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The role of spinal cord activation before neurectomy in the development of autotomy
Pain, 64 (1996) 161-167 © 1996 Elsevier Science B.V. All rights reserved 0304-3959/96/$15.00
161
PAIN 2876
The role of spinal cord activation before neurectomy in the development of autotomy Hiroshi Asada a,*, Yuzo Yamaguchi a, Sigeru Tsunoda a and Yutaka F u k u d a b a Department of Health Science, College of Integrated Arts and Sciences, Osaka Prefecture University, Gakuencho 1-1, Sakai 593 (Japan) and b Department of Physiology, Osaka University Medical School, Yamadaoka 2-2, Suita 565, (Japan) (Received 13 April 1995, accepted 19 April 1995)
Summary A model of deafferentation pain is provided by sectioning the sciatic and saphenous nerves in the rat and mouse. This procedure leads to self-mutilation of the denervated hindpaw (autotomy). A noxious stimulus to the denervated area before neurectomy is known to enhance the autotomy. To understand the mechanism underlying this enhancement by prior noxious stimuli, we examined the effects of intrathecal (i.t.) injection of substance P (SP) and somatostatin (SOM) on autotomy behavior. These peptides are known to be released from primary afferent terminals in the dorsal horn by noxious stimuli. A single i.t. injection of SP or SOM just before neurectomy dramatically enhanced autotomy behavior in mice. Autotomy was enhanced in a dose-dependent manner with i.t. injection of SP (0.1-20 nmol) 5 min before neurectomy or SOM (0.1-1.0 nmol) 20 min before neurectomy. Autotomy s:ignificantly decreased by extending the interval between i.t. injection of SP or SOM and neurectomy. Intact mice injected with the same doses of SP or SOM showed dose-dependent acute nociceptive responses directed to the hindpaw. The severity of autotomy in neurectomized mice and the duration of acute nociceptive responses induced by the same doses of SP or SOM in intact mice were related. These results suggest that neuropeptides applied to the spinal dorsal horn just before deafferentation induce a state of central neural activation with long-lasting effects on the function of CNS cells. Augmentation of autotomy is a result of this activation which is kept as a 'memory'. Key words: Substance P; Somatostatin; Neurectomy; Autotomy; Intrathecal injection; Memory
Introduction Animals with transected peripheral nerves may develop self-mutilating behavior (autotomy), directed to the denervated areas after a delay of several days to weeks. This behavior is considered to be useful for the study of mechanisms of chronic deafferentation pain. The development of autotomy behavior is affected by noxious stimuli and tissue injury before neurectomy. Noxious chemical (Denni,,; and Melzack 1979; Coderre et al. 1986), heat (Coderre and Melzack 1985, 1986; Asada et al. 1991; Katz et al. 1991) and electrical (Seltzer et al. 1991; Katz et al. 1991) stimuli prior to
* Corresponding author." Hiroshi Asada, Department of Health Sciences, Osaka Prefecture University, Gakuencho 1-1, Sakai 593, Japan. Tel.: (81) 722-52-1161, ext. 2663; FAX: (81) 722-55-2981. SSDI 0304-3959(95)00086-Cb
neurectomy or rhizotomy significantly enhance the development of autotomy following subsequent deafferentation. Noxious stimuli or injury after neurectomy also enhances autotomy, but the effect is significantly less (Coderre and Melzack 1985; Katz et al. 1991). Even though the noxious stimulation prior to neurectomy is only of short duration, its effects on autotomy are long-delayed and manifest itself later as an acceleration of autotomy onset and an increase in its severity. These findings support the notion that noxious stimulation produces long-lasting changes in function in the dorsal horn; such central changes constitute a 'pain memory' (Coderre et al. 1993). A related observation has been reported in man. Specifically, phantom limb pain occurs more often in amputees who had pain in the limb prior to amputation (Melzack 1971). If preamputation pain is experienced at or near the time of amputation, there is a higher probability that it will
162 p e r s i s t i n t h e p h a n t o m l i m b ( J e n s e n e t al. 1985; K a t z e t al. 1990). Noxious stimulation or peripheral injury, including i n f l a m m a t i o n , is k n o w n t o c a u s e t h e r e l e a s e o f s u b stance P (SP) and somatostatin (SOM) from afferent f i b e r t e r m i n a l s in r a t s p i n a l c o r d d o r s a l h o r n ( Y a k s h e t al. 1980; K a n t n e r e t al. 1985; K u r a i s h i e t al. 1985; S a t o h e t al. 1987; M o r t o n e t al. 1989; T i s e o e t al. 1990), If SP and SOM released in the dorsal horn by noxious injury before neurectomy are related to the enhancem e n t o f a u t o t o m y , i n t r a t h e c a l (i.t.) a d m i n i s t r a t i o n o f these peptides before neurectomy should facilitate subsequent autotomy. The present study was aimed at testing this prediction.
Experiment 2
Materials and methods
Experiment 3
This study was carried out in accordance with the guidelines of the Ethics Committee of the International Association for the Study of Pain (Zimmermann 1983) and all procedures were approved by the local Animal Ethics Committee of the Osaka University Medical School.
Intrathecal injection of SP and SOM before neurectomy In experiments 1 and 2, 114 adult male ddY mice weighing 25-40 g were used. Animals were operated under deep pentobarbital anesthesia (80-100 mg/kg, i.p.). SP and SOM (Sigma Chemical, USA) were dissolved in normal saline. Solutions of these drugs were applied according to a slight modification of the method of Hylden and Wilcox (1980). All i.t. injections were made at the L5 and L6 intervertebral space in anesthetized mice using a stainless steel hypodermic needle of 0.3 mm diameter under a stereoscopic microscope. A volume of 2.5 /zl SP, SOM or saline was injected using a 10/~1 Hamilton syringe. At various times (5-40 min) after the i.t. injection of peptide or vehicle, neurectomy was performed. First, the sciatic nerve was exposed in the mid-thigh of the left hindlimb and tightly ligated at two locations 3-5 mm apart. The nerve segment between the ligatures was then removed. Likewise, the saphenous nerve was neurectomized in the leg. The muscles and skin were sutured in layers. The animals were housed 3-5 in a cage with sawdust litter under natural day/night cycle and constant room temperature (24+1°C). Food and water were always available.
