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Antinociceptive effects of intrathecal administration of α-adrenoceptor antagonists and clonidine in the formalin test in the mouse
Neuropharmacology Vol. 32, No. 4, pp. 367-371, 1993 Printed in Great Britain. All rights reserved
0028-3908/93 $6.00+ 0.00 Copyright © 1993Pergamon Press Ltd
ANTINOCICEPTIVE EFFECTS OF INTRATHECAL A D M I N I S T R A T I O N OF c¢-ADRENOCEPTOR ANTAGONISTS A N D CLONIDINE IN THE FORMALIN TEST IN THE MOUSE T. I. KANUI,l A. TJOLSEN,2 A. LUND,2 NORMA MJELLEM-JOLY2 and K. HOLE2 tDepartment of Animal Physiology, University of Nairobi, P.O. Box 30197, Nairobi, Kenya and 2Department of Physiology, University of Bergen, Norway (Accepted 16 November 1992)
Summary--The hypothesis that the descending noradrenergic system tonically inhibits nociception at the spinal level was investigated, using the formalin test in mice. The ct-adrenoceptor agonist clonidine (0.46 and 0.92/tg), injected intrathecally, significantly reduced licking activity in both the early and late phase of the test. The cq-antagonist prazosin (3.75, 7.5 and 15 #g) and the ct2-antagonist yohimbine (7.5 #g) also significantly reduced licking activity in both phases. The smaller doses of yohimbine (1.87 and 3.75 #g) induced an insignificant reduction of licking in the early phase. Except for the largest doses of clonidine (0.92 pg), the drugs used had no effect on the general level of activity and motor performance. These results support previous findings that increased noradrenergic activity in the spinal cord inhibits nociception, however, this inhibition seems not to be tonically active. The mechanisms of the antinociceptive actions of ~t-antagonists are not clear. Key words--intrathecal, noradrenergic, nociception, formalin test, mice.
In thermal tests of nociception, the hot-plate and tail-flick tests, selective lesions of descending serotonergic and noradrenergic pathways have been reported to reduce response latencies to noxious heat stimulation (Archer, Danysz, Johnson, Minor and Post, 1986; Howe, Wang and Yaksh, 1983; Proudfit and Hammond, 1981; Reddy and Yaksh, 1980; Sagen and Proudfit, 1984, 1985; Sugimoto, Kuraishi, Satoh and Takagi, 1986). Intrathecal administration of ~t-adrenoceptor agonists has been reported to induce analgesia in a number of studies (Jones and Gebhart, 1986; Kuraishi, Harada and Takagi, 1979; Kuraishi, Hirota, Satoh and Takagi, 1985; Sagen and Proudfit, 1984). In electrophysiological studies, iontophoresis of noradrenaline caused selective inhibition of nociceptive responses of identified neurones (Belcher, Ryall and Schaffner, 1978; Fleetwood-Walker, Mitchell, Hope, Molony and Iggo, 1985; Headley, Duggan and Griersmith, 1978). Clonidine, an ~adrenoceptor agonist, inhibited C-fibre evoked potentials (Sullivan, Dashwood and Dickenson, 1987). These studies suggest that the descending noradrenergic system tonically inhibits nociception in the spinal cord. However, more recent work (Berge, Garcia-Cabrera and Hole, 1988; Eide and Tjolsen, 1988; Tjolsen, Lund and Hole, 1990) showed that when a thermal stimulus was used in tests of nociception, a reduced response latency may be due to an increase in skin temperature at the onset of stimu-
lation, thus representing an artifact. When the tail flick latencies were corrected for the changes in tail temperature, a tonic descending inhibition, mediated by the noradrenergic system, could not be observed (Tjolsen et al., 1990). Thus, the role of the noradrenergic system in the tonic regulation of nociception should be reexamined also using non-thermal tests of nociception. In the present experiments, the formalin test was used. To study a possible tonic descending inhibition of nociception by the noradrenergic system, the ~adrenoceptor antagonists prazosin and yohimbine were used and, to study a possible antinociception induced by noradrenergic stimulation, the ct-adrenoceptor agonist clonidine was chosen.
METHODS
Animals
Male albino mice ( B O M : N M R I , BOM-MICE, Denmark), weighing 15-19 g at the time of testing, were used. The animals were housed in colony cages 16 mice in each, with free access to food and water. They were maintained in climate- and light-controlled rooms (23-t-I°C, 12/12hr dark/light cycle with lights on at 07.00 hr) for at least 2 weeks prior to the experiments. Testing took place between 09.00 and 17.00 hr. The animals were placed individually in standard macrolone cages (30 x 12 x 13cm), which also served as observation chambers after injection of formalin. They were allowed to adapt to the new 367
368
T.I. KAY:Z:et al.
environment for at least 2 h r before testing. All testing was performed with the observer being unaware of the treatment.
Drugs and administration All drugs were injected intrathecally in a volume of 7 #1 (Hylden and Wilcox, 1980). Clonidine (Boehringer Ingelheim) was dissolved in 0.9% NaC1 and was given in doses of 0, 0.23, 0.46 and 0.92/zg (0, 1.2, 2.3 and 4.6 nmol). Yohimbine and prazosin (both from Research Biochemicals) were dissolved in 50% dimethylsulphoxide (DMSO) in 0.9% NaCI. Yohimbine was administered in doses of 0, 1.87, 3.75, 7.5 #g (0, 5.25, 10.5 and 21.0 nmol) and prazosin in doses of 0, 3.75, 7.5 and 15 #g (0, 9.75, 19.5 and 39.0 nmol). Control animals were injected with the same volume of the appropriate vehicle.
