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Subarachnoid injection of salmon calcitonin does not induce analgesia in rats
European Journal of Pharmacology, 104 (1984) 375-377
375
Elsevier Short communication S U B A R A C H N O I D I N J E C T I O N O F S A L M O N C A L C I T O N I N D O E S N O T I N D U C E A N A L G E S I A IN RATS z. WIESENFELD-HALLIN * and A. PERSSON Department of Clinical Neurophysiologv, Huddinge UniversiO' Hospital, S-141 86 ltuddinge, Sweden
Received 3 July 1984, accepted 16 July 1984
Z. W I E S E N F E L D - H A L L I N and A. PERSSON, Subarachnoid injection of salmon calcitonin does not induce analgesia in rats, European J. Pharmacol. 104 (1984) 375-377. It has been proposed that the polypeptide calcitonin (CT) occurs in the central nervous system and may have a neuromodulatory role in endogenous pain relief pathways. However, recent results suggest that CT is not present in the central nervous system. Intrathecal (IT) injection of CT on the lumbar enlargement of rats caused a reversible increase of the hindpaw lick latency in the hot plate test. No analgesia was observed with the vocalization test to electrical stimulation of the tail. In contrast 10 #g morphine hydrochloride IT caused analgesia in both tests. It is concluded that IT CT does not cause analgesia, but has a reversible blocking effect on motor responses. Calcitonin
Intrathecal
Rats
Analgesia
1. Introduction C a l c i t o n i n (CT) is a p o l y p e p t i d e p r o d u c e d in the t h y r o i d gland of m a m m a l s which causes h y p o c a l c e m i a b y i n h i b i t i o n of the release of calcium f r o m bone. C T is also p r e s e n t in a large n u m b e r of i n f r a m a m m a l i a n species ( M u n s o n , 1976). Clinically, C T is a d m i n i s t e r e d to treat Paget's disease, where b o n e t u r n o v e r is enhanced. T h e p a i n associa t e d with this disease is relieved d u r i n g C T a d m i n istration, b u t the late onset of the analgesia d u r i n g t r e a t m e n t p r o b a b l y simply reflects the i m p r o v e d clinical status ( K a n i s et al., 1974). A p o s s i b l e direct analgesic role of C T in the central nervous system has been i n v e s t i g a t e d in a n i m a l s (Pecile et al., 1975; Bates et al., 1980) a n d m a n ( F a i o l i et al., 1982). The results of these studies suggest that C T m a y act as a central n e u r o m o d u l a t o r of neural activity e v o k e d b y nociceptive stimulation. R e p o r t s of the existence of C T in the p i t u i t a r y g l a n d a n d / o r b r a i n are b a s e d on i m m u n o h i s t o c h e m i c a l or r a d i o i m m u n o a s s a y evidence (see
* To whom all correspondence should be addressed. 0014-2999/84/$03.00 ~c~'~1984 Elsevier Science Publishers B.V.
Olgiati et al., 1983 and R o s e n f e l d et al., 1983 for references). However, no C T in b r a i n was f o u n d in a recent s t u d y with r e c o m b i n a n t D N A t e c h n o l o g y ( R o s e n f e l d et al., 1983). These a u t h o r s p r o p o s e d that ' t h e i m m u n o l o g i c a l d a t a in rats p r o b a b l y reflect either the d e t e c t i o n of a substance cross-reacting with calcitonin antisera, or the b i n d i n g of calcitonin from b l o o d that has e n t e r e d circ u m v e n t r i c u l a r regions of the b r a i n that lack a b l o o d - b r a i n b a r r i e r to p e p t i d e s ' ( R o s e n f e l d et al., 1983). In view of the c o n t r o v e r s y c o n c e r n i n g the presence a n d possible analgesic effect of C T in the central nervous system, the a n t i n o c i c e p t i v e effect of salmon C T (sCT) injected into the suba r a c h n o i d space of rats was examined.