Experiment 1 Previous studies have demonstrated that i.t. administration of SP or SOM in conscious mice induces a characteristic behavior pattern including biting, licking and scratching of the hindquarter. This behavior peaks at 0-5 min (SP) and 15-20 rain (SOM) post-injection, respectively (Seybold et al. 1982; Ohkubo et al. 1990). In this study, neurectomy was performed 5 rain after i.t. injection of SP or 20 min after i.t. injection of SOM. Four doses of SP were given in 4 different groups of animals: 0.1 nmol (SP-0.1, n = 7), 1.0 nmol (SP-1.0, n =9), 10 nmol (SP-10, n = 10) and 20 nmol (SP-20, n = 10). Similarly, four doses of SOM were given: 0.1 nmol (SOM-0.1, n = 9), 1.0 nmol (SOM-1.0, n = 10), 10 nmoi (SOM-10, n = 10) and 20 nmol (SOM-20, n = 10). In a control group, neurectomy was performed 20 min after i.t. injection of normal saline (SAL, n = 12),
We investigated the effect of extending the interval between i.t. injection of the peptides and neurectomy. In the case of SP injection, neurectomy was performed 20 min after i.t. SP injection (10 nmol, SP-10-20MIN, n = 10). In the case of SOM injection, neurectomy was performed 40 min after i.t. SOM injections (10 nmol and 1.0 nmol, SOM-10-40MIN and SOM-1.0-40MIN, n = 10, respectively). Scoring of autotomy behavior. Autotomy was scored daily for the first autotomy onset days after surgery, and then weekly for up to 6 weeks using the scale devised by Wall et al. (1979). A score of 1 was assigned to an injury of one or more claws, and 1 additional point was added when a distal or proximal half toe was injured. Finally, 1-2 points were added in proportion to the severity of the injury to the distal half of the foot. The maximal possible score was 13 points. Onset day of autotomy was defined as the day on which an autotomy score of at least 1 was observed for the first time. If mice did not autotomize by the 42nd day after surgery, an onset day of 43 was assigned.
In Exp. 3, 36 male ddY mice were used. Duration of acute nociceptive responses induced by i.t. injections of SP or SOM was measured in intact awake behaving mice. This provided an index of spinal excitability. Before the i.t. injection, each mouse was adapted for 10 min to an individual round plastic observation chamber (diameter: 20 cm). A mirror was mounted at a 45° angle beneath the transparent floor to allow clear observation of the paw. The i.t. injection of SP or SOM was made according to the method of Hylden and Wilcox (1980). Immediately after i.t. injections (2.5/~1) of SP (0.1, 1.0 and 10 nmol; n = 6, respectively), SOM (0.1, 1.0 and 10 nmol; n = 6, respectively) or saline (n = 5), the mice were placed in the observation chamber. Their behavior was video-taped for 50 min. The accumulated time spent in licking, biting and shaking the hindpaw during each 1-min interval, measured from the video tape, was used as an indicator of nociception. A single observer was used who was blind as to drug assignment.
Statistical analysis Average autotomy scores of a group were compared 42 days postoperatively (6th week) using the Kruskal-Wallis H test (KW test) and ScheffCs procedure (S test) for multiple comparisons. The severity of autotomy behavior was assessed through evaluation of the area under the autotomy score curve from the time of surgery to the 6th postoperative week in each animal. Average autotomy areas and average onset days of autotomy were also compared using the KW test and S test. In Exp. 2, pair-wise comparisons of autotomy scores and onset days were made using the 2-tailed Mann-Whitney U test (U test). In Exp. 3, time courses of nociceptive responses were compared using the Lindquist type I design of ANOVA. The criterion for statistical significance was P < 0.05 in all cases. Results are expressed as mean + SEM.
Results
Experiment 1 The first self-mutilation started 2 days after neurectomy in the SP- and SOM-injected groups. Autotomy usually involved first the nails and progressed to more p r o x i m a l a r e a s o f t h e d e n e r v a t e d h i n d p a w . Fig. 1 A , B show the time course of the development of autotomy i n m i c e w i t h i.t. i n j e c t i o n s o f S P o r S O M b e f o r e
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Fig. 1. A, B: average autotomy scores (+SEM) of mice which were pre-injected i.t. saline, 0.1, 1.0, 10 or 20 nmol SP and SOM, 5 min (in SP-injected groups) or 20 min (in SOM-injected groups) before neurectomy. * P < 0.05; ** P < 0.01; significant difference from the SAL group, • *P < 0.01; significant difference from the SP-0.1 group, * P < 0.05; significant difference from the SP-1.0 group (S-test). C: mean areas ( 5: SEM) under autotomy score curve in A, B are plotted against drug dose. neurectomy. The average scores of the SP-20 (6.9 + 1.7) and SP-10 (7.8 + 1.1) groups at the 6th week were both significantly higher than that of the SAL group (1.2 + 0.3) ( P < 0.05 and P < 0.01; S test, respectively) and the score of the SP-10 group was significantly higher than those of the SP-0.1 (2.2 + 0.8) and SP-1.0 (3.4 + 0.8) groups ( P < 0.01 and P < 0.05; S test, respectively). The average autotomy scores of the SOM-20 (5.2 + 1.0), SOM-10 ( 5 . 4 + 1.1) and SOM-1.0 ( 7 . 9 + 1.1) groups were significantly higher than that of the SAL group (1.2 + 0.3) ( P < 0.01, P < 0.01 and P < 0.05; S test, respectively). Although the scores of the SOM-10 and SOM-20 groups were lower than that of the SOM1.0 group, the difference was not significant ( P > 0.2).
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In Fig. 1C, the areas under each of the autotomy score curves in Fig. 1A,B are plotted against the dose of SP and SOM. The severity of autotomy as revealed by the areas was enhanced in a dose-dependent manner in groups with i.t. injection of SP 5 min before neurectomy (0.1-20 nmol). In the groups with i.t. injection of SOM 20 min before neurectomy, the severity of autotomy was enhanced in a dose-dependent manner when 0.1 and 1.0 nmol SOM were injected, but it decreased when the dose of SOM was increased to 10 and 20 nmol. The onset of autotomy was presented for each of the SAL-, SP- and SOM-injected groups (Fig. 2). The average delay to autotomy onset was significantly shorter in the SP-1.0 (14.2 + 4.7 days, P < 0.05, S test), SP-10 (9.0 + 2.0 days, P < 0.01, S test) and SP-20 (9.7 + 3.7 days, P < 0.01, S test) groups than in the SAL group (26.7 + 3.9 days). The average onset day of autotomy in the SOM-1.0 (6.1 + 1.3 days), SOM-10 (4.7 + 1.1 days) and SOM-20 (8.5 + 3.9 days) groups was significantly shorter than in the SAL group (26.7 + 3.9 days) ( P < 0.01, in all cases).
Experiment 2
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Fig. 2. Average (+ SEM) onset delays of autotomy (score > 1) of each group in Fig. 1A, B. *P < 0.05; **P <0.01; significant difference from the SAL group (S-test).
