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Formalin-induced nociceptive responses in diabetic mice
Neuroscience Letters, 149 (1993) 161-164 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940193/$ 06.00
161
NSL 09227
Formalin-induced nociceptive responses in diabetic mice Junzo Kamei, Hideki Hitosugi and Yutaka K a s u y a Department of Pharmacology, Faculty of Pharmaceutical Sciences, Hoshi University, Tokyo (Japan) (Received 18 August 1992; Revised version received 14 October 1992; Accepted 15 October 1992)
Key words: Diabetes; Nociception; Formalin; Substance P; Naltrindole; Spantide In non-diabetic mice, s.c. injection of formalin to the hindpaw had a biphasic effect: an immediate nociceptive response (first-phase) followed by a tonic response (second-phase). However, only the immediate nociceptive response was observed in diabetic mice. The duration of the first-phase response was significantly longer in diabetic mice than in non-diabetic mice. In diabetic mice, when spantide, an antagonist of substance P, reduced the duration of the nociceptive response in the first-phase to the levels that were observed in non-diabetic mice, the second-phase response appeared. The second phase also became apparent in diabetic mice after pretreatment with naltrindole (3 mg/kg), an antagonist of ~-opioid receptors. These results suggest that a negative control system, which is mediated by ~-opioid receptors and links substance P with somatostatin-mediated nociceptive transmission, may inhibit the formalin-induced second-phase of the nociceptive response in diabetic mice.
The formalin test has been widely accepted as a model of chemogenic pain [2, 4, 15, 16, 21]. Injection of diluted formalin into an animal's hindpaw produces a biphasic, i.e., early (first-phase) and late (second-phase), nociceptive response [2, 4, 15, 16, 21]. Kuraishi et al. [13] and Kantner et al. [12] showed that substance P (SP) and somatostatin (SST) were released from the dorsal horn upon injection of formalin into the hindpaw. Furthermore, Ohkubo et al. [16] suggested that SP and SST participate in the first and second phases of the formalininduced nociceptive response, respectively. We previously suggested that diabetes significantly enhances the nociceptive neurotransmission involving SP, but not that involving SST, in the spinal cord [10, 11]. To our knowledge, no study has been conducted in the mouse to examine the effect of diabetes in this biphasic nociceptive response. On the other hand, we demonstrated that the normally redundant ~-opioid receptor-mediated endogenous antinociceptive system is enhanced in diabetic mice [6-8]. This ~-opioid receptor-mediated endogenous antinociceptive system may have a modulatory effect on the formalin-induced biphasic antinociceptive response in diabetic mice. Thus, in the present study, we investigated the effect of diabetes on formalin-induced biphasic nociceptive response and its modulation by 8-opioid receptors. Correspondence: J. Kamei, Department of Pharmacology, Faculty of Pharmaceutical Sciences, Hoshi University, 4-41, Ebara 2-chome, Shinagawa-ku, Tokyo 142, Japan. Fax: (81) (3) 3787-0036.