2. Materials and methods M a l e S p r a g u e - D a w l e y rats were i m p l a n t e d with an i n t r a t h e c a l (IT) catheter t e r m i n a t i n g on the l u m b a r e n l a r g e m e n t ( Y a k s h a n d R u d y , 1976). N o c i c e p t i o n was assessed with the hot plate a n d vocalization ( D i c k e n s o n et al., 1979) tests. T h e l a t e n c y to licking the p a w of a h i n d l i m b was
376
m e a s u r e d on the h o t p l a t e (54.5 ° C , 19 cm d i a m e ter), which was covered with a clear plastic cylind e r (22 c m high, 21 cm d i a m e t e r ) d u r i n g testing. T h e a n i m a l s were r e m o v e d from the hot p l a t e if they did not r e s p o n d within 40 s. The threshold of v o c a l i z a t i o n following electric shocks to the tail was also measured. The rats were u n r e s t r a i n e d a n d h o u s e d in i n d i v i d u a l p e r s p e x cages (20 cm × 30 cm). Two needle electrodes c o n n e c t e d to flexible leads were inserted into the tail a n d were held in place by elastoplast. Electrical s t i m u l a t i o n consisting of single 500 ms trains of 50 Hz, 1 ms pulses was delivered through a c o n s t a n t c u r r e n t s t i m u l a t o r (Grass). T h e c u r r e n t was increased from 0 in 0.25 m A steps until threshold for vocalization was reached. The threshold was tested every 5 min until a stable baseline of 4 or 5 consecutive values n o t v a r y i n g b y m o r e than 0.25 m A was o b t a i n e d . T h e n o x i o u s stimulus was t e r m i n a t e d when vocaliz a t i o n threshold was reached. Synthetic s C T (Sigma) dissolved in 10/~1 buffer (2 mg acetic acid, 2 m g N a acetate a n d 7.5 m g NaC1 dissolved in 1 ml sterile water) or the buffer a l o n e was injected IT, followed b y a 10 t~l saline flush. M o r p h i n e h y d r o c h l o r i d e (10 jag, 1 # g / m l ) was injected IT, followed b y a 10/~1 saline flush in o r d e r to establish that differences in results between the two tests of n o c i c e p t i o n are not d u e to, for example, the s t i m u l a t i o n of t h e r m o r e c e p t o r s in the hot p l a t e test a n d electrical s t i m u l a t i o n in the v o c a l i z a t i o n test a n d also to exclude the possibility that the two tests are not equally sensitive for analgesia. C o n t r o l rats were injected with equal volumes of saline. T h e d a t a were statistically e v a l u a t e d with the M a n n - W h i t n e y U-test.
3. Results T h e results for s C T on the hot p l a t e test are p r e s e n t e d in fig. 1A. A d o s e - d e p e n d e n t , reversible increase in l a t e n c y to licking a h i n d p a w was observed. However, it was also n o t e d that the a n i m a l s were agitated a n d vocalized d u r i n g e x p o s u r e to the hot plate post-injection. F u r t h e r m o r e , they res p o n d e d vigorously to p i n c h i n g the toes of the h i n d l i m b . N o obvious a t a x i a was observed, b u t the
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5
6
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5
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B
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Fig. 1. (A) T h e effect of 0.2 /*g ( , ) , I /*g (O) s C T or vehicle ( O ) o n the l a t e n c y to licking a h i n d p a w o n the hot p l a t e test. R e s p o n s e latencies at p r e - i n j e c t i o n a n d 24 h p o s t - i n j e c t i o n are also p r e s e n t e d . T h e vertical lines are s t a n d a r d e r r o r s of the m e a n (S.E.M.). n = 8 in e a c h g r o u p . S i g n i f i c a n t d i f f e r e n c e s b e t w e e n the s C A a n d saline g r o u p s are i n d i c a t e d with -/~ (P < 0.05) a n d -A,~ (P < 0.01). (B) T h e effect of 1/*g s C T (0) o r vehicle ( O ) o n the c u r r e n t for v o c a l i z a t i o n t h r e s h o l d to electri cal s h o c k s a p p l i e d to the tail. P r e - i n j e c t i o n values are p r e s e n t e d a l o n g with the S.E.M. n = 7 in e a c h g r o u p .