Fig. 3 shows the effect on autotomy scores of extending the delay between neurectomy and SP or SOM injection. The average autotomy score in the 6th week of the SP-10-20MIN group (1.7 + 0.4), which received neurectomy 20 rain after SP injection, was significantly lower than that of the SP-10-5MIN group (7.8 + 1.1) which received neurectomy 5 rain after the injection
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Fig. 3. Average autotomy scores (+ SEM) of mice which received neurectomy at different times after i.t. injection of 10 nmol SP, 1.0 nmol SOM or 10 nmol SOM. The curves for the SP-10-5MIN, SOM-I.0-20MIN and SOM-10-20MIN group are, respectively, the same as the SP-10, SOM-1.0 and SOM-10 group shown in Fig. 1A, B. A: ** P < 0.01; significant difference from the SP-10-5MIN group. B: ** P < 0.01; significant difference from the SOM-1.0-20MIN group. C: no significant difference (P > 0.1) (U test).
( P < 0.01, U t e s t ) (Fig. 3A). L i k e w i s e , u s i n g 1.0 n m o l o f S O M , t h e a v e r a g e a u t o t o m y s c o r e at t h e 6th w e e k o f t h e S O M - 1 . 0 - 4 0 M I N g r o u p ( 1 . 0 + 0 . 3 ) , was signifi-
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165 the SOM-10-40MIN group (3.1 + 0.4) was lower than that of the SOM-10-20MIN group (5.4 + 1.1). However the difference in the scores was not statistically significant (P > 0.1, U test) (F'ig. 3C). There was no statistical significant difference in the pair-wise comparisons of autotomy onset days in the SP-10-5MIN (9.0 _+2.0 clays) and SP-10-20MIN (9.5 _+ 3.6 days) group, in the SOM-1.0-20MIN (6.1 _+ 1.3 days) and SOM-1.0-40MIN (2L7.3_+6.3 days) group, and in the SOM-10-20MIN (4.7+ 1.1 days) and SOM-1040MIN (6.6 _+ 1.6 days) group.
Experiment 3 Fig. 4 shows the average duration of responses (licking, biting or shaking the hindpaw) for each 5 min of observation after i.t. injection of SP or SOM (n = 6). Although licking and biting responses directed to the abdomen, thigh and other regions were also frequently observed, we only coded responses directed to the hindpaw. Intrathecal injection of saline had no apparent effect on the behavior of mice. In the mice injected with 10 nmol SP, these responses were observed fi:equently between 0 and 5 rain after injection. In subsequent periods, these responses became less frequent until about 30 min after SP injection when they ceased altogether. When the dose of SP was decreased, responses for 0-5 min decreased in a dose-dependent manner. ANOVA revealed significant effects of SP dose (0.1, 1.0 and 10 nmol SP injection) (F2,15 = 4.31, P < 0.05), t i m e (F7,10 5 = 16,23, P < 0.001), and the SP dose by time interaction (F14,105 = 4.14, P < 0.01). Mice injected with 10 nmol SOM showed a behavioral syndrome similar to that of SP. However, the period showing these responses was longer than that after SP injections. Responses were intermittently observed for about 40 rain after SOM injection and subsequently almost disappeared. In the case of 1.0 nmol injection of SOM, responses were similarly observed intermittently for about 20 min after SOM injection. ANOVA revealed significant effect of time (F7,105 = 2.62, P < 0.05) but did not reveal significant effects of SOM dose (0.1, 1.0 and 10 nmol SOM injection) (P > 0.1) and the SOM dose by time interaction (P > 0.2).
Discussion
Single i.t. injections of SP and SOM just before neurectomy dramatically enhanced autotomy. This suggests that the enhancement of autotomy by noxious stimulation before neurectomy is at least partly associated with CNS mechanisms triggered by the release of SP and SOM in the spinal cord dorsal horn.
Nociceptive behaviors including biting, scratching and licking of the hindquarters, are dose-dependently elicited after i.t. injections of SP or SOM in mice (Hylden and Wilcox 1981, 1983; Gamse and Saria 1986; Ohkubo et al. 1990) and rats (Seybold et al. 1982; Matsumura et al. 1985). The i.t. application of SP (Woolf and Wiesenfeld-Hallin 1986) and SOM (Wiesenfeld-Hallin 1985) produces prolonged enhancement of the excitability of the flexion reflex and the responses of nociceptive neurons (Hylden and Wilcox 1981; Seybold et al. 1982; Ohkubo et al. 1990). These facts suggest that the nociceptive behaviors induced by i.t. injection of SP and SOM reflect the activity level of spinal neurons. We measured the duration of these responses to the hindraw after i.t. SP and SOM injections at the same dose. Our results suggest that these durations reflect the period of activated hyperexcitable neurons in the spinal cord that mediete input from the hindpaw to higher levels in the CNS. This hyperexcitability is able to interact with neurectomy-evoked activities to influence subsequent autotomy. The result of Exp. 3 indicates that increased activity of neurons in the spinal dorsal horn would occur for at least 5 min after i.t. SP injection (10 nmol), and for at least 20 min after i.t. SOM injection (1 or 10 nmol). Thus, in the SP-10-5MIN, SOM-1.0-20MIN and SOM10-20MIN groups, in which neurectomy was done during the evoked activation of spinal neurons, the development of autotomy was dramatically enhanced. Conversely, the activation of neurons in the dorsal horn would already have decreased, or disappeared, about 20 min after i.t. injection of SP (10 nmol) or about 40 min after i.t. injection of SOM (1.0 nmol). When neurectomy was performed at these intervals (SP-1020MIN and SOM-1.0-40MIN groups), there was relatively little effect on autotomy. These facts support the hypothesis that the level of activity of neurons in the dorsal horn just before or during neurectomy plays an important role in the expression of autotomy. Evidence for the role of central activation before deafferentation is presented in several recent reports. Repetitive activity in afferent C fibers before neurectomy, especially at a 'wind-up' frequency, augmented the response of some nociceptive dorsal horn neurons and substantially enhanced the severity of autotomy (Seltzer et al. 1991). Nociceptive responses observed 30-60 min after injection of formalin (phase II responses) are not eliminated by complete anesthetic blockade of the injected area at the time of formalin injection. However, they are significantly reduced by spinal anesthesia induced immediately prior to formalin injection. Spinal anesthesia administered 5 min after formalin injection is also ineffective (Coderre et al. 1990). Local anesthetic applied to the peripheral nerves prior to nerve section significantly reduced autotomy in rats (Gonzalez-Darder et al. 1986; Seltzer et