Male ICR mice (Tokyo Animal Laboratory Inc., Tokyo, Japan), weighing about 20 g at the beginning of the experiments, were used. They had free access to solid food (MF; Oriental Yeast Co., Tokyo, Japan) and water in an animal room which was maintained at 22 + I°C with a 12-h light-dark cycle. Animals were rendered diabetic by an injection of streptozotocin (STZ; 200 mg/kg i.v.) prepared in 0.1 N citrate buffer at pH 4.5. Agematched control mice were injected with the vehicle alone. The experiments were conducted 2 weeks after injection of vehicle or STZ. Mice with serum glucose levels above 400 mg/dl were considered diabetic. The experiment was performed by the method described by Shibata et al. [20]. Each mouse was acclimated to an acrylic observation chamber (32 x 23 x 17 cm 3) for at least 5 min before the injection of formalin. Twentyfive fll of a 0.5% solution of formalin in 0.9% saline were injected into the dorsal surface of the right hindpaw. Immediately after the injection, each animal was returned to the chamber and its nociceptive responses were recorded for 30 min. The accumulated response time (s), namely the duration of biting and licking of the injected paw, was measured for each 5-min block. Data are expressed as means + S.E. Data were subjected to an analysis of variance and Newman-Keul's test for multiple comparisons. The following drugs were used: [D-Arg~, D-Yrp 7'9, LeuU]-substance P (spantide; Peptide Institute, Osaka, Japan); naltrindole hydrochloride (Research Biochemical Inc., Natick, MA, USA). Both drugs were dissolved
162
in 0.9% saline and injected either i.t. or s.c. The i.t. injection was performed by the method described by Hylden and Wilcox [5]. Spantide and naltrindole were administered 10 min and 30 min, respectively, before the injection of formalin. The time courses of the nociceptive responses are shown in Fig. 1A. In non-diabetic mice, s.c. injection of 0.5% formalin into the hindpaw caused an acute, immediate nociceptive response, namely biting and licking, which lasted about 5 min (first-phase response). The second-phase response then began and lasted about 20 min. In diabetic mice, although s.c. injection of formalin into the hindpaw caused the first-phase of nociceptive response, the second phase of the nociceptive response was barely observed (Fig. 1A,B). However, the duration of the nociceptive response in the first phase was significantly longer in diabetic mice than in non-diabetic mice (Fig. 1B). Intrathecal injection of spantide (1 and 3/lg), an antagonist of SP receptors [3, 5, 18, 19], dose-dependently reduced the first phase of the formalin-induced nocicep-
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Fig. 2. Effects of i.t. administration of spantide (1 ,ug, hatched column: 3 ,ug, dotted column) on the formalin-induced two-phase nociceptive response in diabetic mice (A) and non-diabetic mice (B). Data are expressed as the total duration of response during the first (0-5 min) and second (10-30 min) phases. Each column represents the mean + S.E. (n = 6). "P < 0.05, when compared to the saline-treated group {open column).
A
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Fig. 1. A: time course of the two-phase nociceptive response induced by 0.5% formalin injected into the hindpaw of non-diabetic (©) and diabetic (e) mice. Each point represents the mean _+ S.E. (n = 6) of the duration o f licking and biting responses per 5-rain period. B: duration of nociceptive responses induced by an injection of formalin in nondiabetic (open column) and diabetic (hatched column) mice. Data are expressed as the total duration of responses during the first (0-5 min) and the second (10 30 min) phases. Each column represents the mean + S.E. (n = 6). *P < 0.05, when compared to the value for non-diabetic mice.
tive response in non-diabetic mice (Fig. 2B). In fact, the effect of 3/lg of spantide was significantly different from that of saline (Fig. 2B). However, spantide had no significant effect on the second-phase in non-diabetic mice (Fig. 2B). Spantide (1 and 3 ~tg, i.t.) also dose-dependently reduced the first-phase in diabetic mice (Fig. 2A). In contrast to its effect in non-diabetic mice, the second-phase of the formalin-induced nociceptive response in diabetic mice was significantly increased when spantide (1 and 3 /,tg) was injected i.t. I0 min before formalin injection. Naltrindole (3 mg/kg, s.c.) had no significant effect on the first phase, but significantly increased the second phase of the formalin-induced nociceptive response in diabetic mice (Fig. 3A). In non-diabetic mice, naltrindole (3 mg/kg, s.c.) had no significant effect on either the first or second phase (Fig. 3B). It has been suggested that SP and SST are involved in nociceptive transmission during the first and second phases, respectively, of the formalin-induced nociceptive response in mice [16]. The present finding that the duration of the first phase of the formalin-induced nociceptive response was significantly greater in diabetic mice than in non-diabetic mice, supports our previous sugges-
163
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200-
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first S
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--O
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_1_ 100--
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firs
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phase
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Fig. 3 Effects of s.c. administration of naltrindole (1 mg/kg, hatched column; 3 mg/kg, dotted column) on the formalin-induced two-phase nociceptive response in diabetic mice (A) and non-diabetic mice (B). Data are expressed as the total duration of responses during the first (0-5 min) and second (1~30 min) phases. Each column represents the mean + S.E. (n = 6). "P < 0.05, when compared to the saline-treated group (open column).
