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Augmented analgesic effects of enkephalinase inhibitors combined with transcranial electrostimulation
Life Sciences, Vol. 44, pp. 1371-1376 Printed in the U.S.A.
AUGMENTED
ANALGESIC
EFFECTS
OF
WITH TRANSCRANIAL D.H.
Malin,
J.R.
Lake,
R.F.
Pergamon Press
ENKEPHALINASE INHIBITORS ELECTROSTIMULATION Hamilton*
and M.H.
COMBINED
Skolnick*
P r o g r a m s in B e h a v i o r a l and B i o l o g i c a l Sciences U n i v e r s i t y of H o u s t o n - Clear Lake Houston, Texas 77058 and * U n i v e r s i t y of Texas N e u r o p h y s i o l o g y R e s e a r c h Center Houston, Texas 77030 (Received in final form March 3, 1989) Summary The a n a l g e s i c effects of v e r y low current t r a n s c r a n i a l e l e c t r o s t i m u l a t i o n are n a l o x o n e r e v e r s i b l e and thus p r e s u m a b l y m e d i a t e d by e n d o g e n o u s o p i o i d activity. The p r e s e n t e x p e r i m e n t s i n d i c a t e that b l o c k i n g e n k e p h a l i n a s e a c t i v i t y by i.c.v, t h i o r p h a n or i.p. a c e t o r p h a n results in an i n c r e a s e d a n a l g e s i c effect of e l e c t r o s t i m u l a t i o n as m e a s u r e d by the 50°C wet tail flick test. In the case of each drug, rats r e c e i v i n g b o t h d r u g and e l e c t r o s t i m u l a t i o n d i s p l a y e d s i g n i f i c a n t l y more a n a l g e s i a than rats r e c e i v i n g e l e c t r o s t i m u l a t i o n and i n j e c t i o n v e h i c l e alone, rats r e c e i v i n g d r u g and sham s t i m u l a t i o n or rats r e c e i v i n g v e h i c l e and sham stimulation.
Low current t r a n s c r a n i a l e l e c t r o s t i m u l a t i o n (TE) involves the a p p l i c a t i o n of e x t r e m e l y low (I0 ~A), pulsed, chargeb a l a n c e d current across the head t h r o u g h electrodes of o p p o s i t e p o l a r i t y a t t a c h e d to low i m p e d a n c e sites in the external ear. The applied current level of 10 ~ A is w e l l b e l o w the flinchstartle t h r e s h o l d (25-30 DA) of the rat (i). It is also well b e l o w the c u r r e n t levels m o s t c o m m o n l y e m p l o y e d in e l e c t r o a c c u p u n c t u r e or t r a n s c u t a n e o u s e l e c t r i c a l nerve s t i m u l a t i o n (TENS) studies in the rat (i). TE at a f r e q u e n c y of ]0 Hz induced a n a l o x o n e - r e v e r s i b l e a n a l g e s i a in the 50°C w e t tail flick test (1,2) as well as a n a l o x o n e - r e v e r s i b l e r e d u c t i o n in opiate a b s t i n e n c e syndrome (2,3). Since the TE e f f e c t s are p r e s u m a b l y m e d i a t e d by release of e n d o g e n o u s opioids, it was h y p o t h e s i z e d that these effects m i g h t be i n c r e a s e d b y c o n c o m i t a n t a d m i n i s t r a t i o n of drugs that p r o t e c t c e r t a i n e n d o g e n o u s o p i o i d s from rapid e n z y m a t i c destruction.
