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Induction of tolerance and withdrawal in rats receiving morphine in the spinal subarachnoid space
European Journal o f Pharmacology, 42 (1977) 275--284
275
© Elsevier/North-Holland Biomedical Press
I N D U C T I O N O F T O L E R A N C E A N D W I T H D R A W A L IN R A T S R E C E I V I N G M O R P H I N E I N T H E SPINAL S U B A R A C H N O I D SPACE TONY L. YAKSH *, RANDALL L. KOHL and THOMAS A. RUDY School o f Pharmacy, University o f Wisconsin, Madison, Wi. 53706, U.S.A.
Received 28 July 1976, revised MS received 17 November 1976, accepted 10 December 1976
T.L. YAKSH, R.L. KOHL and T.A. RUDY, Induction o f tolerance and withdrawal in rats receiving morphine in the spinal subarachnoid space, European J. Pharmacol. 42 (1977) 275--284. Rats implanted with chronic catheters in the spinal subarachnoid space were given twice daily injections for 7 days of morphine sulfate, either intrathecaliy into the lumbar subarachnoid space (15 or 50 pg) or i.p. (20 mg/ kg). The development of tolerance, as manifested in a reduction of the analgetic efficacy of these injections on the hot plate and tail flick, occurred in a dose dependent fashion over a period of 7 days. At this time, injections of i.p. morphine into animals which had received spinal morphine and vice versa revealed the existence of a two way cross tolerance between spinal and systemically administered morphine. Injection of naloxone into the spinal cord of animals exposed to i.p. morphine or conversely, i.p. naloxone in animals tolerant to intrathecal morphine, yielded a hyperreflexia and extreme sensitivity to handling. Other signs commonly observed in precipitated withdrawal, however, such as wet shakes and weight loss, were not observed. Spinal cord
Morphine
Tolerance
Withdrawal
1. I n t r o d u c t i o n We have r e c e n t l y d e m o n s t r a t e d t h a t in t h e i n t a c t and u n a n e s t h e t i z e d a n i m a l , m o r p h i n e , applied via a c a t h e t e r c h r o n i c a l l y inserted into t h e l u m b a r s u b a r a c h n o i d space, can p r o d u c e a d o s e < l e p e n d e n t a n d p h a r m a c o l o g i c a l l y specific (i.e., s t e r e o s p e c i f i c a n d n a l o x o n e reversible) e l e v a t i o n in t h e analgetic t h r e s h o l d as d e f i n e d b y t h e tail flick, h o t p l a t e , f o r c e p s p i n c h and s h o c k t i t r a t i o n tests ( Y a k s h and R u d y , 1 9 7 6 a ) . A l t h o u g h a direct a c t i o n of n a r c o t i c s o n cord f u n c t i o n has b e e n p o s i t e d o n t h e basis o f a v a r i e t y o f e x p e r i m e n t s carried o u t in spinal animals (for e x a m p l e , Irwin et al., 1 9 5 1 ; Koll et al., 1 9 6 3 ; Martin et al., 1 9 6 4 ; Besson et al., 1 9 7 3 ; K i t a h a t a et al., 1 9 7 4 ; LeBars et al., 1 9 7 5 ; J u r n a a n d Grossman, 1976), our experiments with intrathecal * Present address: Department of Anatomy and Embryology, University College London, London WC1E 6BT, England.
Naloxone
a d m i n i s t r a t i o n indicate t h a t t h e a l t e r a t i o n in c o r d f u n c t i o n b y t h e local a c t i o n o f n a r c o t i c s has relevance t o t h e analgetic r e s p o n s e o f t h e i n t a c t , b e h a v i n g , animal. In p r e v i o u s experim e n t s , for e x a m p l e , we have s h o w n t h a t n a l o x o n e given i n t o t h e spinal s u b a r a c h n o i d s p a c e can significantly a n t a g o n i z e t h e analgesia p r o d u c e d b y s y s t e m i c a l l y a d m i n i s t e r e d m o r p h i n e ( Y a k s h and R u d y , 1 9 7 7 a ) . Such o b s e r v a t i o n s suggest t h a t at least a f r a c t i o n o f t h e analgetic e f f e c t o b s e r v e d f o l l o w i n g p e r i p h e r a l a d m i n i s t r a t i o n of a n a r c o t i c m a y result f r o m its local e f f e c t o n a spinal m e c h a nism. In t h e p r e s e n t e x p e r i m e n t s , we s o u g h t t o examine further the characteristics of the m o r p h i n e - s e n s i t i v e analgetic s y s t e m l o c a t e d in t h e spinal c o r d b y observing: (1) t o w h a t degree a n i m a l s m a d e t o l e r a n t t o i n t r a t h e c a l l y a d m i n i s t e r e d m o r p h i n e w o u l d d i s p l a y an analgetic r e s p o n s e to s y s t e m i c a l l y administ e r e d m o r p h i n e and vice versa, i.e. cross toler-
276
T.L. YAKSH ET AL.
ance; and (2) the nature of the precipitated withdrawal response evoked by the administration of naloxone into the spinal subarachnoid space of animals made tolerant to systemic morphine as well as the response observed when systemically administered naloxone was given to animals made tolerant to intrathecally administered morphine.
2. Materials and methods
2.1. General procedures Rats were implanted with chronic catheters constructed from PE-10 polyethylene tubing. The tip of each catheter rested at the rostral face of the lumbar enlargement in the spinal subarachnoid space. Details of the catheter construction and implantation are given elsewhere (Yaksh and Rudy, 1976b). Following a 7--14 day recovery period, rats were assigned to I of 7 groups (see below). Injections into the spinal subarachnoid space (intrathecal) were made in volumes of 10 pl (5 pl of drug followed by 5 pl of control vehicle) delivered over approximately 30--45 sec with a manually turned, micrometer~lriven syringe. Solutions for intrathecal injection were always made up in a balanced ion solution consisting of NaC1 7.46 rag; KC1 0.19 mg; MgC12 0.19 mg and CaC12 0.14 mg per 1000 ml of distilled water. 2 doses of morphine sulfate were
employed 15 pg (3 pg/pl) and 50 pg (10pg/ pl). The respective osmolarity of the two solutions was 262 and 270 mosms. Systemic injections (i.p.) were made with morphine sulfate (20 mg/ml/kg) dissolved in 0.9% saline. When control injections were made, the respective vehicles were employed. To develop tolerance by either systemic or spinal administration, morphine was injected by either route in accordance with the group designation (see below) twice a day at 0600 and at 1800 for 7 days. This period was observed to yield a complete tolerance to both spinally and systemically administered morphine in all doses employed as indicated by the absence of any analgetic activity.
2.2. Experimental design 2.2.1. Tolerance development In general, groups received either systemic morphine or intrathecal morphine for 7 days, twice a day as described above. On the morning of the 8th day, the animals received their usual treatment. 6 h later, animals which had received intrathecal morphine during the tolerance period were given systemic morphine and vice versa. The groups and their respective dose regimens are given in table 1. The doses administered were chosen on the basis of previous work which demonstrated that 15 pg was the minimum dose which produced a near maximum detectable effect on
TABLE 1 Listing of group treatments used in the testing of cross tolerance between systemically and intrathecally administered morphine on the hot plate and tail flick tests. Group
n1
Pretreatment (7 days)
Test (8th day)
A B C D E F G
4 4 6 5 5 5 6
Systemic control Systemic control Systemic morphine (20 mg/kg) Systemic morphine (20 mg/kg) Intrathecal control Intrathecal morphine (15/~g) Intrathecal morphine (50 pg)
Intrathecal morphine (15 pg) Intrathecal morphine (50 pg) Intrathecal morphine (15 pg) Intrathecal morphine (50 pg) Systemic morphine (20 mg/kg) Systemic morphine (20 mg/kg) Systemic morphine (20 mg/kg)
I Number of animals in group.