tion that STZ-induced diabetes selectively enhances the neurotransmission involving SP in the spinal cord [10, 111. An unexpected, but important, finding in the present study is that s.c. injection of formalin into the hindpaw induced only the first-phase of the nociceptive response in diabetic mice. Furthermore, the second-phase of the nociceptive response appeared in diabetic mice when spantide, an antagonist of SP, was administered i.t. 10 min before the injection of formalin. It is well established that exposure to a stressful experience induces a pronounced antinociception in animals [22, 23]. Previously, we demonstrated that diabetic mice displayed greater swimming stress-induced analgesia, which is primarily mediated by ~-opioid receptors, than non-diabetic mice [8]. Furthermore, the normally redundant 'backup' antinociceptive system, which is also mediated by 6opioid receptors, is enhanced in diabetic mice [6-8]. It would be reasonable to suggest that excessive nociceptive transmission in diabetic mice during the first phase of the response, which might be mediated by SP [16], may be recognized as a stressful experience and, therefore, activate the ~-opioid receptor-mediated endogenous antinociceptive systems. Thus, these ~-opioid receptor-medi-
ated endogenous antinociceptive systems might inhibit the formalin-induced second-phase nociceptive response, which is mediated predominantly by SST, in diabetic mice. This hypothesis is strongly supported by the present result that the second phase of the response appeared in diabetic mice when they had been pretreated with naltrindole, an antagonist of 8-opioid receptors. However, in the present study, the second phase of the response became evident when diabetic mice were pretreated with spantide at a dose of 1/ag, which had no effect on the first-phase response. Furthermore, in diabetic mice, naltrindole significantly increased the duration of the second-phase nociceptive response, whereas it had no significant effects on the duration of the firstphase response. Some reports have suggested that i.t. injection of SP produces the naloxone-sensitive increase in the threshold for pain perception in the response to noxious thermal stimuli, which is mediated mainly by SST [1, 17]. A negative control system may exist which links SP with SST-mediated nociceptive transmission and which probably involves 8-opioid receptors. Our previous reports have shown that diabetic animals exhibited supersensitivity to SP [9] and 8-opioid agonist [7]. Thus, this negative control system may be enhanced in diabetic animals, thereby inhibiting the second phase of the formalin-induced nociceptive response. We would like to thank Ms. Mari Kondoh and Ms. Nana Suzuki for their invaluable technical assistance. 1 Doi, T. and Jurna, I., Intrathecal substance P depresses the tail-flick response antagonism by naloxone, Naunyn-Schmiedeberg's Arch. Pharmacol., 317 (1981) 135 139. 2 Dubuisson, D. and Dennis, S.G., The formalin test: a quantitative study of the analgesic effects of morphine, meperidine, and brain stem stimulation in rats and cats, Pain, 4 (1977) 161-174. 3 Folkers, K., Hakanson, R., Horig, J., Jie-Cheng, X. and Leander, S., Biological evaluation of substance P antagonists, Br. J. Pharmacol., 83 (1984) 449-456. 4 Hunskaar, S., Fasmer, O.B. and Hole, K., Formalin test in mice, a useful technique for evaluating mild analgesia, J. Neurosci. Methods, 14 (1985) 6%76. 5 Hylden, J.L.K. and Wilcox, G.L., Intrathecal morphine in mice: a new technique, Eur. J. Pharmacol., 167 (1980) 313 -316. 