0024-3205/89 $3.00 + .00 Copyright (c) 1989 Pergamon Press plc
1372
Enkephalinase and Electrostimulation
Vol. 44, No. 19, 1989
E n k e p h a l i n a s e is a m e t a l l o e n d o p e p t i d a s e w h i c h r a p i d l y i n a c t i v a t e s e n k e p h a l i n s b y c l e a v a g e of the Gly-Phe b o n d (4). The e n k e p h a l i n a s e i n h i b i t o r t h i o r p h a n g r e a t l y enhances the a n a l g e s i a i n d u c e d by a d m i n i s t r a t i o n of ( D - A l a 2 - M e t 5 ) - e n k e p h a l i n (5). T h i o r p h a n m a y also a m p l i f y the n a l o x o n e - r e v e r s i b l e a n a l g e s i c e f f e c t s of c e r t a i n types of e n v i r o n m e n t a l stimulation. T h i o r p h a n p o t e n t i a t e d the a n a l g e s i a (hot p l a t e test) induced by i m m o b i l i z a t i o n stress in mice (6) as well as the a n a l g e s i a (tail flick test) i n d u c e d by i n e s c a p a b l e f o o t s h o c k in rats (7). Jin, Zhou and Han (8) found that m i c r o i n j e c t i o n of t h i o r p h a n into the nucleus a c c u m b e n s of the rabbit p o t e n t i a t e d e l e c t r o a c u p u n c t u r e i n d u c e d analgesia. T h i o r p h a n has a low p o t e n c y w h e n s y s t e m i c a l l y a d m i n i s t e r e d and it has g e n e r a l l y been a d m i n i s t e r e d i n t r a v e n t r i c u l a r l y or intrathecally. However, its more l i p o p h i l i c derivative, acetorphan, p o t e n t l y inhibits c e r e b r a l e n k e p h a l i n a s e u p o n p a r e n t e r a l a d m i n i s t r a t i o n in mice and rats (9) and in m a n (i0). P a r e n t e r a l a c e t o r p h a n e l i c i t e d a series of n a l o x o n e - r e v e r s i b l e , o p i o i d - l i k e effects in mice and rats r e s e m b l i n g those earlier found w i t h i n t r a c e r e b r a l t h i o r p h a n (9). The p r e s e n t study e v a l u a t e s the s e p a r a t e and c o m b i n e d a n a l g e s i c effects of TE and i.c.v, t h i o r p h a n as well as the s e p a r a t e and c o m b i n e d effects of TE and i.p. acetorphan.
Experiment
i. C o m b i n e d
Effects
Material
of TE and i.c.v.
Thiorphan
and M e t h o d s
The s u b j e c t s w e r e 20 m a l e S p r a g u e - D a w l e y rats, w e i g h i n g 200225 grams. S u b j e c t s were m a i n t a i n e d on ad lib food and water and a 12 hour l i g h t / d a r k cycle. Seven days prior to the experiment, e a c h rat was p l a c e d under Innovar anesthesia, i m p l a n t e d w i t h g o l d - p l a t e d s t a i n l e s s steel e l e c t r o d e s t h r o u g h the apex of the a n t i - h e l i x of each pinna (1), and s t e r e o t a x i c a l l y c a n n u l a t e d in the third ventricle. Each c a n n u l a p l a c e m e n t was s u b s e q u e n t l y c o n f i r m e d by dye injection and h i s t o l o g i c a l examination. On the two days p r e c e d i n g the experiment, each rat was h a b i t u a t e d to a c y l i n d r i c a l p l a s t i c restrainer. E a c h rat was p l a c e d in the r e s t r a i n e r and p r e t e s t e d for n o c i c e p t i v e s e n s i t i v i t y by m e a s u r e m e n t of tail flick l a t e n c y on the 50°C wet tail flick test (average of three trials). The ear e l e c t r o d e s of e a c h rat were then c o n n e c t e d to the s t i m u l a t o r t h r o u g h leads w i t h a 200K ohm r e s i s t o r in series. Ten rats then r e c e i v e d 30 m i n u t e s of TE s t i m u l a t i o n (10 Hz, 10 uA, 2 msec pulse width), w h i l e ten rats (the "sham s t i m u l a t i o n group") r e c e i v e d no stimulation. B e g i n n i n g c o n c u r r e n t l y w i t h TE or sham stimulation, five rats from each g r o u p then r e c e i v e d 250 ~g t h i o r p h a n i.c.v. (Peninsula L a b o r a t o r i e s ) in saline w i t h 3% ethanol. A motor d r i v e n s y r i n g e d e l i v e r e d a g r a d u a l i n f u s i o n of 5 ~ i / m i n of 2.5 m g / m l t h i o r p h a n s o l u t i o n over a 20 m i n u t e period. Five rats from e a c h group w e r e infused only w i t h the i n j e c t i o n v e h i c l e of
Vol. 44, No. 19, 1 9 8 9
Enkephallnase and Electrostlmulatlon
1373
3% ethanol in saline. Immediately following drug and electrostimulation treatment, all rats were retested under "blind" conditions for tail flick latency. Each animal's analgesia score was the percentage increase in tail flick latency from pretest to posttest. These scores were analyzed by two-way analysis of variance. The group receiving combined thiorphan and TE treatment was compared with all other groups according to Dunnett's Procedure for post-hoc comparison of a single treatment group with all others.