TOLERANCE AND SPINAL MORPHINE the tail flick test (vide infra). Throughout these experiments, the animals were housed singly in wire mesh cage. 2.2.2. Withdrawal To study withdrawal induced by systemic and intrathecal naloxone, the animals were continued for 4 days after the test for cross tolerance, receiving the same schedule of injections as they had received prior to the cross tolerance testing. For the precipitated withdrawal, on the 12th day, 6 h after the morning injection, i.e. at 12.00 h, animals receiving systemic morphine were given intrathecal injections of naloxone HC1 (10 pg) while those given intrathecal morphine during the tolerance period were given systemic naloxone (2 mg/kg, i.p.). These doses, in a previous study (Yaksh and Rudy, unpublished data) have been shown to completely reverse the analgetic action of the respective doses of morphine. Following injection, each rat was returned to its home cage. In preliminary experiments, animals not first exposed to the cross tolerance testing were observed to yield the same response during precipitated withdrawal as those which had undergone the cross tolerance testing 4 days previously. The withdrawal data for these animals were thus combined. 2.3. Measurements 2.3.1. Analgesia To measure the development of tolerance, the tail flick response was observed 30 min after each injection according to the schedules described above. This interval represents the time at which the level of analgesia was consistently observed to be maximum and stable. The tail flick consisted of laying the rat's tail across a slit under which was a focussed projection bulb. The response measure was the time between the light being switched on and the tail being briskly moved from the slit. Because of the long term nature of the tests, an 8 rather than 10 sec cut off was employed. When cross tolerance was examined, the hot plate as well as the tail flick was examined. In
277 the hot plate test, the rat was placed on a metal surface (17 X 22 cm) heated to 55 + I°C by a waterbath. The response measure was the time between the animal being placed on the surface and until it licked its hindpaw. Hot plate responses were also measured following the first injection of morphine and on the morning of the 8th day of the tolerance series. Repeated hot plate measures were not taken due to the possible interaction resulting from learning during the repeated trials (Kayan et al., 1969). In the precipitated withdrawal experiments, the presence of the following signs was systematically noted during each 5 min interval following the injection of naloxone: wet shakes, escape behavior (jumping), piloerection, seminal emissions, chromodacryorrhea and diarrhea. Weight loss occurring over a three hour period after the injection of naloxone was also examined. Descriptions of these withdrawal signs have been published elsewhere (Wei et al., 1973b). The precipitated withdrawal of rats made tolerant to systemic morphine is often accompanied by a marked increase in motor activity within the cage. When the cage was opened slightly, animals would often make an apparent effort to climb or jump through the small opening. This behavior, for purposes of description, was labeled as escape behavior. The incidence of withdrawal induced aggression was also examined by placing an untreated rat of approximately the same b o d y weight in the cage of the withdrawn rat 45 min after the injection of naloxone. The untreated rat was left in the cage for 5 min and the withdrawn rat scored for the presence or absence of aggressive behavior by noting whether it assumed the characteristic aggressive posture (i.e. erect, in a " b o x i n g " stance with its forepaws slightly extended). Active signs of aggression such as squeaking and biting attempts were c o m m o n l y associated with such behavior. 2.4. Statistics To permit comparison between animals, tail flick and hot plate response times were
278
T.L. Y A K S H E T AL.
converted to m a x i m u m (M.P.E.) where:
percent
effects
M.P.E. = postdrug response latency -- predrug response latency X 100 cut off time -- p r e d r u g r e s p o n s e latency
As noted previously, the cut off time for the hot plate and tail flick was 30 and 8 sec, respectively. Confidence intervals (95%) for group data were calculated as described by Goldstein (1964), while the quantal withdrawal data were compared by means of the X 2 statistic.
3. Results
3.1. Tolerance development In the present experiments, we observed that the twice daily administration of intrathecal morphine yields a clear time and dosedependent reduction in the analgetic effects of morphine. Fig. 1 presents the time course of this tolerance development as measured by the tail flick response with the tolerance
I00-
effect appearing most rapidly after about 3 days, for the lower dose (15 pg), while the higher dose (50 pg) required approximately 5 days. That this tolerating effect of intrathecal morphine was not limited to a spinal reflex is evidenced in table 2 by the fact that on the 8th day the analgetic effect of intrathecal morphine measured on the hot plate was significantly reduced as compared to the response observed after the first injection of the series. Although the fact that the degree of tolerance was clearly dependent upon the intrathecal dose of morphine, we considered the possibility that a portion of the effect might be due simply to some non-specific action resulting from the repeated injection of vehicle into the subarachnoid space. To examine this possibility, three animals were tested on the hot plate and tail flick following the administration of 15 #g of intrathecally administered morphine. These animals then received twice daily injections of vehicle intrathecally. On the morning of the 8th day, these same animals received an injection of intrathecal morphine (15pg) instead of the vehicle. The results of these experiments are shown in table 3. As can be seen there was no detectable effect on the analgetic potency of intrathecal morphine resulting from these control intrathecal injec-
r
~
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]20mg/kg
i,p.
"-...
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n 50
TABLE 2 \
\
i
50p.q S.A.
\
~\
T h e d e v e l o p m e n t of t o l e r a n c e t o t h e a n a l g e t i c a c t i o n o f m o r p h i n e a d m i n i s t e r e d e i t h e r s y s t e m i c a l l y or intrat h e c a l l y as m e a s u r e d b y t h e h o t plate t e s t .
\,
H o t plate r e s p o n s e 1
J5~g S.A.S.' ~
Day I
Day 8
9 4 . 2 ± 3.9 100.0
22.4 + 12.8 2 16.3 • 13.9 2
98.2 ± 6.4
13.8 -+ 11.2 2
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ODAYS
Fig. 1. T h e t i m e c o u r s e o f t h e d e v e l o p m e n t o f tolera n c e t o m o r p h i n e given i n t o t h e spinal s u b a r a c h n o i d s p a c e (S.A.S.; 15/~g: o, 50 p g : e ) or m o r p h i n e a d m i n istered s y s t e m i c a l l y (i.p.; 20 m g / k g : i ) as m a n i f e s t e d b y t h e e f f e c t o f s u c h i n j e c t i o n s o n t h e tail flick r e s p o n s e (M.P.E.). E a c h curve r e p r e s e n t s t h e m e a n a n d S.E.M. o f 5 (S.A.S., 1 5 ) ; 6 (S.A.S. 5 0 ) a n d 11 (i.p.) a n i m a l s , r e s p e c t i v e l y .
Intrathecal morphine 15 p g 50pg Systemic morphine 20 m g / k g
1 T h e h o t plate r e s p o n s e w a s m e a s u r e d o n t h e m o r n ing o f t h e 1st a n d 8 t h d a y i m m e d i a t e l y b e f o r e a n d 30 m i n a f t e r t h e i n t r a t h e c a l or s y s t e m i c i n j e c t i o n o f m o r p h i n e . T h e h o t plate r e s p o n s e is e x p r e s s e d as M.P.E. w i t h S.E.M. 2 p < 0.05. T e s t u s e d a m a t c h e d pairs t-statistic.
279
TOLERANCE AND SPINAL MORPHINE TABLE 3 The effect of control solutions administered intrathecally twice daily for 7 days on the analgetic action of intrathecally administered morphine.
Tail flick Hot plate
Test I 1
Test II
91.2 ~ 6.3 89.9 ~ 9.6
98.2 ÷ 1.6 86.4 -+ 11.4
,,; 50
1 Response on test I expressed as M.P.E. to 15 pg of morphine given intrathecally. Test II occurs on the morning of the 8th day and represents the effects on the tail flick and hot plate (15 pg) expressed as M.P.E. 3 animals were used in these experiments.
t i o n s . T h e f a i l u r e o f c o n t r o l i n t r a t h e c a l inject i o n s t o a l t e r t h e a n a l g e t i c p o t e n c y o f syst e m i c m o r p h i n e is s h o w n b y t h e r e s u l t s o b t a i n e d w i t h g r o u p (E) (see b e l o w ) .