6 Kamei, J., Kawashima, N. and Kasuya, Y., Naloxone-induced analgesia in diabetic mice, Eur. J. Pharmacol., 210 (1992) 339-341. 7 Kamei, J., Kawashima, N. and Kasuya, Y., Paradoxical analgesia produced by naloxone in diabetic mice is attributable to supersensitivity of ~;-opioid receptors, Brain Res., in press. 8 Kamei, J., Kawashima, N., Ohhashi, Y. and Kasuya, Y., Effects of diabetes on stress-induced analgesia in mice, Brain Res., 580 (1992) 180-184. 9 Kamei, J., Ogawa, M. and Kasuya, Y., Development of supersensitivity to substance P in the spinal cord of the streptozotocin-induced diabetic mice, Pharmacol. Biochem. Behav., 35 (1990)473-475. 10 Kamei, J., Ogawa, M., Ohhashi, Y. and Kasuya, Y., Alterations in the potassium-evoked release of substance P from the spinal cord of
164
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12
13
14
15
16
streptozotocin-induced diabetic rats in vitro, Gen. Pharmacol., 22 (1991) 1093-1096. Kamei, J., Ohhashi, Y., Aoki, T. and Kasuya, Y., Streptozotocininduced diabetes in mice reduces the nociceptive threshold, as recognized after application of noxious mechanical stimuli but not of thermal stimuli, Pharmacol. Biochem. Behav.. 39 (1991 ) 541-544. Kantner, R.M., Goldenstein, B.D. and Kirby, M.L., Regulatory mechanisms for substance P in the dorsal horn during a nociceptive stimulus: axoplasmic transport vs electrical activity, Brain Res., 385 (1986) 282-290. Kuraishi, Y., Hirota, N., Sato, Y., Hanashima, N., Takagi, H. and Satoh, M., Stimulus specificity of peripherally evoked substance P release from the rabbit dorsal horn in situ, Neuroscience, 30 (1989) 241 250. Mjellem-Joly, N., Lund, A., Berg, O.-G. and Hole, K., Potentiation of a behavioural response in mice by spinal coadministration of substance P and excitatory amino acid agonists, Neurosci. Lett., 133(1991)121 124. Murray, C.W., Porreca, F. and Cowan, A., Methodological refinements to the mouse paw formalin test, J. Pharmacol. Methods, 20 (1988) 175-186. Ohkubo, T., Shibata, M., Takahashi, H. and Inoki, R., Roles of substance P and somatostatin on transmission of nociceptive information induced by formalin in spinal cord, J. Pharmacol. Exp. Ther., 252 (1990) 1261 1268.
17 Rodriguez, R.E. and Rodriguez, F.D., Spinal and supraspinat substance P antinociception: synergistic interaction. In J.L. Henry, R. Couture, A.C. Cuello, G. Pelletier, R. Ouirion and D. Regoli (Eds.), Substance P and Neurokinins, Springer, New York, 1987, pp. 331 333. 18 Sakurada, T., Yamada, T., Sakurada, S., Kisara, K. and Ohba, M., Substance P analogues containing o-histidine antagonize the behavioral effect of intrathecally co-administered substance P in mice, Eur. J. Pharmacol., 174 (1989) 153 160. 19 Sakurada, T., Yamada, T., Tan-no, K., Manome, Y., Sakurada, S., Kisara, K. and Ohba, M., Differential effects of substance P analogs on neurokinin 1 receptor agonists in the mouse spinal cord, J. Pharmacol. Exp. Ther., 259 (1991) 205 210. 20 Shibata, M., Ohkubo, T.. Takahashi, H. and Inoki, R., Modified formalin test: Characteristic biphasic pain response, Pain, 38 (1989) 347 352. 21 Sugimoto, M., Kuraishi, Y., Satoh, M. and Takagi, H., Involvement of medullary opioid-peptidergic and spinal noradrenergic systems in the regulation of formalin-induced persistent pain, Neuropharmacology, 25 (1986) 481485. 22 Terman, G.W., Shavit, Y., Lewis, J.W., Cannon, J.T. and Liebeskind, J.C., Intrinsic mechanisms of pain inhibition: activation by stress, Science, 226 (1984) 1270 1277. 23 Watkins, L.R. and Mayer, D.J., The organization of endogenous opiate and nonopiate pain control system, Science, 216 (1982) 1185 t192.