eo
60 5O
_z w
>.
= ~
40
z(~ <. c0 30 +, ,o_ ~
10
--
a,
o -10 ElectrosUmulation injection
Sham Vehicle
TE Vehicle
Sham Thlorphen
TE •.
Figure
Thlorphan p ( . 0 1 vs aii other groups
1
Separate and combined effects of low current transcranial electrostimulation (TE) and 250 ~g thiorphan i.c.v, on percentage increase in 50°C wet tail flick latency (mean ~ SEM)
Results The results are shown in Fig. i. ANOVA indicated a significant electrostimulation effect (TE vs. sham), F(I,16) = 9.29, p < .01, and a significant drug effect (thiorphan vs. vehicle), F(I,16) = 29.54, p < .01. The interaction effect was not significant, F(I,16) = 0.78, NS. The group receiving both TE and thiorphan had a significantly greater percentage increase in tail flick latency than any of the other groups, p < .01, according to Dunnett's Procedure. Experiment
2. Combined Effects Methods
of TE and i.p. Acetorphan
and Materials
The subjects were 84 male Sprague-Dawley rats weighing 180220 grams. Each rat was implanted with ear electrodes under
1374
Enkephalinase and Electrostimulation
Vol. 44, No. 19, 1989
h a l o t h a n e a n e s t h e s i a and s u b s e q u e n t l y h a b i t u a t e d and p r e t e s t e d for tail flick l a t e n c y as in E x p e r i m e n t i. F o r t y - t w o rats then r e c e i v e d i.p. i n j e c t i o n s of 15 m g / k g a c e t o r p h a n (donated by J.C. Schwartz, INSERM, Paris) in a v e h i c l e of ethanol ( 1 0 % ) - c r e m o p h o r EL (10%)-saline (80%). F o r t y - t w o rats r e c e i v e d the i n j e c t i o n v e h i c l e alone. B e g i n n i n g 5 m i n u t e s after injection, half of the a c e t o r p h a n r e c i p i e n t s and half of the v e h i c l e r e c i p i e n t s r e c e i v e d 30 min. of TE as in E x p e r i m e n t i. The r e m a i n i n g half of each group r e c e i v e d 30 min. of sham s t i m u l a t i o n only. All rats w e r e then i m m e d i a t e l y r e t e s t e d under "blind" c o n d i t i o n s for tail flick l a t e n c y and their a n a l g e s i a scores were e x p r e s s e d as p e r c e n t a g e i n c r e a s e from p r e t e s t to p o s t t e s t latencies. These scores were a n a l y z e d by the same p r o c e d u r e s e m p l o y e d in E x p e r i m e n t i.
5o z
40
o
-lO ElectrosUmulation Injection
Sham Vehicle
TE Vehicle
Sham Acetorphan
TE Acetorphan
• o p ( . 0 1 vs all other groups
Figure
2
S e p a r a t e and c o m b i n e d effects of low current t r a n s c r a n i a l electrostimulation (TE) and 15 m g / k g a c e t o r p h a n i.p. on p e r c e n t a g e increase in 50°C wet tail flick latency (mean + SEM).