Nociceptive testing Testing was started 5 min after injection of drug and was performed in a sound-attenuated room, maintained between 22.5 and 23.0°C. One animal was tested at a time. Formalin 5% (i.e. formaldehyde 2%) in 0.9% NaC1 was injected subcutaneously dorsally in a hindpaw in a volume of 20/zl. The animal was then returned to its home cage and the time spent licking/biting the injected paw was scored for 1 hr. Scoring was performed in 12 blocks of 5 min and the data was recorded as total licking time in the early phase (0-10 min) and the late phase (10-60 min after injection of formalin.
RESULTS
The formalin test All three doses of clonidine used (0.23, 0.46 and 0.92/~g) significantly reduced licking of the hind paw in the early phase (ANOVA: F(3.24)=43.39, P < 0.001). In the late phase there was an overall dose-dependent reduction of licking (ANOVA: F(3.:4) = 59.28, P < 0.001), however, the smallest dose of clonidine failed to reduce the licking response (Fig. 1). Prazosin (Fig. 2) significantly reduced hind-paw licking in the early phase (ANOVA: F~3.24~= 19.31, P < 0.001), as well as in tile late phase (ANOVA: F0,24) = 63.98, P < 0.001). Yohimbine (Fig. 3) produced an overall reduction of licking in the early phase (ANOVA: F(3,24)= 3.53, P < 0.02); however, only the largest dose (7.5 ltg) had a significant effect (P = 0 . 0 1 , t-test subsequent to ANOVA). In the late phase, yohimbine produced a dose-dependent reduction of licking (ANOVA: Ft3,z4) = 9.17, P = 0.001).
Colonic temperature There was an overall reduction of colonic temperature by clonidine (ANOVA: F~3' 24~= 6.17, P = 0.003). However, subsequent data analysis revealed that only the largest dose of clonidine (0.92 ~tg) produced a significant reduction of colonic temperature (P = 0.005, t-test subsequent to ANOVA) (Fig. 4). Prazosin also induced an overall reduction of colonic temperature (ANOVA: F13.24)= 2.99,
100SO;
Assessment of sensorimotor performance and activity Assessment of the effect of the drug on sensorimotor performance and general motor activity was based on an arbitrary scale of 0--4 as follows: immobility--0, distinctly a b n o r m a l - - l , moderate--2, slightly abnormal--3 and n o n n a l ~ . Activity was similarly scored: sleep-like state-4), alert but not active---l, little activity--2, moderate activity--3, active most of the time--4.
Early phase
60" 40" "~ O
20"
,~ _c o
Late phase 300
,.I
Measurement of body temperature Deep body temperature was measured in the colon with an electronic thermometer, the probe inserted 2 cm from the anus. The temperature was determined at the beginning and the end of each testing session.
200 100
I
0.23 Statistical analysis The data were analysed by analysis of variance (ANOVA) or by Student's t-test subsequent to ANOVA, when the comparison was restricted to two means. Activity and sensorimotor performance data were analysed using Kruskal-Wallis ANOVA by ranks. Significance was accepted at the 5% level.
0.46
0.92
Clonldlne (Pg) Fig. 1. Effect of intrathecal administration of clonidine on hind-paw licking in the early (0-10 rain) and the late phase (10~0min after injection of formalin). Results are presented as means _+SEM (indicated by open symbols) (N = 7 in each group). *P < 0.05, ***P < 0.001 (Student's t-test compared to controls).
ct-Adrenoceptors and nociception Early phase
100 80 60
"G 0 m
'mOO
40 0
20 0
:E
I
I
I
7.5
15
a g~
450 ~
Late phase
o ,J
350
`m`m`m
250
0
3.75
Prazosin (pg)
Fig. 2. Effect of intrathecal administration of prazosin on hind-paw licking in the early and the late phase. Results are presented as means ___SEM (indicated by open symbols) (N = 7 in each group). *P < 0.05, ***P < 0.001 (Student's t-test compared to controls). P = 0.05) but only the largest dose (15/tg) produced hypothermia (P < 0.05, t-test). Yohimbine had no significant effect on colonic temperature (ANOVA: F(3.z4) = 1.69, P = 0.19).
1978), supporting the hypothesis that stimulation of spinal ct-adrenoceptors produces antinociception. These results not only provide more evidence that clonidine has an antinociceptive effect on phasic pain, that in the early phase of the formalin test is probably due to direct stimulation of nociceptors but also provide evidence that clonidine has antinociceptive activity in the late phase of this test. The pain in this late phase is probably due to inflammation (Hunskaar and Hole, 1987). However, a somewhat larger dose of clonidine (double the smallest dose used) was required to produce analgesia in this phase. The literature reviewed above and the results presented here support the existence of a spinal site of action for clonidine. Prazosin and yohimbine, ~t-adrenoceptor antagonists, induced analgesia in mice at very small doses. Both antagonists had antinociceptive actions in both the early and the late phase of the formalin test. The antinociceptive effect of yohimbine was weaker than that of prazosin in both phases. The present results are in agreement with earlier studies which suggested that ~t-adrenoceptor blockers, phenoxybenzamine, phentolamine and tolazoline (Cicero, Meyer and Smithloff, 1974; Elliot, Spiehler and Navarro, 1976), had antinociceptive effects in the writhing and tailflick tests. Yohimbine has been demonstrated to have antinociceptive activity in the hot-plate test (Denis, Melzack, Gutman and Boucher, 1980). The largest dose of cionidine and prazosin induced significant hypothermia. Yohimbine did not cause an
.~
Activity and motor performance
Yohimbine and prazosin had no significant effect on activity and motor performance. Clonidine, however, caused a significant reduction of activity at 0.92/~g, where the mean activity score was reduced from 3.7 in controls to 1.7 (Kruskal-Wallis ANOVA by ranks: H(3.2s)= 17.67, P < 0.001), Mann-Whitney U-test: vehicle vs 0.92/zg, P < 0.05).