rats injected with sCT d i s p l a y e d a slow righting r e s p o n s e when p l a c e d on their backs. The vocaliz a t i o n test does n o t require the a n i m a l to execute a m o t o r response involving the h i n d limbs or tail. In fig. 1B is illustrated the lack of analgesic effect of 1 t~g sCT or vehicle IT on the vocalization threshold. T h e effect of 10 /~g IT m o r p h i n e on the hot plate test is shown in fig. 2A. Significantly elevated response latency was o b s e r v e d at 1 a n d 2 h postinjection. T h e a n i m a l s were placid while being tested a n d the righting response was n o r m a l after being p l a c e d on their backs. The results for the vocalization test are shown in fig. 2B. T h e time course of
377
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I
I
1
2
3
2B
.
sponse to a dose of IT morphine that has been shown to have a submaximal analgesic effect (Yaksh and Rudy, 1977). The motor system was not affected by this does of morphine, which is in agreement with previous observations (Yaksh and Rudy, 1977). In conclusion: the present results indicate that CT does not seem to have a neuromodulatory effect on the endogenous pain relief pathways at spinal level.
Acknowledgement This work was supported by a grant from the Folksam Insurance Company and research funds of the Karolinska Institute.
1
I
I
1
2
3
HOURS
Fig. 2. The effect of 10 /zg morphine (e) or saline ((3) on the latency to licking a hind paw on the hot plate test (A) and vocalization threshold to electrical shocks applied to the tail (B). Pre-injection values are presented along with the S.E.M. n = 5 in each group. Significance values are indicated the same as in fig. 1A.
the analgesia was similar to that in the hot plate test.
4. D i s c u s s i o n I T s C T Hoes n o t c a u s e a n a l g e s i a in r a t s b u t h a s a reversible blocking effect on the motor system, as d e m o n s t r a t e d b y a s l o w e d r i g h t i n g r e s p o n s e . I n previous animal studies with intraventricularly administered sCT the possibility was not excluded that the behavioral tests of analgesia may have required the performance of motor responses that w e r e b l o c k e d b y s C T ( P e c i l e e t al., 1975; B a t e s et al., 1980). T h e a n a l g e s i a o b s e r v e d a f t e r i n j e c t i o n o f s C T into the s u b a r a c h n o i d space in t e r m i n a l c a n c e r p a t i e n t s is p r o b a b l y d u e t o n o n s p e c i f i c e f f e c t s . T h i s is t h e m o s t likely e x p l a n a t i o n f o r t h e r e l i e f o f p a i n in the trigeminal region within 5 m i n after i n j e c t i n g s C T i n t o t h e l u m b a r a r e a ( c a s e n o . 4 in F r a i o l i et al., 1982). The adequacy of our two testing methods for d e t e c t i n g a n a l g e s i a w a s d e m o n s t r a t e d b y t h e re-
References
Bates, R.F.L., G.A. Buckley, R.M. Eglen and R.J.Strettle, 1980, Comparison of the analgesic effects of subcutaneous and intracerebroventricular injection of calcitonin on acetic acid induced abdominal constrictions in the mouse, Br. J. Pharmacol. 74, 279P. Dickenson, A.H., J.-L. Oliveras and J.-M. Besson, 1979, Role of the nucleus raphe magnus in opiate analgesia as studied by the microinjection technique in the rat, Brain Res. 170, 96. Fraioli, F., A. Fabbri, L. Gnessi, C. Moretti, C. Santoro and M. Felici, 1982, Subarachnoid injection of salmon calcitonin induces analgesia in man, European J. Pharmacol. 78, 381. Kanis, J.A., D.B. Horn, R.D.M. Scott and G.A. Strong, 1974, Treatment of Paget's disease of bone with synthetic salmon calcitonin, Br. Med. J. 3, 727. Munson, P.L., 1976, Physiology and pharmacology of thyrocalcitonin, in: Handbook of Physiology, Vol. 7, ed. G.D. Aurbach (Amer. Physiol. Soc., Washington) p. 443. Olgiati, V.R., F. Guidobono, C. Netti and A. Pecile, 1983, Localization of calcitonin binding sites in rat central nervous system: evidence of its neuroactivity, Brain Res. 265, 209. Pecile, A., S. Ferri, P.C. Braga and V.R. Olgiati, 1975, Effects of intracerebroventricular calcitonin in the conscious rabbit, Experientia 31, 332. Rosenfeld, M.G., J.-J. Mermod, S.G. Amara, L.W. Swanson, P.E. Sawchenko, J. Rivier, W.W. Vale and R.M. Evans, 1983, Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing, Nature 304, 129. Yaksh, T.L. and T.A. Rudy, 1976, Chronic catheterization ot the spinal subarachnoid space, Physiol. Behav. 17, 1031. Yaksh, T.L. and T.A. Rudy, 1977, Studies on the direct spihal action of narcotics in the production of analgesia in the rat, J. Pharmacol. Exp. Ther. 202, 411.