161
NSL 09227
Formalin-induced nociceptive responses in diabetic mice Junzo Kamei, Hideki Hitosugi and Yutaka K a s u y a Department of Pharmacology, Faculty of Pharmaceutical Sciences, Hoshi University, Tokyo (Japan) (Received 18 August 1992; Revised version received 14 October 1992; Accepted 15 October 1992)
Key words: Diabetes; Nociception; Formalin; Substance P; Naltrindole; Spantide In non-diabetic mice, s.c. injection of formalin to the hindpaw had a biphasic effect: an immediate nociceptive response (first-phase) followed by a tonic response (second-phase). However, only the immediate nociceptive response was observed in diabetic mice. The duration of the first-phase response was significantly longer in diabetic mice than in non-diabetic mice. In diabetic mice, when spantide, an antagonist of substance P, reduced the duration of the nociceptive response in the first-phase to the levels that were observed in non-diabetic mice, the second-phase response appeared. The second phase also became apparent in diabetic mice after pretreatment with naltrindole (3 mg/kg), an antagonist of ~-opioid receptors. These results suggest that a negative control system, which is mediated by ~-opioid receptors and links substance P with somatostatin-mediated nociceptive transmission, may inhibit the formalin-induced second-phase of the nociceptive response in diabetic mice.
The formalin test has been widely accepted as a model of chemogenic pain [2, 4, 15, 16, 21]. Injection of diluted formalin into an animal's hindpaw produces a biphasic, i.e., early (first-phase) and late (second-phase), nociceptive response [2, 4, 15, 16, 21]. Kuraishi et al. [13] and Kantner et al. [12] showed that substance P (SP) and somatostatin (SST) were released from the dorsal horn upon injection of formalin into the hindpaw. Furthermore, Ohkubo et al. [16] suggested that SP and SST participate in the first and second phases of the formalininduced nociceptive response, respectively. We previously suggested that diabetes significantly enhances the nociceptive neurotransmission involving SP, but not that involving SST, in the spinal cord [10, 11]. To our knowledge, no study has been conducted in the mouse to examine the effect of diabetes in this biphasic nociceptive response. On the other hand, we demonstrated that the normally redundant ~-opioid receptor-mediated endogenous antinociceptive system is enhanced in diabetic mice [6-8]. This ~-opioid receptor-mediated endogenous antinociceptive system may have a modulatory effect on the formalin-induced biphasic antinociceptive response in diabetic mice. Thus, in the present study, we investigated the effect of diabetes on formalin-induced biphasic nociceptive response and its modulation by 8-opioid receptors. Correspondence: J. Kamei, Department of Pharmacology, Faculty of Pharmaceutical Sciences, Hoshi University, 4-41, Ebara 2-chome, Shinagawa-ku, Tokyo 142, Japan. Fax: (81) (3) 3787-0036.
Male ICR mice (Tokyo Animal Laboratory Inc., Tokyo, Japan), weighing about 20 g at the beginning of the experiments, were used. They had free access to solid food (MF; Oriental Yeast Co., Tokyo, Japan) and water in an animal room which was maintained at 22 + I°C with a 12-h light-dark cycle. Animals were rendered diabetic by an injection of streptozotocin (STZ; 200 mg/kg i.v.) prepared in 0.1 N citrate buffer at pH 4.5. Agematched control mice were injected with the vehicle alone. The experiments were conducted 2 weeks after injection of vehicle or STZ. Mice with serum glucose levels above 400 mg/dl were considered diabetic. The experiment was performed by the method described by Shibata et al. [20]. Each mouse was acclimated to an acrylic observation chamber (32 x 23 x 17 cm 3) for at least 5 min before the injection of formalin. Twentyfive fll of a 0.5% solution of formalin in 0.9% saline were injected into the dorsal surface of the right hindpaw. Immediately after the injection, each animal was returned to the chamber and its nociceptive responses were recorded for 30 min. The accumulated response time (s), namely the duration of biting and licking of the injected paw, was measured for each 5-min block. Data are expressed as means + S.E. Data were subjected to an analysis of variance and Newman-Keul's test for multiple comparisons. The following drugs were used: [D-Arg~, D-Yrp 7'9, LeuU]-substance P (spantide; Peptide Institute, Osaka, Japan); naltrindole hydrochloride (Research Biochemical Inc., Natick, MA, USA). Both drugs were dissolved