369
Early phase
4O
| Late
DISCUSSION
These results support earlier investigations indicating that intrathecal administration of cionidine, an ~-agonist, has a strong antinociceptive effect (Bentley, Copeland and Starr, 1977; Milne, Cervenko, Jhamandas and Sutak, 1985; Tj~lsen et al., 1990). They also are in accord with electrophysiological results showing that clonidine reduces C-fibre evoked activity (Sullivan et al., 1987). Intrathecal administration of noradrenaline, preferentially stimulating ~-adrenergic receptors (Aran and Proudfit, 1990; De Lander and Hopkin, 1987; Kuraishi et al., 1979, 1985; Osgood, Carr, Kazianis, Kemp, Atchison and Szytelbein, 1990), has been shown to inhibit nociceptive neurones in the spinal cord (Belcher et al., 1978; Fleetwood-Walker et al., 1985; Headley et al., NP 32/4~E
400
phase `m
o
A
200
0 0
u
i
i
1.87
3.75
7.5
Yohlmblne (pg)
Fig. 3. Effect of intrathecal administration of yohimbine on hind-paw licking in the early and late phase. Results are presented as means + SEM (indicated by open symbols) (N = 7 in each group). *P < 0.05 (Student's t-test compared to controls).
T. I. KANUIet al.
370 0.4 ~
Clonidine
0
-0.8 I - 1.2
~"~ I
0
-=
I
//
0.46
I
0.92
Prazosin
1.8 P
I
0.23
0.8 A
o Q. E
I,-
0 -0.8 0
-1.8
I
I
6.75
/I/
I
7.5
15
1.8 --~ Yohimblne .0 ¢: 0 0 0
&
0.8
0
1.87
3.75
7.5
Y o h i m b l n e (pg)
Fig. 4. Effect of intrathecal administration of clonidine, prazosin and yohimbine on colonic temperature, given as difference in temperature before injection of formalin and after the end of testing. Results are presented as means + SEM (indicated by open symbols) (N = 7 in each group). *P < 0.05 (Student's t-test compared to controls).
alteration of colonic temperature, suggesting that spinal ~2-adrenoceptors do not play an important role in thermoregulation. Only the largest dose of clonidine significantly reduced activity and motor performance, while yohimbine and prazosin had no effect. Preliminary experiments indicated that larger doses of all the three drugs induced sensorimotor deficits and muscle flaccidity, in agreement with an earlier investigation (Milne et al., 1985). Recently, it was observed that a reduced skin temperature may reduce the licking response in the late phase in the formalin test (Rosland, 1991), probably due to reduced inflammation. The reduced licking response was not observed in the early phase and in the late phase, the maximum licking response was reduced only at ambient temperatures lower than approximately 23°C. In the present study, a reduced licking response was observed in the early phase of the test, as well as for doses that did not significantly affect body temperature. Although it cannot be ex-
cluded that reduced skin temperature partly contributed to the reduction in the licking response, it seems that such a mechanism cannot explain the results. In the experiments described here, the preferential ~2-agonist clonidine and the ~rantagonist prazosin produced similar effects, reducing the nociceptive response in the formalin test. The antinociceptive effect of the ~2-antagonist yohimbine was considerably less pronounced. The results thus fit the hypothesis that both subclasses of ~-adrenergic receptors may be involved in nociception, mediating opposite effects. This would form a parallel to the findings that ~1- and ~2-adrenoceptors seem to mediate facilitation and attenuation of the motor responses in the spinal cord, respectively (Tanabe, Ono and Fukuda, 1990). However, in the constant temperature hot plate test, no antinociceptive effect of intrathecal injection of prazosin was found (Takano and Yaksh, 1992). When administered intraperitoneally, prazosin was ineffective in the formalin test in the rat (Tasker, Connell and Yole, 1992). In both studies, prazosin blocked the antinociceptive effect of ~-agonists (ST91 and methoxamine, respectively). Hence, the picture is very complex, and the existing knowledge is not sufficient to build a simple model for the role of the ~-adrenergic receptors in spinal nociceptive processing. It has to be discussed in terms of pre- and postsynaptic effects on the cells in the system. From the present data, it seems that the antinociceptive effects observed in the formalin test in mice are not seriously confounded by effects of motor or autonomic functions. Patch-clamp techniques have indicated that ~rreceptors are located postsynaptically and have depolarizing functions, while ~2-receptors have hyperpolarizing properties but may be located both pre- and postsynaptically (Nicoll, Malenka and Kauer, 1990). A hyperpolarization due to stimulation of postsynaptic ~2-receptors is a possible model for the observed antinociception induced by clonidine. The mechanism of the antinociceptive effect of ~antagonists in tonic pain suggested by the present results is not clear and further research is required for its elucidation. In conclusion, the study shows that clonidine, administered intrathecally, also has an antinociceptive effect in the formalin test. The ~-antagonist prazosin also showed a potent antinociceptive effect. No indication of a tonic antinociceptive activity of the descending noradrenergic systems was observed. Acknowledgements--T. I. Kanui was on leave of absence
from the University of Nairobi, Kenya and would like to thank the Vice-Chancellorfor granting leave and NORAD for financial support.