375
Elsevier Short communication S U B A R A C H N O I D I N J E C T I O N O F S A L M O N C A L C I T O N I N D O E S N O T I N D U C E A N A L G E S I A IN RATS z. WIESENFELD-HALLIN * and A. PERSSON Department of Clinical Neurophysiologv, Huddinge UniversiO' Hospital, S-141 86 ltuddinge, Sweden
Received 3 July 1984, accepted 16 July 1984
Z. W I E S E N F E L D - H A L L I N and A. PERSSON, Subarachnoid injection of salmon calcitonin does not induce analgesia in rats, European J. Pharmacol. 104 (1984) 375-377. It has been proposed that the polypeptide calcitonin (CT) occurs in the central nervous system and may have a neuromodulatory role in endogenous pain relief pathways. However, recent results suggest that CT is not present in the central nervous system. Intrathecal (IT) injection of CT on the lumbar enlargement of rats caused a reversible increase of the hindpaw lick latency in the hot plate test. No analgesia was observed with the vocalization test to electrical stimulation of the tail. In contrast 10 #g morphine hydrochloride IT caused analgesia in both tests. It is concluded that IT CT does not cause analgesia, but has a reversible blocking effect on motor responses. Calcitonin
Intrathecal
Rats
Analgesia
1. Introduction C a l c i t o n i n (CT) is a p o l y p e p t i d e p r o d u c e d in the t h y r o i d gland of m a m m a l s which causes h y p o c a l c e m i a b y i n h i b i t i o n of the release of calcium f r o m bone. C T is also p r e s e n t in a large n u m b e r of i n f r a m a m m a l i a n species ( M u n s o n , 1976). Clinically, C T is a d m i n i s t e r e d to treat Paget's disease, where b o n e t u r n o v e r is enhanced. T h e p a i n associa t e d with this disease is relieved d u r i n g C T a d m i n istration, b u t the late onset of the analgesia d u r i n g t r e a t m e n t p r o b a b l y simply reflects the i m p r o v e d clinical status ( K a n i s et al., 1974). A p o s s i b l e direct analgesic role of C T in the central nervous system has been i n v e s t i g a t e d in a n i m a l s (Pecile et al., 1975; Bates et al., 1980) a n d m a n ( F a i o l i et al., 1982). The results of these studies suggest that C T m a y act as a central n e u r o m o d u l a t o r of neural activity e v o k e d b y nociceptive stimulation. R e p o r t s of the existence of C T in the p i t u i t a r y g l a n d a n d / o r b r a i n are b a s e d on i m m u n o h i s t o c h e m i c a l or r a d i o i m m u n o a s s a y evidence (see
* To whom all correspondence should be addressed. 0014-2999/84/$03.00 ~c~'~1984 Elsevier Science Publishers B.V.