166 al. 1991). Clinical r e p o r t s show t h a t if p a i n is experie n c e d in t h e limb at o r n e a r the t i m e o f a m p u t a t i o n , t h e n t h e r e is a high p r o b a b i l i t y t h a t p h a n t o m limb p a i n will occur ( J e n s e n et al. 1985; K a t z a n d M e l z a c k 1990). I n o u r e x p e r i m e n t s t h e effect of i.t. p r e - i n j e c t i o n o f SP on a u t o t o m y was d o s e - d e p e n d e n t , b u t t h e e n h a n c e m e n t o f a u t o t o m y by i.t. injection o f S O M s h o w e d a slight r e d u c t i o n at h i g h e r d o s e (Fig. 1, right panel). This m a y b e d u e to a n e u r o t o x i c o r a n a l g e s i c effect of h i g h - d o s e S O M ( G o n z a l e z - D a r d e r et al. 1986; G a u m a n n et al. 1988, 1989). In t h e following, w e p r o p o s e a p o s s i b l e m e c h a n i s m for the p h e n o m e n a t h a t we have observed. Noxious s t i m u l a t i o n o r injury c a u s e s t h e r e l e a s e of SP, S O M a n d excitatory a m i n o acids ( E A A ) within t h e d o r s a l horn, t h e r e l e a s e o f SP m a y itself cause t h e r e l e a s e of E A A (Smullin et al. 1990). T o g e t h e r , t h e s e n e u r o n a l t r a n s m i t t e r s p r o d u c e activation of spinal d o r s a l h o r n n e u r o n s via N - m e t h y l - t > a s p a r t i c acid ( N M D A ) a n d p e p t i d e r e c e p t o r activation, which triggers f u n c t i o n a l c h a n g e s in m e m b r a n e excitability t h r o u g h i n t e r a c t i o n s with s e c o n d m e s s e n g e r systems such as p r o t e i n kinases (Wilcox 1991). T h e i.t. a p p l i c a t i o n o f the n e u r o p e p t i d e s SP a n d S O M p r o d u c e s t r a n s i e n t e n h a n c e m e n t o f spinal excitability b u t it n e v e r triggers t h e a u t o t o m y b e h a v i o r w i t h o u t d e a f f e r e n t a t i o n ( S e y b o l d et al. 1982). M o r e over, t h e r e is only little a u t o t o m y w h e n d e a f f e r e n t a t i o n is p e r f o r m e d s u b s e q u e n t l y ( g r o u p s S P - 1 0 - 2 0 M I N a n d S O M - 1 . 0 - 4 0 M I N ) . T h e c e n t r a l c h a n g e s t h a t yield enh a n c e d a u t o t o m y r e q u i r e s i m u l t a n e o u s actions of i.t. SP o r S O M excitation a n d w h a t e v e r c h a n g e is t r i g g e r e d at t h e t i m e of t h e n e u r e c t o m y itself, p e r h a p s ' i n j u r y d i s c h a r g e ' ( W a l l et al. 1985; S e l t z e r et al. 1991; W o o l f 1991). B o t h o f t h e s e effects a r e n e e d e d to leave t h e c e n t r a l c h a n g e s as p a i n ' m e m o r y ' . T h e s e f u n c t i o n a l c e n t r a l c h a n g e s m a y b e m a i n t a i n e d for days o r l o n g e r as ' m e m o r y ' in spinal c o r d d o r s a l h o r n n e u r o n r e l a t e d to t h e s u b s e q u e n t l y d e n e r v a t e d area. T h e s e c h a n g e s m a y l e a d to i n c r e a s e d s p o n t a n e o u s activity in t h e spinal c o r d ( L o m b a r d et al. 1983; A p k a r i a n et al. 1984; A s a d a et al. 1990). In a d d i t i o n , following n e u r e c t o m y , s p o n t a n e o u s activity g e n e r a t e d f r o m n e u r o m a a n d d o r s a l r o o t g a n g l i o n m a y evoke d y s e s t h e t i c o r p a i n f u l s e n s a t i o n s r e f e r r e d to t h e d e n e r v a t e d region. T h e c o i n c i d e n c e o f t h e s e activities i n c r e a s e s the severity o f a u t o t o m y .
Acknowledgements W e wish to t h a n k Dr. G a r y J. B e n n e t t , Dr. M a r s h a l l Devor, Dr. Y o s h i o S h i g e n a g a a n d Dr. Kenji K a w a k i t a for t h e i r constructive c o m m e n t s d u r i n g t h e writing o f t h e m a n u s c r i p t a n d for E n g l i s h revision.
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167 information related to pain in the spinal dorsal horn, Brain Res., 325 (1985) 294-298. Lombard, M.C. and Larabi, Y., Electrophysiological study of cervical dorsal horn cells in partially deafferented rats. In: J.J. Bonica, U. Lindblom and A. Iggo (Eds.), Advances in Pain Research and Therapy, Vol. 5, Raven Press, New York, 1983, pp. 147-154. Matsumura, H., Sakurada, T., Hara, A., Sakurada, S. and Kisara, K., Characterization of the hyperalgesic effect induced by intrathecal injection of substance P, Neuropharmacology, 24 (1985) 421-426. Melzack, R., Phantom limb pain: implications for treatment of pathologic pain, Anesthesiology, 35 (1971) 409-419. Morton, C.R., Hutchison, W.D., Hendry, I.A. and Duggan, A.W., Somatostatin: evidence for a role in themal nociception, Brain Res., 488 (1989) 89-96. Ohkubo, T., Shibata, M., Takahashi, H. and Inoki, R., Roles of substance P and somatostatin on transmission of nociceptive information induced by formalin in spinal cord, J. Pharmacol. Exp. Ther., 252 (1990) 12611-1268. Satoh, M., Kuraishi, Y., Hirota, N., Suzuki, H. and Takagi, Involvement of somatostatin in heat-nociceptive transmission at the spinal dorsal horn and inhilgitory mechanisms on it, Pain, 4 (1987) $240. Seltzer, Z., Beilin, B.Z., Ginzburg, R., Paran, Y. and Shimko, T., The role of injury discharge in the induction of neuropathic pain behavior in rats, Pain, 46 (1991) 327-336. Seybold, V.S., Hylden, J.L.K and Wilcox, G.L., Intrathecal substance P and somatostatin in rats: behaviors indicative of sensation, Peptides, 3 (1982) 49--54. Smullin, D.H., Skilling, S.R. and Larson, A.A., Interaction between substance P, calcitonin gene related peptide, taurine and excitatory amino acids in the spinal cord, Pain, 42 (1990) 93-10l. Tiseo, P.J., Adler, M.W. and Liu-Chen, L., Differential release of
substance P and somatostatin in the rat spinal cord in response to noxious cold and heat; effect of dynorphin A(1-17), J. Pharmacol. Exp. Ther., 252 (1990) 539-545. Wall, P.D., Scadding, J.W. and Tomkiewitz, M.M., The production and prevention of experimental anesthesia dolorosa, Pain, 6 (1979) 175-185. Wall, P.D. and Woolf, C.J., The brief and the prolonged facilitatory effects of unmyelinated afferent input on the rat spinal cord are independently influenced by peripheral nerve injury, Neuroscience, 17 (1985) 1199-1206, Wiesenfeld-Hallin, Z., Intrathecal somatostatin modurates spinal sensory reflex mechanisms: behavioral and electrophysiological studies, Neurosci. Lett., 62 (1985) 69-74. Wilcox, G.L., Excitatory neurotransmitters ad pain, In: M.R. Bond, J.E. Charlton and C.J. Woolf (Eds.), Pain Research and Clinical Management, Vol. 4, Proc. Vlth World Congress on Pain, Elsevier, Amsterdam, 1991, pp. 97-117. Woolf, C.J., Central mechanisms of acute pain. In: M.R. Bond, J.E. Charlton and C.J. Woolf (Eds.), Pain Research and Clinical Management, Vol. 4, Proc. Vlth World Congress on Pain, Elsevier, Amsterdam, 1991, pp. 25-34. Woolf, C.J. and Wiesenfeld-Hallin, Z., Substance P and calcitonin gene-related peptide synergistically modulate the gain of the nociceptive flexor withdrawal reflex in the rat, Neurosci. Lett., 66 (1986) 226-230. Yaksh, T.L., Jessell, T.M., Gamse, R., Mudge, A.W. and Leeman, S.E., Intrathecal morphine inhibits substance P release from mammalian spinal cord in vivo, Nature (Lond.), 286 (1980) 155157. Zimmermann, M., Ethical guidelines for investigations of experimental pain in conscious animals, Pain, 16 (1983) 109-110.