Results The results are shown in Fig. 2. ANOVA indicated a s i g n i f i c a n t e l e c t r o s t i m u l a t i o n effect (TE vs. sham), F(I,80) = 13.75, p < .01, and a s i g n i f i c a n t d r u g effect (acetorphan vs. vehicle), F(I,80) = 31.93, p < .01. The i n t e r a c t i o n effect was not significant, F(I,80) = 0.06, NS. The group r e c e i v i n g b o t h TE and a c e t o r p h a n had a s i g n i f i c a n t l y greater p e r c e n t a g e i n c r e a s e in tail flick latency than any of the other groups, p < .01, a c c o r d i n g to D u n n e t t ' s Procedure.
Vol. 44, No. 19, 1 9 8 9
Enkephalinase and Electrostimulation
1375
General Discussion The results suggest that the analgesic effects of TE can be markedly intensified by concomitant administration of enkephalinase inhibitors by either an intraventricular or intraperitoneal route. In the past, the analgesic effects of acetorphan have generally been tested by intravenous injection (9), but the present study suggests that such effects can be readily obtained through intraperitoneal administration. Metenkephalin is also metabolized by an aminopeptidase (ii). It may be of interest in the future to determine whether the effects of TE and thiorphan can be potentiated by aminopeptidase inhibitors such as bestatin. Bestatin has been shown to act synergistically with thiorphan in protecting met-enkephalin released from the spinal cord (12). The current findings, along with the naloxone-reversibility of TE effects (1,3), are consistant with the hypothesis that TE actions are mediated, at least in part, by endogenous opioid release. Enkephalinase inhibitors have potential value as analgesic agents, but their actions are limited by the preexisting rate of enkephalin release. Stimulation methods such as TE might insure a sufficient degree of endogenous opioid stimulation so that there would be a meaningful functional consequence of enkephalinase inhibition. TE, with its subsensory i0 UA currents, might be a particularly nonaversive and safe method of achieving such stimulation. The present results raise the possibility that TE may have value in treating chronic pain, especially in combination with drugs that prolong or intensify its effects, such as thiorphan and acetorphan. Aknowledgements Thanks to J.C. Schwartz, INSERM, Paris, for donation of acetorphan. Thanks to Paul Prasco, Doreen Brubaker, David Fowler, and Phillip Jenkins for skilled technical assistance. Supported by a grant from American Health Services Corporation. References I. 2. 3. 4. 5. 6. 7.
M.H. SKOLNICK, O. WILSON, R.F. HAMILTON, C.D. COLLARD, L. HUDSON-HOWARD, C. HYMEL and D.H. MALIN, Journal of Stereotactic and Functional Neurosurgery (in press) J.B. MURRAY, F.C. SCHWEITZER, G.G. LOCKE, L.A. SWEET, S.G. CRON and D.H. MALIN, Neuroscience Abstracts 1 3 1 3 0 4 (1987) D.H. MALIN, J.B. MURRAY, G.P. CRUCIAN, F.C. SCHWEITZER, R.E. COOK and M.H. SKOLNICK, Biological Psychiatry 2__44886-890 (1988) B. MALFROY, J.P. SWERTS, A. GUYON, B.P. ROQUES and J.C. SCHWARTZ, Nature 2 7 6 5 2 3 - 5 2 6 (1978) B.P. ROQUES, M.C. FOURNIE-ZALUSKI, E. SOROCA, J.M. LECOMTE, B. MALFROY, C. LLORENS and J.C. SCHWARTZ, Nature 2 8 8 2 8 6 - 2 8 8 (1980) R. GREENBERG and E.H. O'KEEFE, Life Sciences 3_!li185-i188 (1982) R.E. CHIPKIN, M.B. LATRANYI and L.C. IORIO, Life Sciences 31 1189-1192 (1982)
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Enkephalinase and Electrostimulation
Vol. 44, No. 19, 1989
W.-Q. JIN, Z.-F. ZHOU and J.-S. HAN, Brain Research 380 317-324 (1980) 9. J.-M. LECOMTE, J. COSTENTIN, A. VLAICULESCU, P. CHAILLET, H. MARCARIS-COLLADO, C. LLORENS-CORTES, M. LEBOYER and J.C. SCHWARTZ, Journal of Pharmacology and Experimental Therapeutics 237 937-944 (1986) i0. M.G. SPILLANTINI, P. GEPPETTI, M. FANCIULLACCI, S. MICHELACCI, J.M. LECOMTE and F. SICUTERI, European Journal of Pharmacology 125 147-150 (1986) Ii. J.C. SCHWARTZ, Trends Neurosci. 6 45-48 (1983) 12. S. BURGOIN, D. LE BARS, F. ARTUAD, A.-M. CLOT, R. BOUBOUTOU, M.-C. FOURNIE-ZALUSKI, B.P. ROQUES, M. HAMON and F. CESSELIN, Journal of Pharmacology and Experimental Therapeutics 238 360-366 (1986)
AUGMENTED
ANALGESIC
EFFECTS
OF
WITH TRANSCRANIAL D.H.
