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Modified formalin test: characteristic biphasic pain response
347
Pain, 38 (1989) 347-352 Elsevier
PAIN 01453
Modified formalin test: characteristic biphasic pain response Manabu
Shibata,
Tsuyako
Ohkubo,
Hiroshi
Takahashi
and Reizo Inoki
*
Department of Pharmacology, Fukuoka Dental College, 700 Ta, Sawara-ku, Fukuoka 814-01 (Japan), and * Department of Pharmacology, Faculty of Dentistry, Osaka University, Suita 565, Osuka (Japan) (Received
20 December
1988, revision
received 5 April 1989, accepted
11 April 1989)
Summary
A modified formalin test in mice was investigated. The pain response curve induced by 0.5% formalin was biphasic, having 2 peaks, from 0 to 5 min (first phase) and from 15 to 20 min (second phase). A low concentration of formalin was used, allowing the effects of weak analgesics to be detected. Centrally acting drugs such as narcotics inhibited both phases equally. Peripherally acting drugs such as aspirin, oxyphenbutazone, hydrocortisone and dexamethasone only inhibited the second phase. Aminopyrine and mefenamic acid which acted on both central and peripheral sites inhibited both phases, but the second phase was inhibited by lower doses. Thus, this method enables one to easily distinguish the site of action of analgesics. Furthermore, pain response in the first phase was inhibited by capsaicin-treated desensitization and Des-Arg9-(Leu*)-bradykinin (bradykinin inhibitor). The second phase was inhibited by compound 48/80 pretreatment, indomethacin and bradykinin inhibitor. Therefore, it is suggested that substance P and bradykinin participate in the manifestation of the first phase response, and histamine, serotonin, prostaglandin and bradykinin are involved in the second phase. These results indicate that the first and second phase responses induced by formalin have distinct characteristic properties, and it is a very useful method for examining pain, nociception and its modulation by pharmacological or other means. Key words:
Formalin
test; Analgesics;
Substance
P; Bradykinin;
Introduction The formalin test was introduced by Dubuisson and Dennis [3] and the subcutaneous injection of formalin produced a biphasic pain response in rats. They noted that this method required little or no restraint and the pain stimulus bore a resemblance to most clinical pain. Formalin is a useful tool for obtaining neurogenic inflammation [l] and continuous pain. We previously modified the formalin test in mice, using the forepaw as the site of formalin injection, and simplified the pain rating scale by means of using only licking behavior
Correspondence to: Tsuyako Ohkubo, Ph.D., Department of Pharmacology, Fukuoka Dental College, 700 Ta, Sawara-ku, Fukuoka 814-01, Japan. 0304-3959/89/$03.50
0 1989 Elsevier Science Publishers
Prostaglandin
of the injected paw as an indicator of pain response [14]. Consequently, pain assessment was very easily performed without the experimenter’s subjective assessment and skill. A low concentration of formalin was used, allowing detection of the analgesic effects of weak non-narcotic analgesics. Hunskaar et al. [5] also reported the formalin test which used the same pain rating scale and the hind paw of mice. As licking the forepaw as an indication of pain response may be confounded with the normal grooming response [2], in the present study we made use of the hind paw of mice. Formalin produced a distinct biphasic response, and it has been reported that the action of analgesics differs in the early phase and the late phase [5,6,14]. Although it is conceivable that this might be due to the differential properties of these
B.V. (Biomedical
Division)
34X
2 phase responses, little is known about the features of the formalin test as the screening method and the mechanism of manifestation of formalininduced pain. We examined the effects of many narcotic and non-narcotic analgesics on the biphasic pain response, and report here its usefulness for differentiating the site of action (central versus peripheral) of various analgesics. In addition, participation of various chemical mediators in the manifestation of formalin pain was investigated. In particular, capsaicin was used to determine the influence of C fibers and substance P in the production of formalin-induced pain response.
obtain obvious biphasic and reproducible ~c’spon~ ses. 25 1-11of 0.5% sterile formalin was administered into the right hind paw. Each aninlnl W:I\ then returned to the chamber and pain response was recorded for a period of 30 min. The summation of time (in seconds) spent in licking and biting responses of the injected paw during each 5 min block was measured as an indicator of pain response. Statistical analysis Significant differences dent’s I test.