3.2. Cross tolerance As s h o w n in fig. 2, for g r o u p s (A) a n d (B), t h e d a i l y i n j e c t i o n o f saline (i.p.) h a d no e f f e c t o n t h e a n a l g e t i c r e s p o n s e o b s e r v e d foll o w i n g t h e i n t r a t h e c a l i n j e c t i o n o f e i t h e r 15 o r 50 pg o f m o r p h i n e , b o t h d o s e s still r e s u l t ing in t h e m a x i m u m d e t e c t a b l e a n a l g e t i c e f f e c t o n b o t h t h e h o t p l a t e a n d t a i l f l i c k . If, h o w e v e r , t h e a n i m a l s r e c e i v e d a 7 d a y pret r e a t m e n t o f m o r p h i n e (20 m g / k g , i.p.), t h e analgetic response measured on both the hot p l a t e a n d t a i l flick was s i g n i f i c a n t l y r e d u c e d as c o m p a r e d t o t h e a p p r o p r i a t e c o n t r o l g r o u p ( g r o u p (C) w i t h g r o u p (A) a n d g r o u p (D) w i t h g r o u p (B). C o m p a r i s o n o f g r o u p (C) w i t h g r o u p (D) i n d i c a t e s t h a t t h e s y s t e m i c a d m i n i s tration of m o r p h i n e p r o d u c e d a relative degree of tolerance with the higher dose of in t r a t h e c a l m o r p h i n e y i e l d i n g a g r e a t e r elevat i o n in t h e a n a l g e t i c t h r e s h o l d t h a n t h e l o w e r dose. The effects of pretreating the animals with intrathecal m o r p h i n e twice daily for 7 days on the analgetic action of systemic morphine are s h o w n in fig. 3. As s h o w n b y t h e r e s u l t s o b t a i n e d in g r o u p (E), t h e c o n t r o l a d m i n i s t r a t i o n of vehicle i n t o t h e s p i n a l s u b a r a c h n o i d
I ~ '
Control
Control
Mor (15p.g)
Mor(50#gJ
Mor Mot (20 rnq/kg) (20mg/kQ)
Mor (15/=g)
Mor(50~.g)
Fig. 2. Cross tolerance experiments wherein the animals were systemically pretceated for 7 days with either saline (control), or morphine (Mot 20 mg/kg) i.p. (upper abscissa). On the 8th day, the animals were treated as described in the text with intrathecal morphine (S.A.S.)in doses of either 15 pg (Mor 15 pg) or 50 pg (Mor 50 pg) (lower abscissa) and the effects on tail flick (left bar) and hot plate (right bar) examined. Results are presented in terms of the M.P.E. The number of animals in each group is given in table 1. The vertical bar indicates the mean and 95% confidence interval.
I00
J~
T
J/ /,
o~ 5C
H i
Control
Mot (20 mg/k9)
Mor (15/~g) Mor (20 mg/k9)
Mor (50/~ g} Mor (20 mg/kg)
Fig. 3. Cross tolerance experiments wherein the animals were pretreated for 7 days with intrathecal morphine (S.A.S.) in doses of 15 (Mor 15 pg) or 50 (Mot 50 pg) pg (upper abscissa). On the 8th day, the animals received, systemically, morphine in a dose of 20 mg/kg (lower abscissa). All details are the same as indicated in fig. 2.
280
T.L. YA.KSH ET AL.
s p a c e f o r this p e r i o d p r o d u c e d n o d e t e c t a b l e decrement in the analgetic action of systemic m o r p h i n e ( 2 0 m g / k g , i.p.). T h e a d d i t i o n o f morphine twice daily for 7 days to the intrathecal injectate, however, produced a dosedependent reduction in the degree of analgesia p r o d u c e d b y t h i s d o s e o f s y s t e m i c m o r p h i n e o n t h e 8 t h d a y . As s h o w n b y t h e r e s u l t s of group (F) and (G), the twice daily administ r a t i o n of 15 pg of m o r p h i n e i n t r a t h e c a l l y p r o d u c e d a slight, b u t n o n - s i g n i f i c a n t reduct i o n in the analgesia p r o d u c e d b y the systemic dose of m o r p h i n e ; whereas the 50 pg dose p r o d u c e d a significant d e c r e m e n t in the ability of systemically administered morphine to elevate the nociceptive threshold.
3.3. Precipitated withdrawal Table 4 summarizes the effect of systemic naloxone in animals showing complete tolera n c e t o t h e a n a l g e t i c e f f e c t s o f 15 o r 50 pg o f morphine sulfate administered intrathecally and the effects of intrathecal naloxone administered to animals tolerant to the anatgetic e f f e c t s o f s y s t e m i c m o r p h i n e ( 2 0 m g / k g , i.p.). As c a n b e s e e n , t h e s y m p t o m s d i s p l a y e d b y the tolerant groups undergoing naloxone-precipitated withdrawal under either paradigm w e r e q u a l i t a t i v e l y s i m i l a r . In b o t h p a r a d i g m s , n a l o x o n e - p r e c i p i t a t e d w i t h d r a w a l was a c c o m p a n i e d b y a s i g n i f i c a n t d e c r e a s e i n t a i l flick l a t e n c y a n d a clear i n c i d e n c e o f j u m p i n g o r
TABLE 4 A summary of the precipitated withdrawal signs observed in animals made tolerant to intrathecally or systemically administered morphine. Withdrawal signs 1
Systemic morphine vs. systemic naloxone
Systemic naloxone (2 mg/kg i.p.) vs. intrathecal morphine
Group
0 mg/kg
2 mg/kg
0 pg (G)
15 ~g (F)
50 pg (G)
0 mg/kg (A, B)
20 mg/kg (C, D)
n4 Seminal emission Piloerection Escape behavior (jumping) Diarrhea Chromodacryorrhea Withdrawal aggression Weight loss 5
15 4 0 0
15 14 2 11 2 14 2
5 0 1
5 1 1
6 2 4
8 2 1
11 2 9 2
Tail flick 6 Hotplate 7
1 0 0 +2.2 ÷ 0.4 +0.6 ± 0.1 +2.1 ± 0.8
15 2 14 2 12 2 --6.3 + 0.8 3 --1.8 ÷ 0.9 3 --3.5 ± 1.4 3
0 0 0 0 --0.9 *- 1.6 +0.2 ± 0.6 --1.6 + 4.2
3 3 0 2 --1.2 + 1.8 --0.8 + 0.4 --3.6 ± 2.1
Intrathecal naloxone (10 pg) vs. systemic morphine
6 2 6 2 0 62 --1.9 -+ 1.8 --2.3 ± 0.6 3 --2.1 ± 1.9
0 1 0 0 --0.6 ÷ 1.3 +0.8 ± 1.3 +3.8 + 2.1
10 2 10 2 3 10 2 2.4 ± 1.9 --3.2 ± 0.3 3 --2.8 ± 2.4
1 Withdrawal signs were monitored at 5 min intervals for the 1st h after naloxone. For the purposes of presentation, the table presents a summary of the response observed during the 1st h. The signs are described in the text. 2 p < 0.05 difference as compared to controls using a X2 contingency table. a p < 0.05 difference using a t-test. 4 The number of animals in each drug group. s Mean with S.E.M. weight loss observed during the 3 h interval after the injection of naloxone. 6 Data given in terms of the mean difference with 95% confidence interval in tail flick latency observed before and after the administration of naloxone. 7 Data given in terms of the mean difference with 95% confidence interval in hot plate response latency observed before and after the administration of naloxone.
TOLERANCE AND SPINAL MORPHINE escape-like behavior. Consonant with the enhanced tail flick response, the flexor reflex in response to hindpaw pinch was noticeably more vigorous. Though difficult to quantify, the animals were clearly more responsive to external stimuli. Puffs of air or stroking the fur with the point of a pencil, normally innocuous stimuli, commonly elicited vigorous squeaking and biting behavior. Though diarrhea was c o m m o n l y observed, it appeared to be transient and minimal. The limited and variable effect on b o d y weight resulting from the precipitated withdrawal by naloxone confirms that such losses were slight. In contrast, in a control group of 15 unimplanted animals, which received 7 twice daily injections of morphine sulfate (20 mg/kg) the systemic administration of naloxone (2 mg/kg) yielded clear signs of withdrawal, the incidence of which were statistically significant (table 4). The incidence of aggressive behavior as indicated in table 4 was remarkable in all groups with the exception of the controls. Although the measure was taken at 45 min after the administration of morphine, considerable rearing and squeaking was observed during the 5 min interval the test rat was in the withdrawn animal's cage. Withdrawal-induced aggression, as we observed in these experiments following spinal antagonism has also been reported following the systemic administration of naloxone to rats tolerated to systemic morphine (c.f. Lal, 1976). The resemblance of such species-specific behavior to shock-induced aggression confirms the apparent stressful or aversive nature of this particular form of withdrawal.
4. Discussion By their direct administration into the lumbar region of the subarachnoid space, we have shown that the local action of narcotics in the spinal cord will produce a dose-dependent analgesia which is stereospecific and subject to antagonism by systemically administered naloxone (Yaksh and Rudy, 1976a).