Malin,
J.R.
Lake,
R.F.
Pergamon Press
ENKEPHALINASE INHIBITORS ELECTROSTIMULATION Hamilton*
and M.H.
COMBINED
Skolnick*
P r o g r a m s in B e h a v i o r a l and B i o l o g i c a l Sciences U n i v e r s i t y of H o u s t o n - Clear Lake Houston, Texas 77058 and * U n i v e r s i t y of Texas N e u r o p h y s i o l o g y R e s e a r c h Center Houston, Texas 77030 (Received in final form March 3, 1989) Summary The a n a l g e s i c effects of v e r y low current t r a n s c r a n i a l e l e c t r o s t i m u l a t i o n are n a l o x o n e r e v e r s i b l e and thus p r e s u m a b l y m e d i a t e d by e n d o g e n o u s o p i o i d activity. The p r e s e n t e x p e r i m e n t s i n d i c a t e that b l o c k i n g e n k e p h a l i n a s e a c t i v i t y by i.c.v, t h i o r p h a n or i.p. a c e t o r p h a n results in an i n c r e a s e d a n a l g e s i c effect of e l e c t r o s t i m u l a t i o n as m e a s u r e d by the 50°C wet tail flick test. In the case of each drug, rats r e c e i v i n g b o t h d r u g and e l e c t r o s t i m u l a t i o n d i s p l a y e d s i g n i f i c a n t l y more a n a l g e s i a than rats r e c e i v i n g e l e c t r o s t i m u l a t i o n and i n j e c t i o n v e h i c l e alone, rats r e c e i v i n g d r u g and sham s t i m u l a t i o n or rats r e c e i v i n g v e h i c l e and sham stimulation.
Low current t r a n s c r a n i a l e l e c t r o s t i m u l a t i o n (TE) involves the a p p l i c a t i o n of e x t r e m e l y low (I0 ~A), pulsed, chargeb a l a n c e d current across the head t h r o u g h electrodes of o p p o s i t e p o l a r i t y a t t a c h e d to low i m p e d a n c e sites in the external ear. The applied current level of 10 ~ A is w e l l b e l o w the flinchstartle t h r e s h o l d (25-30 DA) of the rat (i). It is also well b e l o w the c u r r e n t levels m o s t c o m m o n l y e m p l o y e d in e l e c t r o a c c u p u n c t u r e or t r a n s c u t a n e o u s e l e c t r i c a l nerve s t i m u l a t i o n (TENS) studies in the rat (i). TE at a f r e q u e n c y of ]0 Hz induced a n a l o x o n e - r e v e r s i b l e a n a l g e s i a in the 50°C w e t tail flick test (1,2) as well as a n a l o x o n e - r e v e r s i b l e r e d u c t i o n in opiate a b s t i n e n c e syndrome (2,3). Since the TE e f f e c t s are p r e s u m a b l y m e d i a t e d by release of e n d o g e n o u s opioids, it was h y p o t h e s i z e d that these effects m i g h t be i n c r e a s e d b y c o n c o m i t a n t a d m i n i s t r a t i o n of drugs that p r o t e c t c e r t a i n e n d o g e n o u s o p i o i d s from rapid e n z y m a t i c destruction.