Olgiati et al., 1983 and R o s e n f e l d et al., 1983 for references). However, no C T in b r a i n was f o u n d in a recent s t u d y with r e c o m b i n a n t D N A t e c h n o l o g y ( R o s e n f e l d et al., 1983). These a u t h o r s p r o p o s e d that ' t h e i m m u n o l o g i c a l d a t a in rats p r o b a b l y reflect either the d e t e c t i o n of a substance cross-reacting with calcitonin antisera, or the b i n d i n g of calcitonin from b l o o d that has e n t e r e d circ u m v e n t r i c u l a r regions of the b r a i n that lack a b l o o d - b r a i n b a r r i e r to p e p t i d e s ' ( R o s e n f e l d et al., 1983). In view of the c o n t r o v e r s y c o n c e r n i n g the presence a n d possible analgesic effect of C T in the central nervous system, the a n t i n o c i c e p t i v e effect of salmon C T (sCT) injected into the suba r a c h n o i d space of rats was examined.
2. Materials and methods M a l e S p r a g u e - D a w l e y rats were i m p l a n t e d with an i n t r a t h e c a l (IT) catheter t e r m i n a t i n g on the l u m b a r e n l a r g e m e n t ( Y a k s h a n d R u d y , 1976). N o c i c e p t i o n was assessed with the hot plate a n d vocalization ( D i c k e n s o n et al., 1979) tests. T h e l a t e n c y to licking the p a w of a h i n d l i m b was
376
m e a s u r e d on the h o t p l a t e (54.5 ° C , 19 cm d i a m e ter), which was covered with a clear plastic cylind e r (22 c m high, 21 cm d i a m e t e r ) d u r i n g testing. T h e a n i m a l s were r e m o v e d from the hot p l a t e if they did not r e s p o n d within 40 s. The threshold of v o c a l i z a t i o n following electric shocks to the tail was also measured. The rats were u n r e s t r a i n e d a n d h o u s e d in i n d i v i d u a l p e r s p e x cages (20 cm × 30 cm). Two needle electrodes c o n n e c t e d to flexible leads were inserted into the tail a n d were held in place by elastoplast. Electrical s t i m u l a t i o n consisting of single 500 ms trains of 50 Hz, 1 ms pulses was delivered through a c o n s t a n t c u r r e n t s t i m u l a t o r (Grass). T h e c u r r e n t was increased from 0 in 0.25 m A steps until threshold for vocalization was reached. The threshold was tested every 5 min until a stable baseline of 4 or 5 consecutive values n o t v a r y i n g b y m o r e than 0.25 m A was o b t a i n e d . T h e n o x i o u s stimulus was t e r m i n a t e d when vocaliz a t i o n threshold was reached. Synthetic s C T (Sigma) dissolved in 10/~1 buffer (2 mg acetic acid, 2 m g N a acetate a n d 7.5 m g NaC1 dissolved in 1 ml sterile water) or the buffer a l o n e was injected IT, followed b y a 10 t~l saline flush. M o r p h i n e h y d r o c h l o r i d e (10 jag, 1 # g / m l ) was injected IT, followed b y a 10/~1 saline flush in o r d e r to establish that differences in results between the two tests of n o c i c e p t i o n are not d u e to, for example, the s t i m u l a t i o n of t h e r m o r e c e p t o r s in the hot p l a t e test a n d electrical s t i m u l a t i o n in the v o c a l i z a t i o n test a n d also to exclude the possibility that the two tests are not equally sensitive for analgesia. C o n t r o l rats were injected with equal volumes of saline. T h e d a t a were statistically e v a l u a t e d with the M a n n - W h i t n e y U-test.