281 These findings have been extended in the present experiments to indicate that this pharmacological action of morphine in the spinal cord of the intact rat shows the development of tolerance following repeated administration. These findings are in accord with the work of Martin and colleagues (1964) in which tolerance to the antireflexive actions of narcotics has been shown in chronically spinal dogs. Importantly, in the present experiments, the repeated intrathecal administration of morphine was shown to reduce the analgetic effects of a test dose of systemically administered morphine. These results, suggesting the existence of a cross tolerance offer evidence that the local action of narcotics in the spinal cord, presumably in the dorsal horn where local stereospecific narcotic binding has been demonstrated (Pert et al., 1975; Atweh and Kuhar, 1976; LaMotte et al., 1976), plays a role in mediating the analgesia resulting from systemically administered narcotics. As such these observations are consistent with our previous observation that naloxone, administered into the spinal subarachnoid space will produce a dose~lependent antagonis~n of the analgesia evoked by systemically administered morphine (Yaksh and Rudy, 1977b). That the analgetic action of narcotics is not uniquely mediated by an action in the spinal cord has been unequivocally shown by the fact that the intracerebral administration of narcotics, either into the ventricles or into specific brain loci in a variety of species, will produce analgesia (Tsou and Jang, 1964; Herz et al., 1970; Watanabe, 1971; Jacquet and Lajtha, 1973; Sharpe et al., 1974; Pert and Yaksh, 1974; Yasksh et al., 1976). That this observed action within the brain also has relevance to the analgesia evoked b y systemic morphine is supported first by the observation that intracranially administered opiate antagonists will attenuate analgesia resulting from systemically administered opiate agonists (Tsou, 1963; Herz et al., 1972; Pert and Yaksh, unpublished data) and secondly, by studies where tolerance produced by intra-
282 cranially administered morphine will attenuate the analgesia resulting from systemically administered narcotics, i.e. cross tolerance (Herz and Teschemacher, 1973; Jacquet and Lajtha, 1976). T h e results, therefore, of such experiments listed above i.e., the intracerebral and intrathecal administration of agonists, the attenuation by intracerebral or intrathecal antagonists and the observation of cross tolerance with systemic morphine jointly offer substantial support for the suggestion that both spinal and supraspinal narcotic-sensitive systems play an interactive role in mediating the analgetic action of systemic narcotics (Yaksh and R u d y , 1977b). The intracerebral administration of opiate antagonists to animals having been made tolerant to systemic injections of morphine produces many of the signs c o m m o n l y observed when the antagonist is injected systemically (Eidelberg and Barstow, 1971; Herz et al., 1972; Wei et al., 1973a, 1975; Blasig et al., 1975). In contrast, the withdrawal phenomena observed when the pharmacological antagonism was limited to the spinal cord (intrathecal naloxone) in animals having received systemic morphine or when the tolerance was limited to the spinal cord (intrathecal morphine) was basically limited to signs of hyperreflexia, as indicated by a significantly reduced tail flick latency and marked agitation, suggest an increased sensitivity to otherwize innocuous stimuli. The lack of other withdrawal symptoms following intrathecal naloxone cannot be ascribed to a peculiarity of the schedule of morphine treatments (c.f. Blasig et al., 1973). As noted in table 4, all of the sequelia of precipitated withdrawal were observed in the control groups following the systemic administration of the opiate antagonist. While it is conceivable that a higher dose of intrathecal naloxone might have elicited other effects, we point out that the dose employed was amply sufficient to antagonize the analgetic effects of systemic morphine. Furthermore, as noted previously, in preliminary experiments doses as high as 40 pg
T.L. YAKSH ET AL. failed to elicit any qualitatively different effects than those observed at the lower dose. The enhanced behavioral responsiveness and the exaggerated flexor reflexes we observed in the present experiments appear similar to the descriptions given for the precipitated withdrawal of dogs with chronic spinal sections (Martin et al., 1964, 1974). This markedly enhanced sensitivity of the animals to peripheral stimuli may in part account for the incidence of clear escape behavior produced by intrathecal withdrawal with systemically tolerant animals and vice versa. It seems probable from these data that the extreme hypersensitivity observed following the precipitated withdrawal of animals made previously tolerant to systemic morphine (c.f. Tilson et al., 1973) may in part be mediated b y a spinal system made hyperreactive. As with the phenomena of analgesia, however, it seems likely that supraspinal systems are also involved, as signs of agitation can also be elicited by focal administration of naloxone into the brain of morphine tolerant rats (Wei et al., 1973, 1975). Although the jumping elicited b y naloxone administered either intracranially or intrathecally may reflect behavioral agitation, it cannot be presently determined whether the mechanism underlying the agitation is the same. Thus, it might be suggested that the spinal antagonism is permitting the abnormal throughput of sensory information, while the central antagonism is altering some discriminative aspect of the stimulus. It should be noted that because intrathecal naloxone given to systemically tolerated animals or systemic naloxone given to intrathecally tolerated animals did not produce the full range of behavioral effects argues first, that the chronically administered intrathecal morphine did not gain access to the brain in any significant quantities and secondly, that the effects of intrathecal naloxone were limited predominantly to the spinal cord. These functional observations confirm the results of previous experiments with radiolabeled naloxone in which it was shown that the gradient of distribution of naloxone up the spi-
TOLERANCE AND SPINAL MORPHINE nal cord from the catheter tip was relatively steep, extending no more than 3--4 cm. As the catheter extends 8.5 cm caudal to the cisternal membrane, the likelihood that the injectate would reach the brain appears small. Moreover, brain levels of radioactivity following such an injection of labeled naloxone reached barely above background levels of d e t e c t i o n , e v e n in w h o l e b r a i n s a m p l e s ( Y a k s h and Rudy, 1976b).
References Atweh, S.F. and M.J. Kuhar, 1976, Autoradiographic localization of opiate receptors in rat brain. I. Spinal cord and lower medulla, Brain Res. (in press). Besson, J.M., M. Wyon-Maillard, J.M. Benoist, C. Conseiller and K. Hamann, 1973, Effects of phenoperidine on lamine V cells in the cat dorsal horn, J. Pharmacol. Exptl. Therap. 1 8 7 , 2 3 9 . Blasig, J., A. Herz, K. Rheinhold and J. Zieglganzberger, 1973, Development of physical dependence on morphine in respect to time and degree and quantification of the precipitated withdrawal, Psychopharmacology 33, 19. Eidelberg, E. and C.A. Barstow, 1971, Morphine tolerance and dependence induced by intraventricular injection, Science 174, 74. Golstein, A., 1964, Biostatistics (MacMillan Co., New York). Herz, A., K. Albus, J. Metys, P. Schubert and H. Teschemacher, 1970, On the central sites for the antinonciceptive action of morphine and fetanyl, Neuropharmacology 9 , 5 3 9 . Herz, A. and H. Teschemacher, 1973, Development of tolerance to the antinociceptive effect of morphine after intraventricular injection, Experientia 64, 64. Herz, A., H. Teschemacher, K. Albus and J. Zieglansberger, 1972, Morphine abstinence syndrome in rabbits precipitated by injection of morphine antagonists into the ventricular system and restricted parts of it, Psychopharmacologia (Berlin) 2 6 , 2 1 9 . Irwin, S., R. Houde, D. Bennett, L. Hendershot and M.H. Seevers, 1951, The effects of morphine methadone and meperidine on some reflex responses of spinal animals to nociceptive stimulation, J. Pharmacol. Exptl. Therap. 101, 132. Jacquet, Y.F. and A. Lajtha, 1973, Morphine action at central nervous system sites in rat: analgesia or hyperalgesia depending on site and dose, Science 182,490.