0024-3205/89 $3.00 + .00 Copyright (c) 1989 Pergamon Press plc
1372
Enkephalinase and Electrostimulation
Vol. 44, No. 19, 1989
E n k e p h a l i n a s e is a m e t a l l o e n d o p e p t i d a s e w h i c h r a p i d l y i n a c t i v a t e s e n k e p h a l i n s b y c l e a v a g e of the Gly-Phe b o n d (4). The e n k e p h a l i n a s e i n h i b i t o r t h i o r p h a n g r e a t l y enhances the a n a l g e s i a i n d u c e d by a d m i n i s t r a t i o n of ( D - A l a 2 - M e t 5 ) - e n k e p h a l i n (5). T h i o r p h a n m a y also a m p l i f y the n a l o x o n e - r e v e r s i b l e a n a l g e s i c e f f e c t s of c e r t a i n types of e n v i r o n m e n t a l stimulation. T h i o r p h a n p o t e n t i a t e d the a n a l g e s i a (hot p l a t e test) induced by i m m o b i l i z a t i o n stress in mice (6) as well as the a n a l g e s i a (tail flick test) i n d u c e d by i n e s c a p a b l e f o o t s h o c k in rats (7). Jin, Zhou and Han (8) found that m i c r o i n j e c t i o n of t h i o r p h a n into the nucleus a c c u m b e n s of the rabbit p o t e n t i a t e d e l e c t r o a c u p u n c t u r e i n d u c e d analgesia. T h i o r p h a n has a low p o t e n c y w h e n s y s t e m i c a l l y a d m i n i s t e r e d and it has g e n e r a l l y been a d m i n i s t e r e d i n t r a v e n t r i c u l a r l y or intrathecally. However, its more l i p o p h i l i c derivative, acetorphan, p o t e n t l y inhibits c e r e b r a l e n k e p h a l i n a s e u p o n p a r e n t e r a l a d m i n i s t r a t i o n in mice and rats (9) and in m a n (i0). P a r e n t e r a l a c e t o r p h a n e l i c i t e d a series of n a l o x o n e - r e v e r s i b l e , o p i o i d - l i k e effects in mice and rats r e s e m b l i n g those earlier found w i t h i n t r a c e r e b r a l t h i o r p h a n (9). The p r e s e n t study e v a l u a t e s the s e p a r a t e and c o m b i n e d a n a l g e s i c effects of TE and i.c.v, t h i o r p h a n as well as the s e p a r a t e and c o m b i n e d effects of TE and i.p. acetorphan.
Experiment
i. C o m b i n e d
Effects
Material
of TE and i.c.v.