162
in 0.9% saline and injected either i.t. or s.c. The i.t. injection was performed by the method described by Hylden and Wilcox [5]. Spantide and naltrindole were administered 10 min and 30 min, respectively, before the injection of formalin. The time courses of the nociceptive responses are shown in Fig. 1A. In non-diabetic mice, s.c. injection of 0.5% formalin into the hindpaw caused an acute, immediate nociceptive response, namely biting and licking, which lasted about 5 min (first-phase response). The second-phase response then began and lasted about 20 min. In diabetic mice, although s.c. injection of formalin into the hindpaw caused the first-phase of nociceptive response, the second phase of the nociceptive response was barely observed (Fig. 1A,B). However, the duration of the nociceptive response in the first phase was significantly longer in diabetic mice than in non-diabetic mice (Fig. 1B). Intrathecal injection of spantide (1 and 3/lg), an antagonist of SP receptors [3, 5, 18, 19], dose-dependently reduced the first phase of the formalin-induced nocicep-
S
S
-200
'~ 100--
-100
t0
C 0
=
a
O-
first
phase
second phase
-0
S
S
200-
--200
m = o
[ii4 -100
I
c
.2
7 a O-
J
first
phase
second
)hase
-O
Fig. 2. Effects of i.t. administration of spantide (1 ,ug, hatched column: 3 ,ug, dotted column) on the formalin-induced two-phase nociceptive response in diabetic mice (A) and non-diabetic mice (B). Data are expressed as the total duration of response during the first (0-5 min) and second (10-30 min) phases. Each column represents the mean + S.E. (n = 6). "P < 0.05, when compared to the saline-treated group {open column).
A
130-.=_ E
S
200--
|Ioo-
In
"~30_ a
d O-
t I 0--5 Formalin s
3O0-
I I I I l 5 - 1 0 10-15 15-300 2 0 - 2 5 25-30min s
B
-3O0
J
-130
-~ 130-
d
o, ~-~!~
first phase
~* -o
second phase
Fig. 1. A: time course of the two-phase nociceptive response induced by 0.5% formalin injected into the hindpaw of non-diabetic (©) and diabetic (e) mice. Each point represents the mean _+ S.E. (n = 6) of the duration o f licking and biting responses per 5-rain period. B: duration of nociceptive responses induced by an injection of formalin in nondiabetic (open column) and diabetic (hatched column) mice. Data are expressed as the total duration of responses during the first (0-5 min) and the second (10 30 min) phases. Each column represents the mean + S.E. (n = 6). *P < 0.05, when compared to the value for non-diabetic mice.
tive response in non-diabetic mice (Fig. 2B). In fact, the effect of 3/lg of spantide was significantly different from that of saline (Fig. 2B). However, spantide had no significant effect on the second-phase in non-diabetic mice (Fig. 2B). Spantide (1 and 3 ~tg, i.t.) also dose-dependently reduced the first-phase in diabetic mice (Fig. 2A). In contrast to its effect in non-diabetic mice, the second-phase of the formalin-induced nociceptive response in diabetic mice was significantly increased when spantide (1 and 3 /,tg) was injected i.t. I0 min before formalin injection. Naltrindole (3 mg/kg, s.c.) had no significant effect on the first phase, but significantly increased the second phase of the formalin-induced nociceptive response in diabetic mice (Fig. 3A). In non-diabetic mice, naltrindole (3 mg/kg, s.c.) had no significant effect on either the first or second phase (Fig. 3B). It has been suggested that SP and SST are involved in nociceptive transmission during the first and second phases, respectively, of the formalin-induced nociceptive response in mice [16]. The present finding that the duration of the first phase of the formalin-induced nociceptive response was significantly greater in diabetic mice than in non-diabetic mice, supports our previous sugges-
163
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Fig. 3 Effects of s.c. administration of naltrindole (1 mg/kg, hatched column; 3 mg/kg, dotted column) on the formalin-induced two-phase nociceptive response in diabetic mice (A) and non-diabetic mice (B). Data are expressed as the total duration of responses during the first (0-5 min) and second (1~30 min) phases. Each column represents the mean + S.E. (n = 6). "P < 0.05, when compared to the saline-treated group (open column).