161
PAIN 2876
The role of spinal cord activation before neurectomy in the development of autotomy Hiroshi Asada a,*, Yuzo Yamaguchi a, Sigeru Tsunoda a and Yutaka F u k u d a b a Department of Health Science, College of Integrated Arts and Sciences, Osaka Prefecture University, Gakuencho 1-1, Sakai 593 (Japan) and b Department of Physiology, Osaka University Medical School, Yamadaoka 2-2, Suita 565, (Japan) (Received 13 April 1995, accepted 19 April 1995)
Summary A model of deafferentation pain is provided by sectioning the sciatic and saphenous nerves in the rat and mouse. This procedure leads to self-mutilation of the denervated hindpaw (autotomy). A noxious stimulus to the denervated area before neurectomy is known to enhance the autotomy. To understand the mechanism underlying this enhancement by prior noxious stimuli, we examined the effects of intrathecal (i.t.) injection of substance P (SP) and somatostatin (SOM) on autotomy behavior. These peptides are known to be released from primary afferent terminals in the dorsal horn by noxious stimuli. A single i.t. injection of SP or SOM just before neurectomy dramatically enhanced autotomy behavior in mice. Autotomy was enhanced in a dose-dependent manner with i.t. injection of SP (0.1-20 nmol) 5 min before neurectomy or SOM (0.1-1.0 nmol) 20 min before neurectomy. Autotomy s:ignificantly decreased by extending the interval between i.t. injection of SP or SOM and neurectomy. Intact mice injected with the same doses of SP or SOM showed dose-dependent acute nociceptive responses directed to the hindpaw. The severity of autotomy in neurectomized mice and the duration of acute nociceptive responses induced by the same doses of SP or SOM in intact mice were related. These results suggest that neuropeptides applied to the spinal dorsal horn just before deafferentation induce a state of central neural activation with long-lasting effects on the function of CNS cells. Augmentation of autotomy is a result of this activation which is kept as a 'memory'. Key words: Substance P; Somatostatin; Neurectomy; Autotomy; Intrathecal injection; Memory
Introduction Animals with transected peripheral nerves may develop self-mutilating behavior (autotomy), directed to the denervated areas after a delay of several days to weeks. This behavior is considered to be useful for the study of mechanisms of chronic deafferentation pain. The development of autotomy behavior is affected by noxious stimuli and tissue injury before neurectomy. Noxious chemical (Denni,,; and Melzack 1979; Coderre et al. 1986), heat (Coderre and Melzack 1985, 1986; Asada et al. 1991; Katz et al. 1991) and electrical (Seltzer et al. 1991; Katz et al. 1991) stimuli prior to
* Corresponding author." Hiroshi Asada, Department of Health Sciences, Osaka Prefecture University, Gakuencho 1-1, Sakai 593, Japan. Tel.: (81) 722-52-1161, ext. 2663; FAX: (81) 722-55-2981. SSDI 0304-3959(95)00086-Cb
neurectomy or rhizotomy significantly enhance the development of autotomy following subsequent deafferentation. Noxious stimuli or injury after neurectomy also enhances autotomy, but the effect is significantly less (Coderre and Melzack 1985; Katz et al. 1991). Even though the noxious stimulation prior to neurectomy is only of short duration, its effects on autotomy are long-delayed and manifest itself later as an acceleration of autotomy onset and an increase in its severity. These findings support the notion that noxious stimulation produces long-lasting changes in function in the dorsal horn; such central changes constitute a 'pain memory' (Coderre et al. 1993). A related observation has been reported in man. Specifically, phantom limb pain occurs more often in amputees who had pain in the limb prior to amputation (Melzack 1971). If preamputation pain is experienced at or near the time of amputation, there is a higher probability that it will
162 p e r s i s t i n t h e p h a n t o m l i m b ( J e n s e n e t al. 1985; K a t z e t al. 1990). Noxious stimulation or peripheral injury, including i n f l a m m a t i o n , is k n o w n t o c a u s e t h e r e l e a s e o f s u b stance P (SP) and somatostatin (SOM) from afferent f i b e r t e r m i n a l s in r a t s p i n a l c o r d d o r s a l h o r n ( Y a k s h e t al. 1980; K a n t n e r e t al. 1985; K u r a i s h i e t al. 1985; S a t o h e t al. 1987; M o r t o n e t al. 1989; T i s e o e t al. 1990), If SP and SOM released in the dorsal horn by noxious injury before neurectomy are related to the enhancem e n t o f a u t o t o m y , i n t r a t h e c a l (i.t.) a d m i n i s t r a t i o n o f these peptides before neurectomy should facilitate subsequent autotomy. The present study was aimed at testing this prediction.