REFERENCES Aran S. and Proudfit H. K. (1990) Antinociception produced by interaction between intrathecally administered
~-Adrenoceptors and nociception adenosine agonists and norepinephrine. Brain Res. 513: 255-263. Archer T., Danysz W., Johnson G., Minor G. and Post C. (1986) 5-MethoxyoN-N, N-dimethyltryptamine induced analgesia is blocked by alpha-adrenoceptor antagonists in rats. Br. J. Pharmac. 89: 293-298. Belcher G., Ryall R. W. and Schaffner R. (1978) The differential effects of 5-hydroxytryptamine, noradrenaline and raphe stimulation on nociceptive and non-nociceptive dorsal horn interneurones in the cat. Brain Res. 151: 307-321. Bentley G. A., Copeland I. W. and Starr J. (1977) The actions of some ~-adrenoceptor agonists and antagonists in an antinociceptive test in mice. Clin. Exp. Pharmac. Physiol. 4: 405-419. Berge O.-G., Garcia-Cabrera I. and Hole K. (1988) Response latencies in the tail-flick test depend on tail skin temperature. Neurosci. Lett. 86: 284-288. Cicero T. J., Meyer E. R. and Smithloff B. R. (1974) Alpha adrenergic blocking agents: Antinociceptive activity and enhancement of morphine-induced analgesia. J. Pharmac. Exp. Ther. 189:72 82. De Lander G. E. and Hopkin C. J. (1987) Interdependence of spinal adenosinergic, serotonergic and noradrenergic systems mediating antinociception. Neuropharmacology 26: 1791-1794. Dennis S. G., Melzack R., Gutman S. and Boucher F. (1980) Pain modulation by adrenergic agents and morphine as measured by three pain tests. Life Sci. 26: 1247-1259. Eide P. K. and Tjolsen A. (1988) Effects of serotonin receptor antagonists and agonists on the tail-flick response in mice involve altered tail-skin temperature. Neuropharmacology 27: 889-893. Elliott H. W., Spiehler V. and Navarro G. (1976) Effects of naloxone on antinociceptive activity of phenoxybenzamine. Life Sci. 19: 1637-1644. Fleetwood-Walker S. M., Mitchell R., Hope P. J., Molony V. and Iggo A. (1985) An alfa(2)-receptor mediates the selective inhibition by noradrenaline of nociceptive responses of identified dorsal horn neurones. Brain Res. 334: 243-254. Headley P. M., Duggan A. W. and Griersmith B. T. (1978) Selective reduction by noradrenaline and 5-hydroxytryptamine of nociceptive responses of cat dorsal horn neurones. Brain Res. 145: 185-189. Howe J. R., Wang J.-Y. and Yaksh T. L. (1983) Selective antagonism of the antinociceptive effect of intrathecally applied alpha-adrenergic agonists by intrathecal prazosin and intrathecal yohimbine. J. Pharmac. Exp. Ther. 224: 552-558. Hunskaar S. and Hole K. (1987) The formalin test in mice, dissociation between inflammatory and non-inflammatory pain. Pain 30:103-114. Hylden J. L. K. and Wilcox G. L. (1980) Intrathecal morphine in mice: a new technique. Eur. J. Pharmac. 67: 313-316. Jones S. L. and Gebhart G. F. (1986) Characterisation of coeruleospinal inhibition of the tail-flick reflex in the rat: mediation by spinal alpha-adrenoceptors. Brain Res. 364: 315-330.
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Kuraishi Y., Harada Y. and Takagi H. (1979) Noradrenaline regulation of pain transmission in the spinal cord mediated by alpha-adrenoceptors. Brain Res. 174: 333-336. Kuraishi Y., Hirota N., Satoh M. and Takagi H. (1985) Antinociceptive effects of intrathecal opioids, noradrenaline and serotonin in rats: mechanical and thermal algesic tests. Brain Res. 326: 168-171. Milne B., Cervenko F. W., Jhamandas K. and Sutak M. (1985) Intrathecal clonidine: analgesia and effect on opiate withdrawal in the rat. Anaesthesiology 62: 34-38. Nicoll R. A., Malenka R. C. and Kauer J. A. (1990) Functional comparison of neurotransmitter receptor subtypes in mammalian central nervous system. Physiol. Rev. 70: 513-565. Proudfit H. K. and Hammond D. L. (1981) Alterations in nociceptive threshold and morphine-induced analgesia produced by intrathecally administered amine antagonists. Brain Res. 218: 393-399. Osgood P. F., Cart D. B., Kazianis A., Kemp J. W., Atchison N. E. and Szyfelbein S. K. (1990) Antinociception in the rat induced by a cold environment. Brain Res. 507:11-16. Reddy S. V. R. and Yaksh T. L. (1980) Spinal noradrenergic terminal system mediates antinociception. Brain Res. 189: 391-401. Rosland J. H. (1991) The formalin test in mice: the influence of ambient temperature. Pain 45:211-216. Sagen J. and Proudfit H. K. (1984) Effects of intrathecally administered noradrenergic antagonists on nociception in the rat. Brain Res. 310: 295-301. Sagen J. and Proudfit H. K. (1985) Evidence for pain modulation by pre- and postsynaptic noreadrenergic receptors in the medulla oblongata. Brain Res. 331: 285-293. Sugimoto M., Kuraishi Y., Satoh M. and Takagi H. (1986) Involvement of medullary opioid-peptidergic and spinal noradrenergic systems in the regulation of formalin-induced persistent pain. Neuropharmacology 25: 481-485. Sullivan A. F., Dashwood M. R. and Dickenson A. H. (1987) ~t2-Adrenoceptor modulation of nociception in rat spinal cord: location, effects and interactions with morphine. Eur. J. Pharmac. 138: 169-177. Takano Y. and Yaksh T. L. (1992) Characterization of the pharmacology of intrathecally administered alpha-2 agonists and antagonists in rats. J. Pharmac. Exp. Ther. 261: 764-772. Tanabe M., Ono H. and Fukuda H. (1990) Spinal alphasand alpha2-adrenoceptors mediate facilitation and inhibition of spinal motor transmission, respectively. Jap. J. Pharmac. 54: 69-77. Tasker R. A. R., Connell B. J. and Yole M. J. (1992) Systemic injections of alpha-1 adrenergic agonists produce antinociception in the formalin test. Pain 49: 383-391. Tj~lsen A., Lund A. and Hole K. (1990) The role of descending noradrenergic systems in regulation of nociception: the effects of intrathecally administered ~adrenoceptor antagonists and clonidine. Pain 43: 113-120.