Thiorphan
and M e t h o d s
The s u b j e c t s w e r e 20 m a l e S p r a g u e - D a w l e y rats, w e i g h i n g 200225 grams. S u b j e c t s were m a i n t a i n e d on ad lib food and water and a 12 hour l i g h t / d a r k cycle. Seven days prior to the experiment, e a c h rat was p l a c e d under Innovar anesthesia, i m p l a n t e d w i t h g o l d - p l a t e d s t a i n l e s s steel e l e c t r o d e s t h r o u g h the apex of the a n t i - h e l i x of each pinna (1), and s t e r e o t a x i c a l l y c a n n u l a t e d in the third ventricle. Each c a n n u l a p l a c e m e n t was s u b s e q u e n t l y c o n f i r m e d by dye injection and h i s t o l o g i c a l examination. On the two days p r e c e d i n g the experiment, each rat was h a b i t u a t e d to a c y l i n d r i c a l p l a s t i c restrainer. E a c h rat was p l a c e d in the r e s t r a i n e r and p r e t e s t e d for n o c i c e p t i v e s e n s i t i v i t y by m e a s u r e m e n t of tail flick l a t e n c y on the 50°C wet tail flick test (average of three trials). The ear e l e c t r o d e s of e a c h rat were then c o n n e c t e d to the s t i m u l a t o r t h r o u g h leads w i t h a 200K ohm r e s i s t o r in series. Ten rats then r e c e i v e d 30 m i n u t e s of TE s t i m u l a t i o n (10 Hz, 10 uA, 2 msec pulse width), w h i l e ten rats (the "sham s t i m u l a t i o n group") r e c e i v e d no stimulation. B e g i n n i n g c o n c u r r e n t l y w i t h TE or sham stimulation, five rats from each g r o u p then r e c e i v e d 250 ~g t h i o r p h a n i.c.v. (Peninsula L a b o r a t o r i e s ) in saline w i t h 3% ethanol. A motor d r i v e n s y r i n g e d e l i v e r e d a g r a d u a l i n f u s i o n of 5 ~ i / m i n of 2.5 m g / m l t h i o r p h a n s o l u t i o n over a 20 m i n u t e period. Five rats from e a c h group w e r e infused only w i t h the i n j e c t i o n v e h i c l e of
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3% ethanol in saline. Immediately following drug and electrostimulation treatment, all rats were retested under "blind" conditions for tail flick latency. Each animal's analgesia score was the percentage increase in tail flick latency from pretest to posttest. These scores were analyzed by two-way analysis of variance. The group receiving combined thiorphan and TE treatment was compared with all other groups according to Dunnett's Procedure for post-hoc comparison of a single treatment group with all others.
eo
60 5O
_z w
>.
= ~
40
z(~ <. c0 30 +, ,o_ ~
10
--
a,
o -10 ElectrosUmulation injection
Sham Vehicle
TE Vehicle
Sham Thlorphen
TE •.
Figure
Thlorphan p ( . 0 1 vs aii other groups
1
Separate and combined effects of low current transcranial electrostimulation (TE) and 250 ~g thiorphan i.c.v, on percentage increase in 50°C wet tail flick latency (mean ~ SEM)
Results The results are shown in Fig. i. ANOVA indicated a significant electrostimulation effect (TE vs. sham), F(I,16) = 9.29, p < .01, and a significant drug effect (thiorphan vs. vehicle), F(I,16) = 29.54, p < .01. The interaction effect was not significant, F(I,16) = 0.78, NS. The group receiving both TE and thiorphan had a significantly greater percentage increase in tail flick latency than any of the other groups, p < .01, according to Dunnett's Procedure. Experiment
2. Combined Effects Methods
of TE and i.p. Acetorphan
and Materials
The subjects were 84 male Sprague-Dawley rats weighing 180220 grams. Each rat was implanted with ear electrodes under
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h a l o t h a n e a n e s t h e s i a and s u b s e q u e n t l y h a b i t u a t e d and p r e t e s t e d for tail flick l a t e n c y as in E x p e r i m e n t i. F o r t y - t w o rats then r e c e i v e d i.p. i n j e c t i o n s of 15 m g / k g a c e t o r p h a n (donated by J.C. Schwartz, INSERM, Paris) in a v e h i c l e of ethanol ( 1 0 % ) - c r e m o p h o r EL (10%)-saline (80%). F o r t y - t w o rats r e c e i v e d the i n j e c t i o n v e h i c l e alone. B e g i n n i n g 5 m i n u t e s after injection, half of the a c e t o r p h a n r e c i p i e n t s and half of the v e h i c l e r e c i p i e n t s r e c e i v e d 30 min. of TE as in E x p e r i m e n t i. The r e m a i n i n g half of each group r e c e i v e d 30 min. of sham s t i m u l a t i o n only. All rats w e r e then i m m e d i a t e l y r e t e s t e d under "blind" c o n d i t i o n s for tail flick l a t e n c y and their a n a l g e s i a scores were e x p r e s s e d as p e r c e n t a g e i n c r e a s e from p r e t e s t to p o s t t e s t latencies. These scores were a n a l y z e d by the same p r o c e d u r e s e m p l o y e d in E x p e r i m e n t i.