Experiment 2
Materials and methods
Experiment 3
This study was carried out in accordance with the guidelines of the Ethics Committee of the International Association for the Study of Pain (Zimmermann 1983) and all procedures were approved by the local Animal Ethics Committee of the Osaka University Medical School.
Intrathecal injection of SP and SOM before neurectomy In experiments 1 and 2, 114 adult male ddY mice weighing 25-40 g were used. Animals were operated under deep pentobarbital anesthesia (80-100 mg/kg, i.p.). SP and SOM (Sigma Chemical, USA) were dissolved in normal saline. Solutions of these drugs were applied according to a slight modification of the method of Hylden and Wilcox (1980). All i.t. injections were made at the L5 and L6 intervertebral space in anesthetized mice using a stainless steel hypodermic needle of 0.3 mm diameter under a stereoscopic microscope. A volume of 2.5 /zl SP, SOM or saline was injected using a 10/~1 Hamilton syringe. At various times (5-40 min) after the i.t. injection of peptide or vehicle, neurectomy was performed. First, the sciatic nerve was exposed in the mid-thigh of the left hindlimb and tightly ligated at two locations 3-5 mm apart. The nerve segment between the ligatures was then removed. Likewise, the saphenous nerve was neurectomized in the leg. The muscles and skin were sutured in layers. The animals were housed 3-5 in a cage with sawdust litter under natural day/night cycle and constant room temperature (24+1°C). Food and water were always available.
Experiment 1 Previous studies have demonstrated that i.t. administration of SP or SOM in conscious mice induces a characteristic behavior pattern including biting, licking and scratching of the hindquarter. This behavior peaks at 0-5 min (SP) and 15-20 rain (SOM) post-injection, respectively (Seybold et al. 1982; Ohkubo et al. 1990). In this study, neurectomy was performed 5 rain after i.t. injection of SP or 20 min after i.t. injection of SOM. Four doses of SP were given in 4 different groups of animals: 0.1 nmol (SP-0.1, n = 7), 1.0 nmol (SP-1.0, n =9), 10 nmol (SP-10, n = 10) and 20 nmol (SP-20, n = 10). Similarly, four doses of SOM were given: 0.1 nmol (SOM-0.1, n = 9), 1.0 nmol (SOM-1.0, n = 10), 10 nmoi (SOM-10, n = 10) and 20 nmol (SOM-20, n = 10). In a control group, neurectomy was performed 20 min after i.t. injection of normal saline (SAL, n = 12),
We investigated the effect of extending the interval between i.t. injection of the peptides and neurectomy. In the case of SP injection, neurectomy was performed 20 min after i.t. SP injection (10 nmol, SP-10-20MIN, n = 10). In the case of SOM injection, neurectomy was performed 40 min after i.t. SOM injections (10 nmol and 1.0 nmol, SOM-10-40MIN and SOM-1.0-40MIN, n = 10, respectively). Scoring of autotomy behavior. Autotomy was scored daily for the first autotomy onset days after surgery, and then weekly for up to 6 weeks using the scale devised by Wall et al. (1979). A score of 1 was assigned to an injury of one or more claws, and 1 additional point was added when a distal or proximal half toe was injured. Finally, 1-2 points were added in proportion to the severity of the injury to the distal half of the foot. The maximal possible score was 13 points. Onset day of autotomy was defined as the day on which an autotomy score of at least 1 was observed for the first time. If mice did not autotomize by the 42nd day after surgery, an onset day of 43 was assigned.
In Exp. 3, 36 male ddY mice were used. Duration of acute nociceptive responses induced by i.t. injections of SP or SOM was measured in intact awake behaving mice. This provided an index of spinal excitability. Before the i.t. injection, each mouse was adapted for 10 min to an individual round plastic observation chamber (diameter: 20 cm). A mirror was mounted at a 45° angle beneath the transparent floor to allow clear observation of the paw. The i.t. injection of SP or SOM was made according to the method of Hylden and Wilcox (1980). Immediately after i.t. injections (2.5/~1) of SP (0.1, 1.0 and 10 nmol; n = 6, respectively), SOM (0.1, 1.0 and 10 nmol; n = 6, respectively) or saline (n = 5), the mice were placed in the observation chamber. Their behavior was video-taped for 50 min. The accumulated time spent in licking, biting and shaking the hindpaw during each 1-min interval, measured from the video tape, was used as an indicator of nociception. A single observer was used who was blind as to drug assignment.
Statistical analysis Average autotomy scores of a group were compared 42 days postoperatively (6th week) using the Kruskal-Wallis H test (KW test) and ScheffCs procedure (S test) for multiple comparisons. The severity of autotomy behavior was assessed through evaluation of the area under the autotomy score curve from the time of surgery to the 6th postoperative week in each animal. Average autotomy areas and average onset days of autotomy were also compared using the KW test and S test. In Exp. 2, pair-wise comparisons of autotomy scores and onset days were made using the 2-tailed Mann-Whitney U test (U test). In Exp. 3, time courses of nociceptive responses were compared using the Lindquist type I design of ANOVA. The criterion for statistical significance was P < 0.05 in all cases. Results are expressed as mean + SEM.
Results
Experiment 1 The first self-mutilation started 2 days after neurectomy in the SP- and SOM-injected groups. Autotomy usually involved first the nails and progressed to more p r o x i m a l a r e a s o f t h e d e n e r v a t e d h i n d p a w . Fig. 1 A , B show the time course of the development of autotomy i n m i c e w i t h i.t. i n j e c t i o n s o f S P o r S O M b e f o r e
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Fig. 1. A, B: average autotomy scores (+SEM) of mice which were pre-injected i.t. saline, 0.1, 1.0, 10 or 20 nmol SP and SOM, 5 min (in SP-injected groups) or 20 min (in SOM-injected groups) before neurectomy. * P < 0.05; ** P < 0.01; significant difference from the SAL group, • *P < 0.01; significant difference from the SP-0.1 group, * P < 0.05; significant difference from the SP-1.0 group (S-test). C: mean areas ( 5: SEM) under autotomy score curve in A, B are plotted against drug dose. neurectomy. The average scores of the SP-20 (6.9 + 1.7) and SP-10 (7.8 + 1.1) groups at the 6th week were both significantly higher than that of the SAL group (1.2 + 0.3) ( P < 0.05 and P < 0.01; S test, respectively) and the score of the SP-10 group was significantly higher than those of the SP-0.1 (2.2 + 0.8) and SP-1.0 (3.4 + 0.8) groups ( P < 0.01 and P < 0.05; S test, respectively). The average autotomy scores of the SOM-20 (5.2 + 1.0), SOM-10 ( 5 . 4 + 1.1) and SOM-1.0 ( 7 . 9 + 1.1) groups were significantly higher than that of the SAL group (1.2 + 0.3) ( P < 0.01, P < 0.01 and P < 0.05; S test, respectively). Although the scores of the SOM-10 and SOM-20 groups were lower than that of the SOM1.0 group, the difference was not significant ( P > 0.2).