283 Jacquet, Y.F. and A. Lajtha, 1976, The periaqueductal gray: site of morphine analgesia and tolerance as shown by 2-way cross tolerance between systemic and intracerebral injection, Brain Res. 103,501. Jurna, I. and W. Grossman, 1976, The effect of morphine on the activity evoked in the ventrolateral tract axons of the cat spinal cord, Exptl. Brain Res. 2 4 , 4 7 3 . Kayan, S., L.A. Woods and C.L. Mitchell, 1969, Experience as a factor in the development of tolerance to the analgesic effect of morphine, European J. Pharmacol. 6 , 3 3 3 . Kitahata, L.M., Y. Kosaka, A. Taub, K. Bonikos and M. Hoffert, 1974, Lamina specific suppression of dorsal-horn unit activity by morphine sulfate, Anesthesiology 41, 39. Koll, W., J. Haase, G. Block and H. Muhlberg, 1963, The predilective action of small doses of morphine on nociceptive spinal reflexes of low spinal cats, Intern. J. Neuropharmacol. 2, 57. Lal, H., 1975, Narcotic dependence, narcotic action and dopamine receptors, Life Sci. 17,483. LaMotte, C., C. Pert and S.H. Snyder, 1976, Opiate receptor binding in primate spinal cord; distribution and changes after dorsal root section, Proc. Nat. Acad. Sci. (in press). Le Bars, D., D. Menetrey, C. Conseiller and J.M. Besson, 1975, Depressive effects of morphine upon lamina V ceils activities in the dorsal horn of the spinal cat, Brain Res. 9 8 , 2 6 1 . Martin, W.F.,~C.G. Eades, H.F. Fraser and A. Wikler, 1964, Use of hindlimb reflexes of the chronic spinal dog for comparing analgesics, J. Pharmacol. Exptl. Therap. 144, 8. Martin, W.R., C. Eades, W. Thompson, J. Thompson and H. Flanary, 1974, Morphine physical dependence in the dog, J. Pharmacol. Exptl. Therap. 189,759. Pert, A. and T. Yaksh, 1974, Sites of morphine induced analgesia in the primate brain: relation to pain pathways, Brain Res. 8 0 , 1 3 5 . Pert, C.B., M. Kuhar and S. Snyder, 1975, Autoradiographic localization of the opiate receptor in rat brain, Life Sci. 16, 1849. Sharpe, L.G., J.E. Garnett and T.J. Cicero, 1974, Analgesia and hyperreactivity produced by intracranical microinjections of morphine into the periaqueductal gray matter of the rat, Behav. Biol. 11, 303. Tsou, K., 1963, Antagonism of morphine analgesia by the intracerebral microinjection of nalorphine, Acta Physiologica Sinica 2 6 , 3 3 2 . Tsou, K. and C.S. Jang, 1964, Studies on the site of analgesic action of morphine by intracerebral microinjection, Scientia Sinica 13, 1099. Watanabe, H., 1971, The development of tolerance to
284 and of physical dependence on morphine following intraventricular injection in the rat, Japan. J. Pharmacol. 2 1 , 3 8 3 . Wei, E., H. Loh and E. Way, 1973, Brain sites of precipitated abstinence in morphine dependent rats, J. Pharmacol. Exptl. Therap. 185,108. Wei, E., H. Loh and E Way, 1973, Quantitative aspects of precipitated abstinence in morphinedependent rats, J. Pharmacol. Exptl. Therap. 184, 398. Wei, E., S. Sigel, H. Loh and E. Way, 1975, Central sites of naloxone-precipitated shaking in the anesthetized morphine-dependent rat, J. Pharmacol. Exptl. Therap. 195,480. Yaksh, T.L. and T.A. Rudy 1976a, Analgesia mediated by a direct spinal action of narcotics, Science 192,1357.
T.L. YAKSH ET AL. Yaksh, T.L. and T.A. Rudy, 1976b, Chronic catheterization of the spinal subarachnoid space, Physiol. Behav. (in press). Yaksh, T.L. and T.A. Rudy, 1977a, Studies on the direct spinal action of narcotics in the production of analgesia in the rat,J. Pharmacol. Exptl. Therap. (in press). Yaksh, T.L. and T.A. Rudy, 1977b, Narcotic analgetics: CNS sites of action as revealed by intracerebral injection techniques, Pain (in press). Yaksh, T.L., J.C. Yeung and T.A. Rudy, 1976, Systematic examination in the rat of brain sites sensi. tive to the direct application of morphine: Observation of differential effects within the periaqueductal gray, Brain Res. (in press).
275
© Elsevier/North-Holland Biomedical Press
I N D U C T I O N O F T O L E R A N C E A N D W I T H D R A W A L IN R A T S R E C E I V I N G M O R P H I N E I N T H E SPINAL S U B A R A C H N O I D SPACE TONY L. YAKSH *, RANDALL L. KOHL and THOMAS A. RUDY School o f Pharmacy, University o f Wisconsin, Madison, Wi. 53706, U.S.A.
Received 28 July 1976, revised MS received 17 November 1976, accepted 10 December 1976
T.L. YAKSH, R.L. KOHL and T.A. RUDY, Induction o f tolerance and withdrawal in rats receiving morphine in the spinal subarachnoid space, European J. Pharmacol. 42 (1977) 275--284. Rats implanted with chronic catheters in the spinal subarachnoid space were given twice daily injections for 7 days of morphine sulfate, either intrathecaliy into the lumbar subarachnoid space (15 or 50 pg) or i.p. (20 mg/ kg). The development of tolerance, as manifested in a reduction of the analgetic efficacy of these injections on the hot plate and tail flick, occurred in a dose dependent fashion over a period of 7 days. At this time, injections of i.p. morphine into animals which had received spinal morphine and vice versa revealed the existence of a two way cross tolerance between spinal and systemically administered morphine. Injection of naloxone into the spinal cord of animals exposed to i.p. morphine or conversely, i.p. naloxone in animals tolerant to intrathecal morphine, yielded a hyperreflexia and extreme sensitivity to handling. Other signs commonly observed in precipitated withdrawal, however, such as wet shakes and weight loss, were not observed. Spinal cord
Morphine
Tolerance
Withdrawal
1. I n t r o d u c t i o n We have r e c e n t l y d e m o n s t r a t e d t h a t in t h e i n t a c t and u n a n e s t h e t i z e d a n i m a l , m o r p h i n e , applied via a c a t h e t e r c h r o n i c a l l y inserted into t h e l u m b a r s u b a r a c h n o i d space, can p r o d u c e a d o s e < l e p e n d e n t a n d p h a r m a c o l o g i c a l l y specific (i.e., s t e r e o s p e c i f i c a n d n a l o x o n e reversible) e l e v a t i o n in t h e analgetic t h r e s h o l d as d e f i n e d b y t h e tail flick, h o t p l a t e , f o r c e p s p i n c h and s h o c k t i t r a t i o n tests ( Y a k s h and R u d y , 1 9 7 6 a ) . A l t h o u g h a direct a c t i o n of n a r c o t i c s o n cord f u n c t i o n has b e e n p o s i t e d o n t h e basis o f a v a r i e t y o f e x p e r i m e n t s carried o u t in spinal animals (for e x a m p l e , Irwin et al., 1 9 5 1 ; Koll et al., 1 9 6 3 ; Martin et al., 1 9 6 4 ; Besson et al., 1 9 7 3 ; K i t a h a t a et al., 1 9 7 4 ; LeBars et al., 1 9 7 5 ; J u r n a a n d Grossman, 1976), our experiments with intrathecal * Present address: Department of Anatomy and Embryology, University College London, London WC1E 6BT, England.
Naloxone
a d m i n i s t r a t i o n indicate t h a t t h e a l t e r a t i o n in c o r d f u n c t i o n b y t h e local a c t i o n o f n a r c o t i c s has relevance t o t h e analgetic r e s p o n s e o f t h e i n t a c t , b e h a v i n g , animal. In p r e v i o u s experim e n t s , for e x a m p l e , we have s h o w n t h a t n a l o x o n e given i n t o t h e spinal s u b a r a c h n o i d s p a c e can significantly a n t a g o n i z e t h e analgesia p r o d u c e d b y s y s t e m i c a l l y a d m i n i s t e r e d m o r p h i n e ( Y a k s h and R u d y , 1 9 7 7 a ) . Such o b s e r v a t i o n s suggest t h a t at least a f r a c t i o n o f t h e analgetic e f f e c t o b s e r v e d f o l l o w i n g p e r i p h e r a l a d m i n i s t r a t i o n of a n a r c o t i c m a y result f r o m its local e f f e c t o n a spinal m e c h a nism. In t h e p r e s e n t e x p e r i m e n t s , we s o u g h t t o examine further the characteristics of the m o r p h i n e - s e n s i t i v e analgetic s y s t e m l o c a t e d in t h e spinal c o r d b y observing: (1) t o w h a t degree a n i m a l s m a d e t o l e r a n t t o i n t r a t h e c a l l y a d m i n i s t e r e d m o r p h i n e w o u l d d i s p l a y an analgetic r e s p o n s e to s y s t e m i c a l l y administ e r e d m o r p h i n e and vice versa, i.e. cross toler-
276
T.L. YAKSH ET AL.
ance; and (2) the nature of the precipitated withdrawal response evoked by the administration of naloxone into the spinal subarachnoid space of animals made tolerant to systemic morphine as well as the response observed when systemically administered naloxone was given to animals made tolerant to intrathecally administered morphine.