were determined
by Stu-
Results Methods Groups of 7 male ddY mice weighing 20-25 g were used. Chemicals used were: morphine hydrochloride; meperidine hydrochloride (Tanabe, Japan), pentazocine; mefenamic acid (Sankyo, Japan), ethylketocyclazocine; ketocyclazocine (Sterling-Winthrop Res. Inst.), buprenorphine hydrochloride (Ootsuka, Japan), codeine phosphate (Shionogi, Japan), indomethacin (Sumitomo, Japan), bromelain; compound 48/80 (Sigma), aspirin (Ken-ei, Japan), hydrocortisone acetate; dexamethasone acetate (Merck-Banyu, Japan), arninopyrine (Ono, Japan), oxyphenbutazone (Fujisawa, Japan), capsaicin (Merck), Des-Arg’-(Leu’)-bradykinin (Peptide Institute, Japan). Aspirin, mefenamic acid and oxyphenbutazone were suspended in 0.5% tragacanth. Ketocyclazocine was dissolved in a small quantity of acetic acid and then diluted with saline. Capsaicin was dissolved in 50% dimethyl sulfoxide (DMSO). When not specified, other drugs were dissolved in saline and administered 30 min prior to the formalin injection. The observation chamber was a glass cylinder of diameter 20 cm on an acrylic transparent plate floor. Beneath the floor, a large mirror was mounted at a 45 o angle to allow clear observation of the paws of the animal. Each mouse was placed in the chamber more than 5 min prior to the formalin injection in order to allow it to acclimatize to the new environment; this is necessary to
Formalin-induced biphasic pain response and morphine inhibition As shown in Fig. 1 (see pain response curve of control), the subcutaneous injection of 0.5% formalin caused an acute pain response which lasted about 5 min (first phase). Subsequently, this response disappeared for about 5 min and then recurred and lasted about 20 min (second phase). The peak period of the second phase was from 15 to 20 min. When morphine (1, 3, 6 mg/kg) was administered subcutaneously 20 min prior to the formalin
-.-
‘formalin
intectlon
control morphine
(saline. s.c ) ( 1 mg/kg,
SC
min. Fig. 1. Time course of pain response induced by formalin and effect of morphine. Pain response was measured from immediately after intraplantar injection of 25 ~1 of 0.5% formalin. Ordinate (pain response): the time (in set) spent in paw kinking or biting response during each 5 min block. Each value represents the meanfS.E. (n = 7). * P < 0.05, *** P-C 0.005.
a
-o-control ( tragacanlh. -c-aspirin
-.-.-
set 120
s. ,.
( 100 mg/kg.
t ZOOmg/kg,
(300mg/kg.
PO I, ‘.
1
) )
)
90
60
a lormalln
inp?cllon
min.
0
Ob3
0.3
0.1
1
10
3b
Dose
lormalln
l”,eCm”
mm.
Fig. 2. Effects of aspirin and dexamethasone on formalin-induced pain response. Aspirin and dexamethasone were administered 30 min prior to the formalin. Each value represents the meanf S.E. (n = 7). * P < 0.05. * * P -L0.01, * * * P c 0.005.
mglkg
Fig. 3. Dose-response curves of analgesic action of morphine, pentazocine. EKC, KC, codeine, buprenorphine and meperidine in the first phase (F) and the second phase (S) of formalin-induced pain. Percentage of inhibition = [l -duration time (drug-treated)/duration time (vehicle-treated)] x 100. Each point is the mean value (n = 7).
injection, it inhibited the formalin-induced biphasic pain response dose-dependently. Six mg/kg of morphine produced almost complete analgesia. Effects of aspirin and dexamethasone on formalininduced pain Aspirin (100, 200, 300 mg/kg, p.o) inhibited the second phase response dose-dependently (Fig. 2a), but not the first phase. Similarly, subcutaneous injection of dexamethasone (0.25, 0.5, 1 mg/kg) caused an inhibition of the second phase dose-dependently (Fig. 2b). Dose-response curves of various analgesics and anti-inflammatory drugs In order to obtain dose-response curves of analgesics in both the first and second phase, percent inhibition was calculated from the values of saline-treated and drug-treated animals for each
0
i 0.3
-*--.