0028-3908/93 $6.00+ 0.00 Copyright © 1993Pergamon Press Ltd
ANTINOCICEPTIVE EFFECTS OF INTRATHECAL A D M I N I S T R A T I O N OF c¢-ADRENOCEPTOR ANTAGONISTS A N D CLONIDINE IN THE FORMALIN TEST IN THE MOUSE T. I. KANUI,l A. TJOLSEN,2 A. LUND,2 NORMA MJELLEM-JOLY2 and K. HOLE2 tDepartment of Animal Physiology, University of Nairobi, P.O. Box 30197, Nairobi, Kenya and 2Department of Physiology, University of Bergen, Norway (Accepted 16 November 1992)
Summary--The hypothesis that the descending noradrenergic system tonically inhibits nociception at the spinal level was investigated, using the formalin test in mice. The ct-adrenoceptor agonist clonidine (0.46 and 0.92/tg), injected intrathecally, significantly reduced licking activity in both the early and late phase of the test. The cq-antagonist prazosin (3.75, 7.5 and 15 #g) and the ct2-antagonist yohimbine (7.5 #g) also significantly reduced licking activity in both phases. The smaller doses of yohimbine (1.87 and 3.75 #g) induced an insignificant reduction of licking in the early phase. Except for the largest doses of clonidine (0.92 pg), the drugs used had no effect on the general level of activity and motor performance. These results support previous findings that increased noradrenergic activity in the spinal cord inhibits nociception, however, this inhibition seems not to be tonically active. The mechanisms of the antinociceptive actions of ~t-antagonists are not clear. Key words--intrathecal, noradrenergic, nociception, formalin test, mice.
In thermal tests of nociception, the hot-plate and tail-flick tests, selective lesions of descending serotonergic and noradrenergic pathways have been reported to reduce response latencies to noxious heat stimulation (Archer, Danysz, Johnson, Minor and Post, 1986; Howe, Wang and Yaksh, 1983; Proudfit and Hammond, 1981; Reddy and Yaksh, 1980; Sagen and Proudfit, 1984, 1985; Sugimoto, Kuraishi, Satoh and Takagi, 1986). Intrathecal administration of ~t-adrenoceptor agonists has been reported to induce analgesia in a number of studies (Jones and Gebhart, 1986; Kuraishi, Harada and Takagi, 1979; Kuraishi, Hirota, Satoh and Takagi, 1985; Sagen and Proudfit, 1984). In electrophysiological studies, iontophoresis of noradrenaline caused selective inhibition of nociceptive responses of identified neurones (Belcher, Ryall and Schaffner, 1978; Fleetwood-Walker, Mitchell, Hope, Molony and Iggo, 1985; Headley, Duggan and Griersmith, 1978). Clonidine, an ~adrenoceptor agonist, inhibited C-fibre evoked potentials (Sullivan, Dashwood and Dickenson, 1987). These studies suggest that the descending noradrenergic system tonically inhibits nociception in the spinal cord. However, more recent work (Berge, Garcia-Cabrera and Hole, 1988; Eide and Tjolsen, 1988; Tjolsen, Lund and Hole, 1990) showed that when a thermal stimulus was used in tests of nociception, a reduced response latency may be due to an increase in skin temperature at the onset of stimu-
lation, thus representing an artifact. When the tail flick latencies were corrected for the changes in tail temperature, a tonic descending inhibition, mediated by the noradrenergic system, could not be observed (Tjolsen et al., 1990). Thus, the role of the noradrenergic system in the tonic regulation of nociception should be reexamined also using non-thermal tests of nociception. In the present experiments, the formalin test was used. To study a possible tonic descending inhibition of nociception by the noradrenergic system, the ~adrenoceptor antagonists prazosin and yohimbine were used and, to study a possible antinociception induced by noradrenergic stimulation, the ct-adrenoceptor agonist clonidine was chosen.
METHODS
Animals
Male albino mice ( B O M : N M R I , BOM-MICE, Denmark), weighing 15-19 g at the time of testing, were used. The animals were housed in colony cages 16 mice in each, with free access to food and water. They were maintained in climate- and light-controlled rooms (23-t-I°C, 12/12hr dark/light cycle with lights on at 07.00 hr) for at least 2 weeks prior to the experiments. Testing took place between 09.00 and 17.00 hr. The animals were placed individually in standard macrolone cages (30 x 12 x 13cm), which also served as observation chambers after injection of formalin. They were allowed to adapt to the new 367
368
T.I. KAY:Z:et al.
environment for at least 2 h r before testing. All testing was performed with the observer being unaware of the treatment.
Drugs and administration All drugs were injected intrathecally in a volume of 7 #1 (Hylden and Wilcox, 1980). Clonidine (Boehringer Ingelheim) was dissolved in 0.9% NaC1 and was given in doses of 0, 0.23, 0.46 and 0.92/zg (0, 1.2, 2.3 and 4.6 nmol). Yohimbine and prazosin (both from Research Biochemicals) were dissolved in 50% dimethylsulphoxide (DMSO) in 0.9% NaCI. Yohimbine was administered in doses of 0, 1.87, 3.75, 7.5 #g (0, 5.25, 10.5 and 21.0 nmol) and prazosin in doses of 0, 3.75, 7.5 and 15 #g (0, 9.75, 19.5 and 39.0 nmol). Control animals were injected with the same volume of the appropriate vehicle.
Nociceptive testing Testing was started 5 min after injection of drug and was performed in a sound-attenuated room, maintained between 22.5 and 23.0°C. One animal was tested at a time. Formalin 5% (i.e. formaldehyde 2%) in 0.9% NaC1 was injected subcutaneously dorsally in a hindpaw in a volume of 20/zl. The animal was then returned to its home cage and the time spent licking/biting the injected paw was scored for 1 hr. Scoring was performed in 12 blocks of 5 min and the data was recorded as total licking time in the early phase (0-10 min) and the late phase (10-60 min after injection of formalin.