2. Materials and methods
2.1. General procedures Rats were implanted with chronic catheters constructed from PE-10 polyethylene tubing. The tip of each catheter rested at the rostral face of the lumbar enlargement in the spinal subarachnoid space. Details of the catheter construction and implantation are given elsewhere (Yaksh and Rudy, 1976b). Following a 7--14 day recovery period, rats were assigned to I of 7 groups (see below). Injections into the spinal subarachnoid space (intrathecal) were made in volumes of 10 pl (5 pl of drug followed by 5 pl of control vehicle) delivered over approximately 30--45 sec with a manually turned, micrometer~lriven syringe. Solutions for intrathecal injection were always made up in a balanced ion solution consisting of NaC1 7.46 rag; KC1 0.19 mg; MgC12 0.19 mg and CaC12 0.14 mg per 1000 ml of distilled water. 2 doses of morphine sulfate were
employed 15 pg (3 pg/pl) and 50 pg (10pg/ pl). The respective osmolarity of the two solutions was 262 and 270 mosms. Systemic injections (i.p.) were made with morphine sulfate (20 mg/ml/kg) dissolved in 0.9% saline. When control injections were made, the respective vehicles were employed. To develop tolerance by either systemic or spinal administration, morphine was injected by either route in accordance with the group designation (see below) twice a day at 0600 and at 1800 for 7 days. This period was observed to yield a complete tolerance to both spinally and systemically administered morphine in all doses employed as indicated by the absence of any analgetic activity.
2.2. Experimental design 2.2.1. Tolerance development In general, groups received either systemic morphine or intrathecal morphine for 7 days, twice a day as described above. On the morning of the 8th day, the animals received their usual treatment. 6 h later, animals which had received intrathecal morphine during the tolerance period were given systemic morphine and vice versa. The groups and their respective dose regimens are given in table 1. The doses administered were chosen on the basis of previous work which demonstrated that 15 pg was the minimum dose which produced a near maximum detectable effect on
TABLE 1 Listing of group treatments used in the testing of cross tolerance between systemically and intrathecally administered morphine on the hot plate and tail flick tests. Group
n1
Pretreatment (7 days)
Test (8th day)
A B C D E F G
4 4 6 5 5 5 6
Systemic control Systemic control Systemic morphine (20 mg/kg) Systemic morphine (20 mg/kg) Intrathecal control Intrathecal morphine (15/~g) Intrathecal morphine (50 pg)
Intrathecal morphine (15 pg) Intrathecal morphine (50 pg) Intrathecal morphine (15 pg) Intrathecal morphine (50 pg) Systemic morphine (20 mg/kg) Systemic morphine (20 mg/kg) Systemic morphine (20 mg/kg)
I Number of animals in group.
TOLERANCE AND SPINAL MORPHINE the tail flick test (vide infra). Throughout these experiments, the animals were housed singly in wire mesh cage. 2.2.2. Withdrawal To study withdrawal induced by systemic and intrathecal naloxone, the animals were continued for 4 days after the test for cross tolerance, receiving the same schedule of injections as they had received prior to the cross tolerance testing. For the precipitated withdrawal, on the 12th day, 6 h after the morning injection, i.e. at 12.00 h, animals receiving systemic morphine were given intrathecal injections of naloxone HC1 (10 pg) while those given intrathecal morphine during the tolerance period were given systemic naloxone (2 mg/kg, i.p.). These doses, in a previous study (Yaksh and Rudy, unpublished data) have been shown to completely reverse the analgetic action of the respective doses of morphine. Following injection, each rat was returned to its home cage. In preliminary experiments, animals not first exposed to the cross tolerance testing were observed to yield the same response during precipitated withdrawal as those which had undergone the cross tolerance testing 4 days previously. The withdrawal data for these animals were thus combined. 2.3. Measurements 2.3.1. Analgesia To measure the development of tolerance, the tail flick response was observed 30 min after each injection according to the schedules described above. This interval represents the time at which the level of analgesia was consistently observed to be maximum and stable. The tail flick consisted of laying the rat's tail across a slit under which was a focussed projection bulb. The response measure was the time between the light being switched on and the tail being briskly moved from the slit. Because of the long term nature of the tests, an 8 rather than 10 sec cut off was employed. When cross tolerance was examined, the hot plate as well as the tail flick was examined. In
277 the hot plate test, the rat was placed on a metal surface (17 X 22 cm) heated to 55 + I°C by a waterbath. The response measure was the time between the animal being placed on the surface and until it licked its hindpaw. Hot plate responses were also measured following the first injection of morphine and on the morning of the 8th day of the tolerance series. Repeated hot plate measures were not taken due to the possible interaction resulting from learning during the repeated trials (Kayan et al., 1969). In the precipitated withdrawal experiments, the presence of the following signs was systematically noted during each 5 min interval following the injection of naloxone: wet shakes, escape behavior (jumping), piloerection, seminal emissions, chromodacryorrhea and diarrhea. Weight loss occurring over a three hour period after the injection of naloxone was also examined. Descriptions of these withdrawal signs have been published elsewhere (Wei et al., 1973b). The precipitated withdrawal of rats made tolerant to systemic morphine is often accompanied by a marked increase in motor activity within the cage. When the cage was opened slightly, animals would often make an apparent effort to climb or jump through the small opening. This behavior, for purposes of description, was labeled as escape behavior. The incidence of withdrawal induced aggression was also examined by placing an untreated rat of approximately the same b o d y weight in the cage of the withdrawn rat 45 min after the injection of naloxone. The untreated rat was left in the cage for 5 min and the withdrawn rat scored for the presence or absence of aggressive behavior by noting whether it assumed the characteristic aggressive posture (i.e. erect, in a " b o x i n g " stance with its forepaws slightly extended). Active signs of aggression such as squeaking and biting attempts were c o m m o n l y associated with such behavior. 2.4. Statistics To permit comparison between animals, tail flick and hot plate response times were
278
T.L. Y A K S H E T AL.
converted to m a x i m u m (M.P.E.) where:
percent
effects
M.P.E. = postdrug response latency -- predrug response latency X 100 cut off time -- p r e d r u g r e s p o n s e latency
As noted previously, the cut off time for the hot plate and tail flick was 30 and 8 sec, respectively. Confidence intervals (95%) for group data were calculated as described by Goldstein (1964), while the quantal withdrawal data were compared by means of the X 2 statistic.
3. Results
3.1. Tolerance development In the present experiments, we observed that the twice daily administration of intrathecal morphine yields a clear time and dosedependent reduction in the analgetic effects of morphine. Fig. 1 presents the time course of this tolerance development as measured by the tail flick response with the tolerance
I00-
effect appearing most rapidly after about 3 days, for the lower dose (15 pg), while the higher dose (50 pg) required approximately 5 days. That this tolerating effect of intrathecal morphine was not limited to a spinal reflex is evidenced in table 2 by the fact that on the 8th day the analgetic effect of intrathecal morphine measured on the hot plate was significantly reduced as compared to the response observed after the first injection of the series. Although the fact that the degree of tolerance was clearly dependent upon the intrathecal dose of morphine, we considered the possibility that a portion of the effect might be due simply to some non-specific action resulting from the repeated injection of vehicle into the subarachnoid space. To examine this possibility, three animals were tested on the hot plate and tail flick following the administration of 15 #g of intrathecally administered morphine. These animals then received twice daily injections of vehicle intrathecally. On the morning of the 8th day, these same animals received an injection of intrathecal morphine (15pg) instead of the vehicle. The results of these experiments are shown in table 3. As can be seen there was no detectable effect on the analgetic potency of intrathecal morphine resulting from these control intrathecal injec-
r
~
\
u.
]20mg/kg
i,p.
"-...
\
n 50
TABLE 2 \
\
i
50p.q S.A.
\
~\
T h e d e v e l o p m e n t of t o l e r a n c e t o t h e a n a l g e t i c a c t i o n o f m o r p h i n e a d m i n i s t e r e d e i t h e r s y s t e m i c a l l y or intrat h e c a l l y as m e a s u r e d b y t h e h o t plate t e s t .
\,
H o t plate r e s p o n s e 1
J5~g S.A.S.' ~
Day I
Day 8
9 4 . 2 ± 3.9 100.0
22.4 + 12.8 2 16.3 • 13.9 2
98.2 ± 6.4
13.8 -+ 11.2 2
\
ODAYS
Fig. 1. T h e t i m e c o u r s e o f t h e d e v e l o p m e n t o f tolera n c e t o m o r p h i n e given i n t o t h e spinal s u b a r a c h n o i d s p a c e (S.A.S.; 15/~g: o, 50 p g : e ) or m o r p h i n e a d m i n istered s y s t e m i c a l l y (i.p.; 20 m g / k g : i ) as m a n i f e s t e d b y t h e e f f e c t o f s u c h i n j e c t i o n s o n t h e tail flick r e s p o n s e (M.P.E.). E a c h curve r e p r e s e n t s t h e m e a n a n d S.E.M. o f 5 (S.A.S., 1 5 ) ; 6 (S.A.S. 5 0 ) a n d 11 (i.p.) a n i m a l s , r e s p e c t i v e l y .
