i m a r y hyperalgesia.
Effect of capsaicin injection in prazosin-treated rats
vehicle
Prazosin t r e a t m e n t h a d a preventive effect similar to t h a t produced by p h e n t o l a m i n e , since secondary hyperalgesia only developed for one von Frey filament (9.81raN, P < 0 . 0 5 , A N O V A ; Fig. 6). N o significant effect was seen on primary hyper-
100 8060-
4030 lag capsaicin 100
20
0
pre~ap
'
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Fig. 3. Sympathetic postganglionic neuron-sympathectomized rats. Significant increase in mechanical sensitivity when von Frey filaments were applied onto the heel occurred for only one yon Frey filament tested on the capsaicin-injected hind paw (5.89 mN, P < 0.05, two-way repeated measures ANOVA). A significant increase in mechanical sensitivity was observed when the strongest von Frey filament was applied over the injection area on the capsaicin-injected hind paw (P <0.001, two-way repeated measures ANOVA). No significant increase in mechanical sensitivity occurred when von Frey filaments were applied onto the heel of the capsaicin-injected hind paw, or when applied over the injection site on the vehicle-injected side (two-way repeated measures ANOVA). In this figure and in Figs 4-7, injections were done and mechanical testing was performed as described in the legend to Fig. 2.
8060 40200
u
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precap
vehicle 100 8060
algesia in n o r m a l rats ( P < 0.05, t-test). N o significant changes took place on the vehicle-injected side. These results suggest a role of the S P G N in the d e v e l o p m e n t o f secondary hyperalgesia, a n d to a lesser degree in p r i m a r y hyperalgesia.
Effect of eapsaicin injection in rats with decentralized sympathetic postganglionic neurons Capsaicin injection still gave rise to secondary hyperalgesia for all v o n Frey filaments ( P < 0.005, ANOVA; Fig. 4) a n d p r i m a r y hyperalgesia ( P < 0.0005, A N O V A ; Fig. 4) after transection of the preganglionic efferents. N o significant changes took place on the vehicle-injected side. Thus, preganglionic sympathetic activity does n o t seem to be necessary for the d e v e l o p m e n t o f either secondary or p r i m a r y hyperalgesia.
40-
2O
0
precap
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Fig. 4. Decentralization sympathectomized rats. Significant increase in mechanical sensitivity when von Frey filaments were applied onto the heel occurred for all von Frey filaments tested on the capsaicin-injected hind paw (P < 0.005, two-way repeated measures ANOVA). A significant increase in mechanical sensitivity was observed when the strongest von Frey filament was applied over the injection area on the capsaicin-injected hind paw (P <0.0005, two-way repeated measures ANOVA). No significant increase in mechanical sensitivity occurred when von Frey filaments were applied onto the heel, or when applied over the injection site on the vehicle-injected side (two-way repeated measures ANOVA).
287
Sympathetic neurons and secondary hyperalgesia 30 #g capsaicin 100-
8060-
40200
pre~p
'
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601 404
(t-test), when prazosin was injected in the area subsequently injected with capsaicin. Increased mechanical sensitivity was seen for all v o n Frey filaments in the secondary hyperalgesia area a n d over the capsaicin-injected area (all P < 0.0005, A N O V A ; Fig. 8) o n the contralateral vehicle-injected side. These results suggest t h a t the site of s e n s o r y sympathetic interaction is the capsaicin-injected skin. Capsaicin injection into b o t h h i n d paws led to significant increase in mechanical sensitivity, as in n o r m a l rats, w h e n prazosin was injected into the area of secondary hyperalgesia in one h i n d paw a n d vehicle into the c o r r e s p o n d i n g area o n the contralateral paw (data not shown). Increased mechanical sensitivity developed bilaterally, b o t h in the area o f p r i m a r y hyperalgesia a n d in the area of secondary hyperalgesia (all P < 0.0001, A N O V A ; data not shown). Thus, the site of s e n s o r y - s y m p a t h e t i c interaction is n o t the area of secondary hyperalgesia.
30 txg capsaicin
20 100O"
-
-
precap
postcap
Fig. 5. Phentolamine-treated rats. Significant increase in mechanical sensitivity when von Frey filaments were applied onto the heel occurred for only one von Frey filament on the capsaicin-injected hind paw (P < 0.05, two-way repeated measures (ANOVA). A significant increase in mechanical sensitivity was observed when the strongest von Frey filament was applied over the injection area on the capsaicin-injected hind paw (P < 0.01, two-way repeated measures ANOVA). Significant increase in mechanical sensitivity when von Frey filaments were applied onto the heel occurred for only one yon Frey filament on the vehicle-injected side (27.0 mN, P < 0.05, two-way repeated measures ANOVA). No significant increase in mechanical sensitivity took place when von Frey filaments were applied over the injection site on the vehicle-injected side (two-way repeated measures ANOVA).
8060 40200
precap
postcap
vehicle 10080-
algesia (Fig. 6). This indicates a n i n v o l v e m e n t of ~ - a d r e n e r g i c receptors in secondary hyperalgesia, b u t not in p r i m a r y hyperalgesia.