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In Fig. 1C, the areas under each of the autotomy score curves in Fig. 1A,B are plotted against the dose of SP and SOM. The severity of autotomy as revealed by the areas was enhanced in a dose-dependent manner in groups with i.t. injection of SP 5 min before neurectomy (0.1-20 nmol). In the groups with i.t. injection of SOM 20 min before neurectomy, the severity of autotomy was enhanced in a dose-dependent manner when 0.1 and 1.0 nmol SOM were injected, but it decreased when the dose of SOM was increased to 10 and 20 nmol. The onset of autotomy was presented for each of the SAL-, SP- and SOM-injected groups (Fig. 2). The average delay to autotomy onset was significantly shorter in the SP-1.0 (14.2 + 4.7 days, P < 0.05, S test), SP-10 (9.0 + 2.0 days, P < 0.01, S test) and SP-20 (9.7 + 3.7 days, P < 0.01, S test) groups than in the SAL group (26.7 + 3.9 days). The average onset day of autotomy in the SOM-1.0 (6.1 + 1.3 days), SOM-10 (4.7 + 1.1 days) and SOM-20 (8.5 + 3.9 days) groups was significantly shorter than in the SAL group (26.7 + 3.9 days) ( P < 0.01, in all cases).
Experiment 2
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Fig. 2. Average (+ SEM) onset delays of autotomy (score > 1) of each group in Fig. 1A, B. *P < 0.05; **P <0.01; significant difference from the SAL group (S-test).
Fig. 3 shows the effect on autotomy scores of extending the delay between neurectomy and SP or SOM injection. The average autotomy score in the 6th week of the SP-10-20MIN group (1.7 + 0.4), which received neurectomy 20 rain after SP injection, was significantly lower than that of the SP-10-5MIN group (7.8 + 1.1) which received neurectomy 5 rain after the injection
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Fig. 3. Average autotomy scores (+ SEM) of mice which received neurectomy at different times after i.t. injection of 10 nmol SP, 1.0 nmol SOM or 10 nmol SOM. The curves for the SP-10-5MIN, SOM-I.0-20MIN and SOM-10-20MIN group are, respectively, the same as the SP-10, SOM-1.0 and SOM-10 group shown in Fig. 1A, B. A: ** P < 0.01; significant difference from the SP-10-5MIN group. B: ** P < 0.01; significant difference from the SOM-1.0-20MIN group. C: no significant difference (P > 0.1) (U test).
( P < 0.01, U t e s t ) (Fig. 3A). L i k e w i s e , u s i n g 1.0 n m o l o f S O M , t h e a v e r a g e a u t o t o m y s c o r e at t h e 6th w e e k o f t h e S O M - 1 . 0 - 4 0 M I N g r o u p ( 1 . 0 + 0 . 3 ) , was signifi-
,o
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cantly lower than that of the SOM-1.0-20MIN group (7.9 -t- 1.1) ( P < 0.01, U t e s t ) (Fig. 3B). U s i n g 10 n m o l S O M , t h e a v e r a g e a u t o t o m y s c o r e at t h e 6th w e e k o f
10
---
/
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,,
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,
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After injection (min)
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Fig. 4. Mean duration of responses (licking, biting and shaking the hindpaw) per 5 min after i.t. injection of 0.1, 1.0 and 10 nmol SP or SOM (n = 6, respectively).
165 the SOM-10-40MIN group (3.1 + 0.4) was lower than that of the SOM-10-20MIN group (5.4 + 1.1). However the difference in the scores was not statistically significant (P > 0.1, U test) (F'ig. 3C). There was no statistical significant difference in the pair-wise comparisons of autotomy onset days in the SP-10-5MIN (9.0 _+2.0 clays) and SP-10-20MIN (9.5 _+ 3.6 days) group, in the SOM-1.0-20MIN (6.1 _+ 1.3 days) and SOM-1.0-40MIN (2L7.3_+6.3 days) group, and in the SOM-10-20MIN (4.7+ 1.1 days) and SOM-1040MIN (6.6 _+ 1.6 days) group.
Experiment 3 Fig. 4 shows the average duration of responses (licking, biting or shaking the hindpaw) for each 5 min of observation after i.t. injection of SP or SOM (n = 6). Although licking and biting responses directed to the abdomen, thigh and other regions were also frequently observed, we only coded responses directed to the hindpaw. Intrathecal injection of saline had no apparent effect on the behavior of mice. In the mice injected with 10 nmol SP, these responses were observed fi:equently between 0 and 5 rain after injection. In subsequent periods, these responses became less frequent until about 30 min after SP injection when they ceased altogether. When the dose of SP was decreased, responses for 0-5 min decreased in a dose-dependent manner. ANOVA revealed significant effects of SP dose (0.1, 1.0 and 10 nmol SP injection) (F2,15 = 4.31, P < 0.05), t i m e (F7,10 5 = 16,23, P < 0.001), and the SP dose by time interaction (F14,105 = 4.14, P < 0.01). Mice injected with 10 nmol SOM showed a behavioral syndrome similar to that of SP. However, the period showing these responses was longer than that after SP injections. Responses were intermittently observed for about 40 rain after SOM injection and subsequently almost disappeared. In the case of 1.0 nmol injection of SOM, responses were similarly observed intermittently for about 20 min after SOM injection. ANOVA revealed significant effect of time (F7,105 = 2.62, P < 0.05) but did not reveal significant effects of SOM dose (0.1, 1.0 and 10 nmol SOM injection) (P > 0.1) and the SOM dose by time interaction (P > 0.2).
Discussion
Single i.t. injections of SP and SOM just before neurectomy dramatically enhanced autotomy. This suggests that the enhancement of autotomy by noxious stimulation before neurectomy is at least partly associated with CNS mechanisms triggered by the release of SP and SOM in the spinal cord dorsal horn.