3. Results T h e results for s C T on the hot p l a t e test are p r e s e n t e d in fig. 1A. A d o s e - d e p e n d e n t , reversible increase in l a t e n c y to licking a h i n d p a w was observed. However, it was also n o t e d that the a n i m a l s were agitated a n d vocalized d u r i n g e x p o s u r e to the hot plate post-injection. F u r t h e r m o r e , they res p o n d e d vigorously to p i n c h i n g the toes of the h i n d l i m b . N o obvious a t a x i a was observed, b u t the
A 40A
tJ UJ
lit*
>.30 rJ Z nl
"k~
**
~20--I
v' tJ ,.I 1 0 -
I
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1
2
3
4
5
6
I
I
I
I
3 4 HOURS
5
6
gJ'--n 24
B
E
I
I
1
2
Fig. 1. (A) T h e effect of 0.2 /*g ( , ) , I /*g (O) s C T or vehicle ( O ) o n the l a t e n c y to licking a h i n d p a w o n the hot p l a t e test. R e s p o n s e latencies at p r e - i n j e c t i o n a n d 24 h p o s t - i n j e c t i o n are also p r e s e n t e d . T h e vertical lines are s t a n d a r d e r r o r s of the m e a n (S.E.M.). n = 8 in e a c h g r o u p . S i g n i f i c a n t d i f f e r e n c e s b e t w e e n the s C A a n d saline g r o u p s are i n d i c a t e d with -/~ (P < 0.05) a n d -A,~ (P < 0.01). (B) T h e effect of 1/*g s C T (0) o r vehicle ( O ) o n the c u r r e n t for v o c a l i z a t i o n t h r e s h o l d to electri cal s h o c k s a p p l i e d to the tail. P r e - i n j e c t i o n values are p r e s e n t e d a l o n g with the S.E.M. n = 7 in e a c h g r o u p .
rats injected with sCT d i s p l a y e d a slow righting r e s p o n s e when p l a c e d on their backs. The vocaliz a t i o n test does n o t require the a n i m a l to execute a m o t o r response involving the h i n d limbs or tail. In fig. 1B is illustrated the lack of analgesic effect of 1 t~g sCT or vehicle IT on the vocalization threshold. T h e effect of 10 /~g IT m o r p h i n e on the hot plate test is shown in fig. 2A. Significantly elevated response latency was o b s e r v e d at 1 a n d 2 h postinjection. T h e a n i m a l s were placid while being tested a n d the righting response was n o r m a l after being p l a c e d on their backs. The results for the vocalization test are shown in fig. 2B. T h e time course of
377
A ~30 I,LI
lit
)¢~ 2o z w J lO.J I
I
I
1
2
3
2B
.
sponse to a dose of IT morphine that has been shown to have a submaximal analgesic effect (Yaksh and Rudy, 1977). The motor system was not affected by this does of morphine, which is in agreement with previous observations (Yaksh and Rudy, 1977). In conclusion: the present results indicate that CT does not seem to have a neuromodulatory effect on the endogenous pain relief pathways at spinal level.
Acknowledgement This work was supported by a grant from the Folksam Insurance Company and research funds of the Karolinska Institute.
1
I
I
1
2
3
HOURS
Fig. 2. The effect of 10 /zg morphine (e) or saline ((3) on the latency to licking a hind paw on the hot plate test (A) and vocalization threshold to electrical shocks applied to the tail (B). Pre-injection values are presented along with the S.E.M. n = 5 in each group. Significance values are indicated the same as in fig. 1A.
the analgesia was similar to that in the hot plate test.
4. D i s c u s s i o n I T s C T Hoes n o t c a u s e a n a l g e s i a in r a t s b u t h a s a reversible blocking effect on the motor system, as d e m o n s t r a t e d b y a s l o w e d r i g h t i n g r e s p o n s e . I n previous animal studies with intraventricularly administered sCT the possibility was not excluded that the behavioral tests of analgesia may have required the performance of motor responses that w e r e b l o c k e d b y s C T ( P e c i l e e t al., 1975; B a t e s et al., 1980). T h e a n a l g e s i a o b s e r v e d a f t e r i n j e c t i o n o f s C T into the s u b a r a c h n o i d space in t e r m i n a l c a n c e r p a t i e n t s is p r o b a b l y d u e t o n o n s p e c i f i c e f f e c t s . T h i s is t h e m o s t likely e x p l a n a t i o n f o r t h e r e l i e f o f p a i n in the trigeminal region within 5 m i n after i n j e c t i n g s C T i n t o t h e l u m b a r a r e a ( c a s e n o . 4 in F r a i o l i et al., 1982). The adequacy of our two testing methods for d e t e c t i n g a n a l g e s i a w a s d e m o n s t r a t e d b y t h e re-
References
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