6040-
Effect of capsaicin injection in yohimbine-treated rats R a t s treated with y o h i m b i n e developed secondary a n d p r i m a r y hyperalgesia ( b o t h P < 0.005, A N O V A ; Fig. 7) as in n o r m a l rats. T h u s ~2-adrenergic receptors do n o t a p p e a r to be involved in the d e v e l o p m e n t of either secondary or p r i m a r y hyperalgesia.
Effect of capsaicin in rats treated with prazosin in the law Significantly increased mechanical sensitivity was observed for only one v o n Frey filament (9.81 m N , P < 0.05, A N O V A ; Fig. 8), a n d p r i m a r y hyperalgesia was present in the area of the capsaicin injection ( P < 0 . 0 0 0 1 , A N O V A ; Fig. 8) a n d n o t significantly different f r o m t h a t in n o r m a l rats
20-
precap
postcap
Fig. 6. Prazosin-treated rats. Significant increase in mechanical sensitivity when von Frey filaments were applied onto the heel occurred for only one yon Frey filament tested on the capsaicin-injected hind paw (9.81 mN, P < 0.05, twoway repeated measures ANOVA). A significant increase in mechanical sensitivity was observed when the strongest von Frey filament was applied over the injection area on the capsaicin-injected hind paw (P < 0.0001, two-way repeated measures ANOVA). No significant increase in mechanical sensitivity occurred when von Frey filaments were applied onto the heel, or when they were applied over the injection site on the vehicle-injected side (two-way repeated measures ANOVA).
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Is secondary hyperalgesia dependent upon afferent C-fibre input? Capsaicin is a selective activator o f C-fibre m e c h a n o - h e a t nociceptor fibres in rats ~7'53 a n d h u m a n s , 22'25 a n d produces pain a n d hyperalgesia/ allodynia in h u m a n s (e.g. Ref. 21). Secondary hyperalgesia developing after capsaicin injection in h u m a n s has been suggested to be produced by capsaicinevoked C-fibre activity, leading to central sensitization. 2124'26'56 F u r t h e r m o r e , blocking painful foci in some n e u r o p a t h y patients 13 can abolish touch-evoked allodynia, which could c o r r e s p o n d to the secondary hyperalgesia in the present capsaicin injection model, suggesting t h a t ongoing C-fibre input is also necessary for central sensitization in the clinical situation. The present experiments provide confirmation for the suggestion t h a t ongoing C-fibre activity leads to central sensitization 1°'12'36'48'49'5L59'62'63 and
6030 I.tg capsaicin 40
100
20-
80-
o
60
precap postcap Fig. 7. Yohimbine-treated rats. A significant increase in mechanical sensitivity occurred for all yon Frey filaments tested when applied onto the heel on the capsaicin-injected hind paw (P < 0.005 for all yon Frey filaments, two-way repeated measures ANOVA). A significant increase in mechanical sensitivity was also observed when the strongest von Frey filament was applied over the injection area on the capsaicin-injected hind paw (P < 0.0001, two-way repeated measures ANOVA). No significant increase in mechanical sensitivity occurred when von Frey filaments were applied onto the heel on the vehicle-injected side, although an increase (P < 0.0005, two-way repeated measures ANOVA) was seen over the injection site.
4020-
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DISCUSSION Local injection of capsaicin into the g l a b r o u s skin o f the h i n d paw o f the rat was s h o w n to increse mechanical sensitivity b o t h at the site o f injection (i.e. p r i m a r y hyperalgesia) a n d at a site distant from the site of injection (i.e. secondary hyperalgesia). Increased mechanical sensitivity can be detected by applying repetitive mechanical stimulation with yon Frey filaments (weakest forces giving rise to A/~-fibre activity a n d the strongest forces giving rise to A f a n d C-fibre activity as well 28) o n t o a n area o f skin remote to the site o f capsaicin injection a n d for t h e strongest force also in the area o f flare immediately a r o u n d the site o f capsaicin injection (Fig. 1). Since capsaicin induces mechanical hyperalgesia/allodynia in h u m a n s , H'21'26'52the b e h a v i o u r a l response to mechanical s t i m u l a t i o n is suggested to be painful also in rats.
4020 0
precap
postcap
Fig. 8. Prazosin injected in the paw. Significant increase in mechanical sensitivity when yon Frey filaments were applied onto the heel occurred for only one von Frey filament tested when prazosin was injected into the capsaicin injection area (9.81 mN, P < 0.05, two-way repeated measures ANOVA). A significant increase in mechanical sensitivity was observed when the strongest von Frey filament was appfied over the capsaicin injection area on the prazosin-injected hind paw (P < 0.0005, two-way repeated measures ANOVA). Significant increase in mechanical sensitivity after capsaicin injection was observed for all von Frey filaments tested on the vehicle-injected side both over the capsaicin/vehicle injection area and the secondary hyperalgesia area (all P < 0.0005, two-way repeated measures ANOVA). Mechanical testing was performed as described in the legend to Fig. 2.