RESULTS
The formalin test All three doses of clonidine used (0.23, 0.46 and 0.92/~g) significantly reduced licking of the hind paw in the early phase (ANOVA: F(3.24)=43.39, P < 0.001). In the late phase there was an overall dose-dependent reduction of licking (ANOVA: F(3.:4) = 59.28, P < 0.001), however, the smallest dose of clonidine failed to reduce the licking response (Fig. 1). Prazosin (Fig. 2) significantly reduced hind-paw licking in the early phase (ANOVA: F~3.24~= 19.31, P < 0.001), as well as in tile late phase (ANOVA: F0,24) = 63.98, P < 0.001). Yohimbine (Fig. 3) produced an overall reduction of licking in the early phase (ANOVA: F(3,24)= 3.53, P < 0.02); however, only the largest dose (7.5 ltg) had a significant effect (P = 0 . 0 1 , t-test subsequent to ANOVA). In the late phase, yohimbine produced a dose-dependent reduction of licking (ANOVA: Ft3,z4) = 9.17, P = 0.001).
Colonic temperature There was an overall reduction of colonic temperature by clonidine (ANOVA: F~3' 24~= 6.17, P = 0.003). However, subsequent data analysis revealed that only the largest dose of clonidine (0.92 ~tg) produced a significant reduction of colonic temperature (P = 0.005, t-test subsequent to ANOVA) (Fig. 4). Prazosin also induced an overall reduction of colonic temperature (ANOVA: F13.24)= 2.99,
100SO;
Assessment of sensorimotor performance and activity Assessment of the effect of the drug on sensorimotor performance and general motor activity was based on an arbitrary scale of 0--4 as follows: immobility--0, distinctly a b n o r m a l - - l , moderate--2, slightly abnormal--3 and n o n n a l ~ . Activity was similarly scored: sleep-like state-4), alert but not active---l, little activity--2, moderate activity--3, active most of the time--4.
Early phase
60" 40" "~ O
20"
,~ _c o
Late phase 300
,.I
Measurement of body temperature Deep body temperature was measured in the colon with an electronic thermometer, the probe inserted 2 cm from the anus. The temperature was determined at the beginning and the end of each testing session.
200 100
I
0.23 Statistical analysis The data were analysed by analysis of variance (ANOVA) or by Student's t-test subsequent to ANOVA, when the comparison was restricted to two means. Activity and sensorimotor performance data were analysed using Kruskal-Wallis ANOVA by ranks. Significance was accepted at the 5% level.
0.46
0.92
Clonldlne (Pg) Fig. 1. Effect of intrathecal administration of clonidine on hind-paw licking in the early (0-10 rain) and the late phase (10~0min after injection of formalin). Results are presented as means _+SEM (indicated by open symbols) (N = 7 in each group). *P < 0.05, ***P < 0.001 (Student's t-test compared to controls).
ct-Adrenoceptors and nociception Early phase
100 80 60
"G 0 m
'mOO
40 0
20 0
:E
I
I
I
7.5
15
a g~
450 ~
Late phase
o ,J
350
`m`m`m
250
0
3.75
Prazosin (pg)
Fig. 2. Effect of intrathecal administration of prazosin on hind-paw licking in the early and the late phase. Results are presented as means ___SEM (indicated by open symbols) (N = 7 in each group). *P < 0.05, ***P < 0.001 (Student's t-test compared to controls). P = 0.05) but only the largest dose (15/tg) produced hypothermia (P < 0.05, t-test). Yohimbine had no significant effect on colonic temperature (ANOVA: F(3.z4) = 1.69, P = 0.19).
1978), supporting the hypothesis that stimulation of spinal ct-adrenoceptors produces antinociception. These results not only provide more evidence that clonidine has an antinociceptive effect on phasic pain, that in the early phase of the formalin test is probably due to direct stimulation of nociceptors but also provide evidence that clonidine has antinociceptive activity in the late phase of this test. The pain in this late phase is probably due to inflammation (Hunskaar and Hole, 1987). However, a somewhat larger dose of clonidine (double the smallest dose used) was required to produce analgesia in this phase. The literature reviewed above and the results presented here support the existence of a spinal site of action for clonidine. Prazosin and yohimbine, ~t-adrenoceptor antagonists, induced analgesia in mice at very small doses. Both antagonists had antinociceptive actions in both the early and the late phase of the formalin test. The antinociceptive effect of yohimbine was weaker than that of prazosin in both phases. The present results are in agreement with earlier studies which suggested that ~t-adrenoceptor blockers, phenoxybenzamine, phentolamine and tolazoline (Cicero, Meyer and Smithloff, 1974; Elliot, Spiehler and Navarro, 1976), had antinociceptive effects in the writhing and tailflick tests. Yohimbine has been demonstrated to have antinociceptive activity in the hot-plate test (Denis, Melzack, Gutman and Boucher, 1980). The largest dose of cionidine and prazosin induced significant hypothermia. Yohimbine did not cause an
.~
Activity and motor performance
Yohimbine and prazosin had no significant effect on activity and motor performance. Clonidine, however, caused a significant reduction of activity at 0.92/~g, where the mean activity score was reduced from 3.7 in controls to 1.7 (Kruskal-Wallis ANOVA by ranks: H(3.2s)= 17.67, P < 0.001), Mann-Whitney U-test: vehicle vs 0.92/zg, P < 0.05).