Intrathecal morphine 15 p g 50pg Systemic morphine 20 m g / k g
1 T h e h o t plate r e s p o n s e w a s m e a s u r e d o n t h e m o r n ing o f t h e 1st a n d 8 t h d a y i m m e d i a t e l y b e f o r e a n d 30 m i n a f t e r t h e i n t r a t h e c a l or s y s t e m i c i n j e c t i o n o f m o r p h i n e . T h e h o t plate r e s p o n s e is e x p r e s s e d as M.P.E. w i t h S.E.M. 2 p < 0.05. T e s t u s e d a m a t c h e d pairs t-statistic.
279
TOLERANCE AND SPINAL MORPHINE TABLE 3 The effect of control solutions administered intrathecally twice daily for 7 days on the analgetic action of intrathecally administered morphine.
Tail flick Hot plate
Test I 1
Test II
91.2 ~ 6.3 89.9 ~ 9.6
98.2 ÷ 1.6 86.4 -+ 11.4
,,; 50
1 Response on test I expressed as M.P.E. to 15 pg of morphine given intrathecally. Test II occurs on the morning of the 8th day and represents the effects on the tail flick and hot plate (15 pg) expressed as M.P.E. 3 animals were used in these experiments.
t i o n s . T h e f a i l u r e o f c o n t r o l i n t r a t h e c a l inject i o n s t o a l t e r t h e a n a l g e t i c p o t e n c y o f syst e m i c m o r p h i n e is s h o w n b y t h e r e s u l t s o b t a i n e d w i t h g r o u p (E) (see b e l o w ) .
3.2. Cross tolerance As s h o w n in fig. 2, for g r o u p s (A) a n d (B), t h e d a i l y i n j e c t i o n o f saline (i.p.) h a d no e f f e c t o n t h e a n a l g e t i c r e s p o n s e o b s e r v e d foll o w i n g t h e i n t r a t h e c a l i n j e c t i o n o f e i t h e r 15 o r 50 pg o f m o r p h i n e , b o t h d o s e s still r e s u l t ing in t h e m a x i m u m d e t e c t a b l e a n a l g e t i c e f f e c t o n b o t h t h e h o t p l a t e a n d t a i l f l i c k . If, h o w e v e r , t h e a n i m a l s r e c e i v e d a 7 d a y pret r e a t m e n t o f m o r p h i n e (20 m g / k g , i.p.), t h e analgetic response measured on both the hot p l a t e a n d t a i l flick was s i g n i f i c a n t l y r e d u c e d as c o m p a r e d t o t h e a p p r o p r i a t e c o n t r o l g r o u p ( g r o u p (C) w i t h g r o u p (A) a n d g r o u p (D) w i t h g r o u p (B). C o m p a r i s o n o f g r o u p (C) w i t h g r o u p (D) i n d i c a t e s t h a t t h e s y s t e m i c a d m i n i s tration of m o r p h i n e p r o d u c e d a relative degree of tolerance with the higher dose of in t r a t h e c a l m o r p h i n e y i e l d i n g a g r e a t e r elevat i o n in t h e a n a l g e t i c t h r e s h o l d t h a n t h e l o w e r dose. The effects of pretreating the animals with intrathecal m o r p h i n e twice daily for 7 days on the analgetic action of systemic morphine are s h o w n in fig. 3. As s h o w n b y t h e r e s u l t s o b t a i n e d in g r o u p (E), t h e c o n t r o l a d m i n i s t r a t i o n of vehicle i n t o t h e s p i n a l s u b a r a c h n o i d
I ~ '
Control
Control
Mor (15p.g)
Mor(50#gJ
Mor Mot (20 rnq/kg) (20mg/kQ)
Mor (15/=g)
Mor(50~.g)
Fig. 2. Cross tolerance experiments wherein the animals were systemically pretceated for 7 days with either saline (control), or morphine (Mot 20 mg/kg) i.p. (upper abscissa). On the 8th day, the animals were treated as described in the text with intrathecal morphine (S.A.S.)in doses of either 15 pg (Mor 15 pg) or 50 pg (Mor 50 pg) (lower abscissa) and the effects on tail flick (left bar) and hot plate (right bar) examined. Results are presented in terms of the M.P.E. The number of animals in each group is given in table 1. The vertical bar indicates the mean and 95% confidence interval.
I00
J~
T
J/ /,
o~ 5C
H i
Control
Mot (20 mg/k9)
Mor (15/~g) Mor (20 mg/k9)
Mor (50/~ g} Mor (20 mg/kg)
Fig. 3. Cross tolerance experiments wherein the animals were pretreated for 7 days with intrathecal morphine (S.A.S.) in doses of 15 (Mor 15 pg) or 50 (Mot 50 pg) pg (upper abscissa). On the 8th day, the animals received, systemically, morphine in a dose of 20 mg/kg (lower abscissa). All details are the same as indicated in fig. 2.
280
T.L. YA.KSH ET AL.
s p a c e f o r this p e r i o d p r o d u c e d n o d e t e c t a b l e decrement in the analgetic action of systemic m o r p h i n e ( 2 0 m g / k g , i.p.). T h e a d d i t i o n o f morphine twice daily for 7 days to the intrathecal injectate, however, produced a dosedependent reduction in the degree of analgesia p r o d u c e d b y t h i s d o s e o f s y s t e m i c m o r p h i n e o n t h e 8 t h d a y . As s h o w n b y t h e r e s u l t s of group (F) and (G), the twice daily administ r a t i o n of 15 pg of m o r p h i n e i n t r a t h e c a l l y p r o d u c e d a slight, b u t n o n - s i g n i f i c a n t reduct i o n in the analgesia p r o d u c e d b y the systemic dose of m o r p h i n e ; whereas the 50 pg dose p r o d u c e d a significant d e c r e m e n t in the ability of systemically administered morphine to elevate the nociceptive threshold.
3.3. Precipitated withdrawal Table 4 summarizes the effect of systemic naloxone in animals showing complete tolera n c e t o t h e a n a l g e t i c e f f e c t s o f 15 o r 50 pg o f morphine sulfate administered intrathecally and the effects of intrathecal naloxone administered to animals tolerant to the anatgetic e f f e c t s o f s y s t e m i c m o r p h i n e ( 2 0 m g / k g , i.p.). As c a n b e s e e n , t h e s y m p t o m s d i s p l a y e d b y the tolerant groups undergoing naloxone-precipitated withdrawal under either paradigm w e r e q u a l i t a t i v e l y s i m i l a r . In b o t h p a r a d i g m s , n a l o x o n e - p r e c i p i t a t e d w i t h d r a w a l was a c c o m p a n i e d b y a s i g n i f i c a n t d e c r e a s e i n t a i l flick l a t e n c y a n d a clear i n c i d e n c e o f j u m p i n g o r
TABLE 4 A summary of the precipitated withdrawal signs observed in animals made tolerant to intrathecally or systemically administered morphine. Withdrawal signs 1
Systemic morphine vs. systemic naloxone
Systemic naloxone (2 mg/kg i.p.) vs. intrathecal morphine
Group
0 mg/kg
2 mg/kg
0 pg (G)
15 ~g (F)
50 pg (G)
0 mg/kg (A, B)
20 mg/kg (C, D)
n4 Seminal emission Piloerection Escape behavior (jumping) Diarrhea Chromodacryorrhea Withdrawal aggression Weight loss 5
15 4 0 0
15 14 2 11 2 14 2
5 0 1
5 1 1
6 2 4
8 2 1
11 2 9 2
Tail flick 6 Hotplate 7
1 0 0 +2.2 ÷ 0.4 +0.6 ± 0.1 +2.1 ± 0.8
15 2 14 2 12 2 --6.3 + 0.8 3 --1.8 ÷ 0.9 3 --3.5 ± 1.4 3
0 0 0 0 --0.9 *- 1.6 +0.2 ± 0.6 --1.6 + 4.2
3 3 0 2 --1.2 + 1.8 --0.8 + 0.4 --3.6 ± 2.1
Intrathecal naloxone (10 pg) vs. systemic morphine
6 2 6 2 0 62 --1.9 -+ 1.8 --2.3 ± 0.6 3 --2.1 ± 1.9
0 1 0 0 --0.6 ÷ 1.3 +0.8 ± 1.3 +3.8 + 2.1
10 2 10 2 3 10 2 2.4 ± 1.9 --3.2 ± 0.3 3 --2.8 ± 2.4
1 Withdrawal signs were monitored at 5 min intervals for the 1st h after naloxone. For the purposes of presentation, the table presents a summary of the response observed during the 1st h. The signs are described in the text. 2 p < 0.05 difference as compared to controls using a X2 contingency table. a p < 0.05 difference using a t-test. 4 The number of animals in each drug group. s Mean with S.E.M. weight loss observed during the 3 h interval after the injection of naloxone. 6 Data given in terms of the mean difference with 95% confidence interval in tail flick latency observed before and after the administration of naloxone. 7 Data given in terms of the mean difference with 95% confidence interval in hot plate response latency observed before and after the administration of naloxone.