Sympathetic neurons and secondary hyperalgesia
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secondary hyperalgesia, since a capsaicin injection was also sufficient to produce secondary hyperalgesia in the rat.
Can the sympathetic postganglionic neuron modulate secondary hyperalgesia? Recent investigations have shown that a partial nerve injury ls'2°,3s'aT,5°'64and local treatment with the irritant chloroform 3° leads to sympathetically dependent, evoked and spontaneous pain. Furthermore, symptoms in some neuropathy patients are alleviated by sympathectomy. 2'4'~'57'5s Thus, neuropathic pain conditions could, in part, share a common pathophysiology with the present capsaicin model in giving rise to an interaction between sensory and sympathetic fibres, which in turn modulates the afferent activity leading to central sensitization. Sympathectomy completely prevented secondary hyperalgesia in the present study, indicating that the SPGN is necessary for secondary hyperalgesia to develop. An ~:adrenergic mechanism is suggested for capsaicininduced secondary hyperalgesia, since phentolamine and prazosin, but not yohimbine, blocked secondary hyperalgesia. Since sympathetic stimulation can activate nociceptors in traumatized skin or after nerve injury, in cats and rabbits, 4s'47's~ it is postulated that sympathetic terminals, in capsaicin-injected rat skin, act on C-fibre nociceptor afferents to cause central sensitization. The site of sensory-sympathetic interaction for capsaicin-induced secondary hyperalgesia is conceivably the capsaicin injection area, since prazosin also blocked secondary hyperalgesia when injected locally at this site. Alternatively, the sensory-sympathetic interaction could take place via an existing anatomical coupling between sympathetic postganglionic axons and sensory neurons in dorsal root ganglia. 8,34 This possibility is less likely as these sensorysympathetic couplings seem to be very rare in normal rats and take days to develop following nerve injury. The effect of the SPGN on sensory afferents, independent of preganglionic efferent activity, is possibly produced by changes in SPGN varicosities by a local change in milieu initiated by effects on primary sensory afferents (for reference see Ref. 29). Alternatively, a reflex input to the SPGN could be due to direct primary afferent input onto SPGNs. 42'65
Is the secondary hyperalgesia mediated by Aft-fibres? The increased mechanical sensitivity in the area of secondary hyperalgesia was similar for all yon Frey filaments used, whether they provided forces only in the Aft- or also in the AbC-fibre range, 2~ suggesting that Aft-fibres carry the signal leading to secondary hyperalgesia. This is consistent with the proposed mechanism for secondary hyperalgesia whereby ongoing C-fibre activity sensitizes W D R neurons allowing normally innocuous Aft-fibre stimulation to evoke behavioural responses indicative of dysaesthesia/pain. 21's6 If secondary hyperalgesia
Fig. 9. Schematic drawing of proposed mechanisms involved in capsaicin-induced secondary hyperalgesia. Capsaicinevoked sensory nerve activity from the capsaicin-injected area (1) sensitizes WDR neurons in the dorsal horn of the spinal cord (2). The development of secondary hyperalgesia after capsaicin injection is dependent upon the presence of SPGN terminals in the vicinity of the injected area (1) acting directly or indirectly on sensory afferents via an aq-adrenergic receptor. The effect of the SPGN is independent of preganglionic sympathetic outflow. Secondary hyperalgesia evoked by repetitive yon Frey filament stimulation is mediated by uninjured Aft-fibres converging onto sensitized WDR neurons (3).
was C-fibre mediated, it would be expected to give rise to a larger increase in mechanical response frequency as the force was increased into the C-fibre range. Thus, this study is consistent with a contribution of Aft-fibres to secondary hyperalgesia. This conclusion is also consistent with experiments showing that Aft-fibres signal allodynia in neuropathy patients, 7'13'33'39~2'57,6°, since the symptoms of both mechanical allodynia in neuropathy patients as well as secondary mechanical hyperalgesia after a capsaicin injection in humans, 21'56are thought to be carried by Aft-fibres. CONCLUSIONS
The present study shows that activation of C-fibre nociceptors by capsaicin is sufficient to produce secondary hyperalgesia to develop in the rat, corroborating recent findings on secondary hyperalgesia in humans. Furthermore, the results indicate that "activity" in the SPGN independent of preganglionic input to the SPGN and acting via an Ctl-adrenergic receptor in the injected skin, is needed for secondary hyperalgesia to develop. It is hypothesized that an interaction can take place between SPGN terminals and sensory afferents in the area of intense C-fibre nociceptor activation, enabling the amount of primary afferent activity to be regulated, and thus also regulating the amount of central sensitization (primarily W D R neurons 52) that takes place (Fig. 9).
Acknowledgements--Erik Kinnman's fellowship has been funded by the Reflex Sympathetic Dystrophy Syndrome Association. Supported by NIH grant NS21445 and the Peninsula Community Foundation.
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