tion that STZ-induced diabetes selectively enhances the neurotransmission involving SP in the spinal cord [10, 111. An unexpected, but important, finding in the present study is that s.c. injection of formalin into the hindpaw induced only the first-phase of the nociceptive response in diabetic mice. Furthermore, the second-phase of the nociceptive response appeared in diabetic mice when spantide, an antagonist of SP, was administered i.t. 10 min before the injection of formalin. It is well established that exposure to a stressful experience induces a pronounced antinociception in animals [22, 23]. Previously, we demonstrated that diabetic mice displayed greater swimming stress-induced analgesia, which is primarily mediated by ~-opioid receptors, than non-diabetic mice [8]. Furthermore, the normally redundant 'backup' antinociceptive system, which is also mediated by 6opioid receptors, is enhanced in diabetic mice [6-8]. It would be reasonable to suggest that excessive nociceptive transmission in diabetic mice during the first phase of the response, which might be mediated by SP [16], may be recognized as a stressful experience and, therefore, activate the ~-opioid receptor-mediated endogenous antinociceptive systems. Thus, these ~-opioid receptor-medi-
ated endogenous antinociceptive systems might inhibit the formalin-induced second-phase nociceptive response, which is mediated predominantly by SST, in diabetic mice. This hypothesis is strongly supported by the present result that the second phase of the response appeared in diabetic mice when they had been pretreated with naltrindole, an antagonist of 8-opioid receptors. However, in the present study, the second phase of the response became evident when diabetic mice were pretreated with spantide at a dose of 1/ag, which had no effect on the first-phase response. Furthermore, in diabetic mice, naltrindole significantly increased the duration of the second-phase nociceptive response, whereas it had no significant effects on the duration of the firstphase response. Some reports have suggested that i.t. injection of SP produces the naloxone-sensitive increase in the threshold for pain perception in the response to noxious thermal stimuli, which is mediated mainly by SST [1, 17]. A negative control system may exist which links SP with SST-mediated nociceptive transmission and which probably involves 8-opioid receptors. Our previous reports have shown that diabetic animals exhibited supersensitivity to SP [9] and 8-opioid agonist [7]. Thus, this negative control system may be enhanced in diabetic animals, thereby inhibiting the second phase of the formalin-induced nociceptive response. We would like to thank Ms. Mari Kondoh and Ms. Nana Suzuki for their invaluable technical assistance. 1 Doi, T. and Jurna, I., Intrathecal substance P depresses the tail-flick response antagonism by naloxone, Naunyn-Schmiedeberg's Arch. Pharmacol., 317 (1981) 135 139. 2 Dubuisson, D. and Dennis, S.G., The formalin test: a quantitative study of the analgesic effects of morphine, meperidine, and brain stem stimulation in rats and cats, Pain, 4 (1977) 161-174. 3 Folkers, K., Hakanson, R., Horig, J., Jie-Cheng, X. and Leander, S., Biological evaluation of substance P antagonists, Br. J. Pharmacol., 83 (1984) 449-456. 4 Hunskaar, S., Fasmer, O.B. and Hole, K., Formalin test in mice, a useful technique for evaluating mild analgesia, J. Neurosci. Methods, 14 (1985) 6%76. 5 Hylden, J.L.K. and Wilcox, G.L., Intrathecal morphine in mice: a new technique, Eur. J. Pharmacol., 167 (1980) 313 -316. 6 Kamei, J., Kawashima, N. and Kasuya, Y., Naloxone-induced analgesia in diabetic mice, Eur. J. Pharmacol., 210 (1992) 339-341. 7 Kamei, J., Kawashima, N. and Kasuya, Y., Paradoxical analgesia produced by naloxone in diabetic mice is attributable to supersensitivity of ~;-opioid receptors, Brain Res., in press. 8 Kamei, J., Kawashima, N., Ohhashi, Y. and Kasuya, Y., Effects of diabetes on stress-induced analgesia in mice, Brain Res., 580 (1992) 180-184. 9 Kamei, J., Ogawa, M. and Kasuya, Y., Development of supersensitivity to substance P in the spinal cord of the streptozotocin-induced diabetic mice, Pharmacol. Biochem. Behav., 35 (1990)473-475. 10 Kamei, J., Ogawa, M., Ohhashi, Y. and Kasuya, Y., Alterations in the potassium-evoked release of substance P from the spinal cord of
164
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13
14
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