369
Early phase
4O
| Late
DISCUSSION
These results support earlier investigations indicating that intrathecal administration of cionidine, an ~-agonist, has a strong antinociceptive effect (Bentley, Copeland and Starr, 1977; Milne, Cervenko, Jhamandas and Sutak, 1985; Tj~lsen et al., 1990). They also are in accord with electrophysiological results showing that clonidine reduces C-fibre evoked activity (Sullivan et al., 1987). Intrathecal administration of noradrenaline, preferentially stimulating ~-adrenergic receptors (Aran and Proudfit, 1990; De Lander and Hopkin, 1987; Kuraishi et al., 1979, 1985; Osgood, Carr, Kazianis, Kemp, Atchison and Szytelbein, 1990), has been shown to inhibit nociceptive neurones in the spinal cord (Belcher et al., 1978; Fleetwood-Walker et al., 1985; Headley et al., NP 32/4~E
400
phase `m
o
A
200
0 0
u
i
i
1.87
3.75
7.5
Yohlmblne (pg)
Fig. 3. Effect of intrathecal administration of yohimbine on hind-paw licking in the early and late phase. Results are presented as means + SEM (indicated by open symbols) (N = 7 in each group). *P < 0.05 (Student's t-test compared to controls).
T. I. KANUIet al.
370 0.4 ~
Clonidine
0
-0.8 I - 1.2
~"~ I
0
-=
I
//
0.46
I
0.92
Prazosin
1.8 P
I
0.23
0.8 A
o Q. E
I,-
0 -0.8 0
-1.8
I
I
6.75
/I/
I
7.5
15
1.8 --~ Yohimblne .0 ¢: 0 0 0
&
0.8
0
1.87
3.75
7.5
Y o h i m b l n e (pg)
Fig. 4. Effect of intrathecal administration of clonidine, prazosin and yohimbine on colonic temperature, given as difference in temperature before injection of formalin and after the end of testing. Results are presented as means + SEM (indicated by open symbols) (N = 7 in each group). *P < 0.05 (Student's t-test compared to controls).
alteration of colonic temperature, suggesting that spinal ~2-adrenoceptors do not play an important role in thermoregulation. Only the largest dose of clonidine significantly reduced activity and motor performance, while yohimbine and prazosin had no effect. Preliminary experiments indicated that larger doses of all the three drugs induced sensorimotor deficits and muscle flaccidity, in agreement with an earlier investigation (Milne et al., 1985). Recently, it was observed that a reduced skin temperature may reduce the licking response in the late phase in the formalin test (Rosland, 1991), probably due to reduced inflammation. The reduced licking response was not observed in the early phase and in the late phase, the maximum licking response was reduced only at ambient temperatures lower than approximately 23°C. In the present study, a reduced licking response was observed in the early phase of the test, as well as for doses that did not significantly affect body temperature. Although it cannot be ex-
cluded that reduced skin temperature partly contributed to the reduction in the licking response, it seems that such a mechanism cannot explain the results. In the experiments described here, the preferential ~2-agonist clonidine and the ~rantagonist prazosin produced similar effects, reducing the nociceptive response in the formalin test. The antinociceptive effect of the ~2-antagonist yohimbine was considerably less pronounced. The results thus fit the hypothesis that both subclasses of ~-adrenergic receptors may be involved in nociception, mediating opposite effects. This would form a parallel to the findings that ~1- and ~2-adrenoceptors seem to mediate facilitation and attenuation of the motor responses in the spinal cord, respectively (Tanabe, Ono and Fukuda, 1990). However, in the constant temperature hot plate test, no antinociceptive effect of intrathecal injection of prazosin was found (Takano and Yaksh, 1992). When administered intraperitoneally, prazosin was ineffective in the formalin test in the rat (Tasker, Connell and Yole, 1992). In both studies, prazosin blocked the antinociceptive effect of ~-agonists (ST91 and methoxamine, respectively). Hence, the picture is very complex, and the existing knowledge is not sufficient to build a simple model for the role of the ~-adrenergic receptors in spinal nociceptive processing. It has to be discussed in terms of pre- and postsynaptic effects on the cells in the system. From the present data, it seems that the antinociceptive effects observed in the formalin test in mice are not seriously confounded by effects of motor or autonomic functions. Patch-clamp techniques have indicated that ~rreceptors are located postsynaptically and have depolarizing functions, while ~2-receptors have hyperpolarizing properties but may be located both pre- and postsynaptically (Nicoll, Malenka and Kauer, 1990). A hyperpolarization due to stimulation of postsynaptic ~2-receptors is a possible model for the observed antinociception induced by clonidine. The mechanism of the antinociceptive effect of ~antagonists in tonic pain suggested by the present results is not clear and further research is required for its elucidation. In conclusion, the study shows that clonidine, administered intrathecally, also has an antinociceptive effect in the formalin test. The ~-antagonist prazosin also showed a potent antinociceptive effect. No indication of a tonic antinociceptive activity of the descending noradrenergic systems was observed. Acknowledgements--T. I. Kanui was on leave of absence
from the University of Nairobi, Kenya and would like to thank the Vice-Chancellorfor granting leave and NORAD for financial support.