5o z
40
o
-lO ElectrosUmulation Injection
Sham Vehicle
TE Vehicle
Sham Acetorphan
TE Acetorphan
• o p ( . 0 1 vs all other groups
Figure
2
S e p a r a t e and c o m b i n e d effects of low current t r a n s c r a n i a l electrostimulation (TE) and 15 m g / k g a c e t o r p h a n i.p. on p e r c e n t a g e increase in 50°C wet tail flick latency (mean + SEM).
Results The results are shown in Fig. 2. ANOVA indicated a s i g n i f i c a n t e l e c t r o s t i m u l a t i o n effect (TE vs. sham), F(I,80) = 13.75, p < .01, and a s i g n i f i c a n t d r u g effect (acetorphan vs. vehicle), F(I,80) = 31.93, p < .01. The i n t e r a c t i o n effect was not significant, F(I,80) = 0.06, NS. The group r e c e i v i n g b o t h TE and a c e t o r p h a n had a s i g n i f i c a n t l y greater p e r c e n t a g e i n c r e a s e in tail flick latency than any of the other groups, p < .01, a c c o r d i n g to D u n n e t t ' s Procedure.
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General Discussion The results suggest that the analgesic effects of TE can be markedly intensified by concomitant administration of enkephalinase inhibitors by either an intraventricular or intraperitoneal route. In the past, the analgesic effects of acetorphan have generally been tested by intravenous injection (9), but the present study suggests that such effects can be readily obtained through intraperitoneal administration. Metenkephalin is also metabolized by an aminopeptidase (ii). It may be of interest in the future to determine whether the effects of TE and thiorphan can be potentiated by aminopeptidase inhibitors such as bestatin. Bestatin has been shown to act synergistically with thiorphan in protecting met-enkephalin released from the spinal cord (12). The current findings, along with the naloxone-reversibility of TE effects (1,3), are consistant with the hypothesis that TE actions are mediated, at least in part, by endogenous opioid release. Enkephalinase inhibitors have potential value as analgesic agents, but their actions are limited by the preexisting rate of enkephalin release. Stimulation methods such as TE might insure a sufficient degree of endogenous opioid stimulation so that there would be a meaningful functional consequence of enkephalinase inhibition. TE, with its subsensory i0 UA currents, might be a particularly nonaversive and safe method of achieving such stimulation. The present results raise the possibility that TE may have value in treating chronic pain, especially in combination with drugs that prolong or intensify its effects, such as thiorphan and acetorphan. Aknowledgements Thanks to J.C. Schwartz, INSERM, Paris, for donation of acetorphan. Thanks to Paul Prasco, Doreen Brubaker, David Fowler, and Phillip Jenkins for skilled technical assistance. Supported by a grant from American Health Services Corporation. References I. 2. 3. 4. 5. 6. 7.
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W.-Q. JIN, Z.-F. ZHOU and J.-S. HAN, Brain Research 380 317-324 (1980) 9. J.-M. LECOMTE, J. COSTENTIN, A. VLAICULESCU, P. CHAILLET, H. MARCARIS-COLLADO, C. LLORENS-CORTES, M. LEBOYER and J.C. SCHWARTZ, Journal of Pharmacology and Experimental Therapeutics 237 937-944 (1986) i0. M.G. SPILLANTINI, P. GEPPETTI, M. FANCIULLACCI, S. MICHELACCI, J.M. LECOMTE and F. SICUTERI, European Journal of Pharmacology 125 147-150 (1986) Ii. J.C. SCHWARTZ, Trends Neurosci. 6 45-48 (1983) 12. S. BURGOIN, D. LE BARS, F. ARTUAD, A.-M. CLOT, R. BOUBOUTOU, M.-C. FOURNIE-ZALUSKI, B.P. ROQUES, M. HAMON and F. CESSELIN, Journal of Pharmacology and Experimental Therapeutics 238 360-366 (1986)