Nociceptive behaviors including biting, scratching and licking of the hindquarters, are dose-dependently elicited after i.t. injections of SP or SOM in mice (Hylden and Wilcox 1981, 1983; Gamse and Saria 1986; Ohkubo et al. 1990) and rats (Seybold et al. 1982; Matsumura et al. 1985). The i.t. application of SP (Woolf and Wiesenfeld-Hallin 1986) and SOM (Wiesenfeld-Hallin 1985) produces prolonged enhancement of the excitability of the flexion reflex and the responses of nociceptive neurons (Hylden and Wilcox 1981; Seybold et al. 1982; Ohkubo et al. 1990). These facts suggest that the nociceptive behaviors induced by i.t. injection of SP and SOM reflect the activity level of spinal neurons. We measured the duration of these responses to the hindraw after i.t. SP and SOM injections at the same dose. Our results suggest that these durations reflect the period of activated hyperexcitable neurons in the spinal cord that mediete input from the hindpaw to higher levels in the CNS. This hyperexcitability is able to interact with neurectomy-evoked activities to influence subsequent autotomy. The result of Exp. 3 indicates that increased activity of neurons in the spinal dorsal horn would occur for at least 5 min after i.t. SP injection (10 nmol), and for at least 20 min after i.t. SOM injection (1 or 10 nmol). Thus, in the SP-10-5MIN, SOM-1.0-20MIN and SOM10-20MIN groups, in which neurectomy was done during the evoked activation of spinal neurons, the development of autotomy was dramatically enhanced. Conversely, the activation of neurons in the dorsal horn would already have decreased, or disappeared, about 20 min after i.t. injection of SP (10 nmol) or about 40 min after i.t. injection of SOM (1.0 nmol). When neurectomy was performed at these intervals (SP-1020MIN and SOM-1.0-40MIN groups), there was relatively little effect on autotomy. These facts support the hypothesis that the level of activity of neurons in the dorsal horn just before or during neurectomy plays an important role in the expression of autotomy. Evidence for the role of central activation before deafferentation is presented in several recent reports. Repetitive activity in afferent C fibers before neurectomy, especially at a 'wind-up' frequency, augmented the response of some nociceptive dorsal horn neurons and substantially enhanced the severity of autotomy (Seltzer et al. 1991). Nociceptive responses observed 30-60 min after injection of formalin (phase II responses) are not eliminated by complete anesthetic blockade of the injected area at the time of formalin injection. However, they are significantly reduced by spinal anesthesia induced immediately prior to formalin injection. Spinal anesthesia administered 5 min after formalin injection is also ineffective (Coderre et al. 1990). Local anesthetic applied to the peripheral nerves prior to nerve section significantly reduced autotomy in rats (Gonzalez-Darder et al. 1986; Seltzer et
166 al. 1991). Clinical r e p o r t s show t h a t if p a i n is experie n c e d in t h e limb at o r n e a r the t i m e o f a m p u t a t i o n , t h e n t h e r e is a high p r o b a b i l i t y t h a t p h a n t o m limb p a i n will occur ( J e n s e n et al. 1985; K a t z a n d M e l z a c k 1990). I n o u r e x p e r i m e n t s t h e effect of i.t. p r e - i n j e c t i o n o f SP on a u t o t o m y was d o s e - d e p e n d e n t , b u t t h e e n h a n c e m e n t o f a u t o t o m y by i.t. injection o f S O M s h o w e d a slight r e d u c t i o n at h i g h e r d o s e (Fig. 1, right panel). This m a y b e d u e to a n e u r o t o x i c o r a n a l g e s i c effect of h i g h - d o s e S O M ( G o n z a l e z - D a r d e r et al. 1986; G a u m a n n et al. 1988, 1989). In t h e following, w e p r o p o s e a p o s s i b l e m e c h a n i s m for the p h e n o m e n a t h a t we have observed. Noxious s t i m u l a t i o n o r injury c a u s e s t h e r e l e a s e of SP, S O M a n d excitatory a m i n o acids ( E A A ) within t h e d o r s a l horn, t h e r e l e a s e o f SP m a y itself cause t h e r e l e a s e of E A A (Smullin et al. 1990). T o g e t h e r , t h e s e n e u r o n a l t r a n s m i t t e r s p r o d u c e activation of spinal d o r s a l h o r n n e u r o n s via N - m e t h y l - t > a s p a r t i c acid ( N M D A ) a n d p e p t i d e r e c e p t o r activation, which triggers f u n c t i o n a l c h a n g e s in m e m b r a n e excitability t h r o u g h i n t e r a c t i o n s with s e c o n d m e s s e n g e r systems such as p r o t e i n kinases (Wilcox 1991). T h e i.t. a p p l i c a t i o n o f the n e u r o p e p t i d e s SP a n d S O M p r o d u c e s t r a n s i e n t e n h a n c e m e n t o f spinal excitability b u t it n e v e r triggers t h e a u t o t o m y b e h a v i o r w i t h o u t d e a f f e r e n t a t i o n ( S e y b o l d et al. 1982). M o r e over, t h e r e is only little a u t o t o m y w h e n d e a f f e r e n t a t i o n is p e r f o r m e d s u b s e q u e n t l y ( g r o u p s S P - 1 0 - 2 0 M I N a n d S O M - 1 . 0 - 4 0 M I N ) . T h e c e n t r a l c h a n g e s t h a t yield enh a n c e d a u t o t o m y r e q u i r e s i m u l t a n e o u s actions of i.t. SP o r S O M excitation a n d w h a t e v e r c h a n g e is t r i g g e r e d at t h e t i m e of t h e n e u r e c t o m y itself, p e r h a p s ' i n j u r y d i s c h a r g e ' ( W a l l et al. 1985; S e l t z e r et al. 1991; W o o l f 1991). B o t h o f t h e s e effects a r e n e e d e d to leave t h e c e n t r a l c h a n g e s as p a i n ' m e m o r y ' . T h e s e f u n c t i o n a l c e n t r a l c h a n g e s m a y b e m a i n t a i n e d for days o r l o n g e r as ' m e m o r y ' in spinal c o r d d o r s a l h o r n n e u r o n r e l a t e d to t h e s u b s e q u e n t l y d e n e r v a t e d area. T h e s e c h a n g e s m a y l e a d to i n c r e a s e d s p o n t a n e o u s activity in t h e spinal c o r d ( L o m b a r d et al. 1983; A p k a r i a n et al. 1984; A s a d a et al. 1990). In a d d i t i o n , following n e u r e c t o m y , s p o n t a n e o u s activity g e n e r a t e d f r o m n e u r o m a a n d d o r s a l r o o t g a n g l i o n m a y evoke d y s e s t h e t i c o r p a i n f u l s e n s a t i o n s r e f e r r e d to t h e d e n e r v a t e d region. T h e c o i n c i d e n c e o f t h e s e activities i n c r e a s e s the severity o f a u t o t o m y .
Acknowledgements W e wish to t h a n k Dr. G a r y J. B e n n e t t , Dr. M a r s h a l l Devor, Dr. Y o s h i o S h i g e n a g a a n d Dr. Kenji K a w a k i t a for t h e i r constructive c o m m e n t s d u r i n g t h e writing o f t h e m a n u s c r i p t a n d for E n g l i s h revision.
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