TOLERANCE AND SPINAL MORPHINE escape-like behavior. Consonant with the enhanced tail flick response, the flexor reflex in response to hindpaw pinch was noticeably more vigorous. Though difficult to quantify, the animals were clearly more responsive to external stimuli. Puffs of air or stroking the fur with the point of a pencil, normally innocuous stimuli, commonly elicited vigorous squeaking and biting behavior. Though diarrhea was c o m m o n l y observed, it appeared to be transient and minimal. The limited and variable effect on b o d y weight resulting from the precipitated withdrawal by naloxone confirms that such losses were slight. In contrast, in a control group of 15 unimplanted animals, which received 7 twice daily injections of morphine sulfate (20 mg/kg) the systemic administration of naloxone (2 mg/kg) yielded clear signs of withdrawal, the incidence of which were statistically significant (table 4). The incidence of aggressive behavior as indicated in table 4 was remarkable in all groups with the exception of the controls. Although the measure was taken at 45 min after the administration of morphine, considerable rearing and squeaking was observed during the 5 min interval the test rat was in the withdrawn animal's cage. Withdrawal-induced aggression, as we observed in these experiments following spinal antagonism has also been reported following the systemic administration of naloxone to rats tolerated to systemic morphine (c.f. Lal, 1976). The resemblance of such species-specific behavior to shock-induced aggression confirms the apparent stressful or aversive nature of this particular form of withdrawal.
4. Discussion By their direct administration into the lumbar region of the subarachnoid space, we have shown that the local action of narcotics in the spinal cord will produce a dose-dependent analgesia which is stereospecific and subject to antagonism by systemically administered naloxone (Yaksh and Rudy, 1976a).
281 These findings have been extended in the present experiments to indicate that this pharmacological action of morphine in the spinal cord of the intact rat shows the development of tolerance following repeated administration. These findings are in accord with the work of Martin and colleagues (1964) in which tolerance to the antireflexive actions of narcotics has been shown in chronically spinal dogs. Importantly, in the present experiments, the repeated intrathecal administration of morphine was shown to reduce the analgetic effects of a test dose of systemically administered morphine. These results, suggesting the existence of a cross tolerance offer evidence that the local action of narcotics in the spinal cord, presumably in the dorsal horn where local stereospecific narcotic binding has been demonstrated (Pert et al., 1975; Atweh and Kuhar, 1976; LaMotte et al., 1976), plays a role in mediating the analgesia resulting from systemically administered narcotics. As such these observations are consistent with our previous observation that naloxone, administered into the spinal subarachnoid space will produce a dose~lependent antagonis~n of the analgesia evoked by systemically administered morphine (Yaksh and Rudy, 1977b). That the analgetic action of narcotics is not uniquely mediated by an action in the spinal cord has been unequivocally shown by the fact that the intracerebral administration of narcotics, either into the ventricles or into specific brain loci in a variety of species, will produce analgesia (Tsou and Jang, 1964; Herz et al., 1970; Watanabe, 1971; Jacquet and Lajtha, 1973; Sharpe et al., 1974; Pert and Yaksh, 1974; Yasksh et al., 1976). That this observed action within the brain also has relevance to the analgesia evoked b y systemic morphine is supported first by the observation that intracranially administered opiate antagonists will attenuate analgesia resulting from systemically administered opiate agonists (Tsou, 1963; Herz et al., 1972; Pert and Yaksh, unpublished data) and secondly, by studies where tolerance produced by intra-
282 cranially administered morphine will attenuate the analgesia resulting from systemically administered narcotics, i.e. cross tolerance (Herz and Teschemacher, 1973; Jacquet and Lajtha, 1976). T h e results, therefore, of such experiments listed above i.e., the intracerebral and intrathecal administration of agonists, the attenuation by intracerebral or intrathecal antagonists and the observation of cross tolerance with systemic morphine jointly offer substantial support for the suggestion that both spinal and supraspinal narcotic-sensitive systems play an interactive role in mediating the analgetic action of systemic narcotics (Yaksh and R u d y , 1977b). The intracerebral administration of opiate antagonists to animals having been made tolerant to systemic injections of morphine produces many of the signs c o m m o n l y observed when the antagonist is injected systemically (Eidelberg and Barstow, 1971; Herz et al., 1972; Wei et al., 1973a, 1975; Blasig et al., 1975). In contrast, the withdrawal phenomena observed when the pharmacological antagonism was limited to the spinal cord (intrathecal naloxone) in animals having received systemic morphine or when the tolerance was limited to the spinal cord (intrathecal morphine) was basically limited to signs of hyperreflexia, as indicated by a significantly reduced tail flick latency and marked agitation, suggest an increased sensitivity to otherwize innocuous stimuli. The lack of other withdrawal symptoms following intrathecal naloxone cannot be ascribed to a peculiarity of the schedule of morphine treatments (c.f. Blasig et al., 1973). As noted in table 4, all of the sequelia of precipitated withdrawal were observed in the control groups following the systemic administration of the opiate antagonist. While it is conceivable that a higher dose of intrathecal naloxone might have elicited other effects, we point out that the dose employed was amply sufficient to antagonize the analgetic effects of systemic morphine. Furthermore, as noted previously, in preliminary experiments doses as high as 40 pg
T.L. YAKSH ET AL. failed to elicit any qualitatively different effects than those observed at the lower dose. The enhanced behavioral responsiveness and the exaggerated flexor reflexes we observed in the present experiments appear similar to the descriptions given for the precipitated withdrawal of dogs with chronic spinal sections (Martin et al., 1964, 1974). This markedly enhanced sensitivity of the animals to peripheral stimuli may in part account for the incidence of clear escape behavior produced by intrathecal withdrawal with systemically tolerant animals and vice versa. It seems probable from these data that the extreme hypersensitivity observed following the precipitated withdrawal of animals made previously tolerant to systemic morphine (c.f. Tilson et al., 1973) may in part be mediated b y a spinal system made hyperreactive. As with the phenomena of analgesia, however, it seems likely that supraspinal systems are also involved, as signs of agitation can also be elicited by focal administration of naloxone into the brain of morphine tolerant rats (Wei et al., 1973, 1975). Although the jumping elicited b y naloxone administered either intracranially or intrathecally may reflect behavioral agitation, it cannot be presently determined whether the mechanism underlying the agitation is the same. Thus, it might be suggested that the spinal antagonism is permitting the abnormal throughput of sensory information, while the central antagonism is altering some discriminative aspect of the stimulus. It should be noted that because intrathecal naloxone given to systemically tolerated animals or systemic naloxone given to intrathecally tolerated animals did not produce the full range of behavioral effects argues first, that the chronically administered intrathecal morphine did not gain access to the brain in any significant quantities and secondly, that the effects of intrathecal naloxone were limited predominantly to the spinal cord. These functional observations confirm the results of previous experiments with radiolabeled naloxone in which it was shown that the gradient of distribution of naloxone up the spi-
TOLERANCE AND SPINAL MORPHINE nal cord from the catheter tip was relatively steep, extending no more than 3--4 cm. As the catheter extends 8.5 cm caudal to the cisternal membrane, the likelihood that the injectate would reach the brain appears small. Moreover, brain levels of radioactivity following such an injection of labeled naloxone reached barely above background levels of d e t e c t i o n , e v e n in w h o l e b r a i n s a m p l e s ( Y a k s h and Rudy, 1976b).
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