REFERENCES Aran S. and Proudfit H. K. (1990) Antinociception produced by interaction between intrathecally administered
~-Adrenoceptors and nociception adenosine agonists and norepinephrine. Brain Res. 513: 255-263. Archer T., Danysz W., Johnson G., Minor G. and Post C. (1986) 5-MethoxyoN-N, N-dimethyltryptamine induced analgesia is blocked by alpha-adrenoceptor antagonists in rats. Br. J. Pharmac. 89: 293-298. Belcher G., Ryall R. W. and Schaffner R. (1978) The differential effects of 5-hydroxytryptamine, noradrenaline and raphe stimulation on nociceptive and non-nociceptive dorsal horn interneurones in the cat. Brain Res. 151: 307-321. Bentley G. A., Copeland I. W. and Starr J. (1977) The actions of some ~-adrenoceptor agonists and antagonists in an antinociceptive test in mice. Clin. Exp. Pharmac. Physiol. 4: 405-419. Berge O.-G., Garcia-Cabrera I. and Hole K. (1988) Response latencies in the tail-flick test depend on tail skin temperature. Neurosci. Lett. 86: 284-288. Cicero T. J., Meyer E. R. and Smithloff B. R. (1974) Alpha adrenergic blocking agents: Antinociceptive activity and enhancement of morphine-induced analgesia. J. Pharmac. Exp. Ther. 189:72 82. De Lander G. E. and Hopkin C. J. (1987) Interdependence of spinal adenosinergic, serotonergic and noradrenergic systems mediating antinociception. Neuropharmacology 26: 1791-1794. Dennis S. G., Melzack R., Gutman S. and Boucher F. (1980) Pain modulation by adrenergic agents and morphine as measured by three pain tests. Life Sci. 26: 1247-1259. Eide P. K. and Tjolsen A. (1988) Effects of serotonin receptor antagonists and agonists on the tail-flick response in mice involve altered tail-skin temperature. Neuropharmacology 27: 889-893. Elliott H. W., Spiehler V. and Navarro G. (1976) Effects of naloxone on antinociceptive activity of phenoxybenzamine. Life Sci. 19: 1637-1644. Fleetwood-Walker S. M., Mitchell R., Hope P. J., Molony V. and Iggo A. (1985) An alfa(2)-receptor mediates the selective inhibition by noradrenaline of nociceptive responses of identified dorsal horn neurones. Brain Res. 334: 243-254. Headley P. M., Duggan A. W. and Griersmith B. T. (1978) Selective reduction by noradrenaline and 5-hydroxytryptamine of nociceptive responses of cat dorsal horn neurones. Brain Res. 145: 185-189. Howe J. R., Wang J.-Y. and Yaksh T. L. (1983) Selective antagonism of the antinociceptive effect of intrathecally applied alpha-adrenergic agonists by intrathecal prazosin and intrathecal yohimbine. J. Pharmac. Exp. Ther. 224: 552-558. Hunskaar S. and Hole K. (1987) The formalin test in mice, dissociation between inflammatory and non-inflammatory pain. Pain 30:103-114. Hylden J. L. K. and Wilcox G. L. (1980) Intrathecal morphine in mice: a new technique. Eur. J. Pharmac. 67: 313-316. Jones S. L. and Gebhart G. F. (1986) Characterisation of coeruleospinal inhibition of the tail-flick reflex in the rat: mediation by spinal alpha-adrenoceptors. Brain Res. 364: 315-330.
371
Kuraishi Y., Harada Y. and Takagi H. (1979) Noradrenaline regulation of pain transmission in the spinal cord mediated by alpha-adrenoceptors. Brain Res. 174: 333-336. Kuraishi Y., Hirota N., Satoh M. and Takagi H. (1985) Antinociceptive effects of intrathecal opioids, noradrenaline and serotonin in rats: mechanical and thermal algesic tests. Brain Res. 326: 168-171. Milne B., Cervenko F. W., Jhamandas K. and Sutak M. (1985) Intrathecal clonidine: analgesia and effect on opiate withdrawal in the rat. Anaesthesiology 62: 34-38. Nicoll R. A., Malenka R. C. and Kauer J. A. (1990) Functional comparison of neurotransmitter receptor subtypes in mammalian central nervous system. Physiol. Rev. 70: 513-565. Proudfit H. K. and Hammond D. L. (1981) Alterations in nociceptive threshold and morphine-induced analgesia produced by intrathecally administered amine antagonists. Brain Res. 218: 393-399. Osgood P. F., Cart D. B., Kazianis A., Kemp J. W., Atchison N. E. and Szyfelbein S. K. (1990) Antinociception in the rat induced by a cold environment. Brain Res. 507:11-16. Reddy S. V. R. and Yaksh T. L. (1980) Spinal noradrenergic terminal system mediates antinociception. Brain Res. 189: 391-401. Rosland J. H. (1991) The formalin test in mice: the influence of ambient temperature. Pain 45:211-216. Sagen J. and Proudfit H. K. (1984) Effects of intrathecally administered noradrenergic antagonists on nociception in the rat. Brain Res. 310: 295-301. Sagen J. and Proudfit H. K. (1985) Evidence for pain modulation by pre- and postsynaptic noreadrenergic receptors in the medulla oblongata. Brain Res. 331: 285-293. Sugimoto M., Kuraishi Y., Satoh M. and Takagi H. (1986) Involvement of medullary opioid-peptidergic and spinal noradrenergic systems in the regulation of formalin-induced persistent pain. Neuropharmacology 25: 481-485. Sullivan A. F., Dashwood M. R. and Dickenson A. H. (1987) ~t2-Adrenoceptor modulation of nociception in rat spinal cord: location, effects and interactions with morphine. Eur. J. Pharmac. 138: 169-177. Takano Y. and Yaksh T. L. (1992) Characterization of the pharmacology of intrathecally administered alpha-2 agonists and antagonists in rats. J. Pharmac. Exp. Ther. 261: 764-772. Tanabe M., Ono H. and Fukuda H. (1990) Spinal alphasand alpha2-adrenoceptors mediate facilitation and inhibition of spinal motor transmission, respectively. Jap. J. Pharmac. 54: 69-77. Tasker R. A. R., Connell B. J. and Yole M. J. (1992) Systemic injections of alpha-1 adrenergic agonists produce antinociception in the formalin test. Pain 49: 383-391. Tj~lsen A., Lund A. and Hole K. (1990) The role of descending noradrenergic systems in regulation of nociception: the effects of intrathecally administered ~adrenoceptor antagonists and clonidine. Pain 43: 113-120.