4--.-,_, 1
3
‘---+. 10
.&r,
F
30
KJo DOS-2
C?Qo mg,kg
Fig. 4. Dose-response curves of aminopyrine, mefenamic acid, dexamethasone, hydrocortisone, oxyphenbutasone and aspirin in the first phase (F) and the second phase (S) of formalin-induced pain. See Fig. 3 for details.
350
period of O-5 min (F) and 15-20 min (S) after formalin injection. Dose-response relationships of morphine, ethylketocyclazocine (EKC), ketocyclazocine (KC), pentazocine, buprenorphine, meperidine and codeine on the first and second phases were observed (Fig. 3). All of these drugs inhibited both phases equally and dose-dependently. Dose-response curves of aminopyrine, mefenamic acid, aspirin, oxyphenbutazone, dexamethasone and hydrocortisone are shown in Fig. 4. Aminopyrine and mefenamic acid inhibited both phases, but the second phase was inhibited by lower doses than the first phase. In the case of aspirin, oxyphenbutazone, dexamethasone and hydrocortisone, dose-response relationship was obtained only on the inhibition of the second phase, and they had no effect on the first phase pain response.
a 120.
-o-ca~sac,n-treated
go’
sc~ahc
O-5
formaIm
5-10
10-15
15-20
~n,ecf~on
20-25
25-30
min.
Fig. 6. Effects of compound 48/80 pretreatment and combination of indomethacin and bromelain on formalin-induced pain response. Bromelain and indomethacin were administered 90 and 30 min respectively before the formalin. Each value represents themean+SE.(n=7). * P~0.05, ** PiO.01, *** P
-.-solvent-treated SClatlCnerve (Stklrnoperaho” )
SK
T
nerve
i\
< 0.005.
b
(salme,
-.-control -o-
Des.Arg%(
I
PI
)
Leus )-bradykmm
-I-
5-10
IO-15
15-20
20-25
25-30
(150~mol.
!Pl )
(3OOPmol.
”
1
mm.
Fig. 5. Effects of pretreatment with capsaicin and Des-Arg’(Leu’)-bradykinin on formalin-induced pain response. Each value represents the mean& S.E. (n = 7). * * P < 0.01, * * * P
-=c 0.005.
Capsaicin treatment of sciatic nerve and effect of bradykinin inhibitor Sciatic nerves were pretreated with 1.5% capsaicin solution 12 days prior to the experiment. Control mice were treated with sham operation and vehicle (50% DMSO). In mice treated with capsaicin, only the first phase response was inhibited significantly (Fig. 5a) Des-Arg9-(Leu’)-bradykinin, a specific bradykinin inhibitor, was injected intraplantarly 2 min before formalin injection at doses of 150 and 300 pmol. This drug caused an inhibition of both phases of formalin pain (Fig. 5b). Compound 48/80 treatment and combination effect of indomethacin and bromelain Compound 48/80, a histamine and serotonin depletor, was administered by intraplantar injec-
351
tion at a dose of 1.25 pg/paw twice a day for 2 days. Twelve hours after final administration, formalin was injected into the treated paw. Compound 48/80 reduced the second phase response only (Fig. 6a). Effects of indomethacin (5 mg/kg, p.o.) and bromelain (10 mg/kg, i.v.) and a combination of these 2 drugs were observed. To study the effect of such a combination, lower doses of the 2 drugs were chosen. When given alone, indomethacin and bromelain inhibited only the second phase. The effects of combination of these drugs were greater than mere summation on the second phase pain response, especially for the periods of 10-15 min and 20-25 min.
Discussion Among the screening methods of analgesics, there are many which are available to assess analgesic action of narcotics but are unsuitable for weak analgesics such as antipyretics. This modified formalin test can be used to evaluate the analgesic effects regardless of narcotics or nonnarcotics. Antinociceptive drugs are generally classified into central or peripheral according to the site of action. Typical of the former is morphine, and that of the latter is aspirin. Lim et al. [9] reported the method which determined the site of action by means of nociception caused by intra-arterial injection of bradykinin in dogs. This method is superior in estimating the drug’s action sites; however, the technique is difficult. Assessing the effects of various analgesics using the formalin-induced biphasic response, narcotic drugs such as morphine, codeine, EKC, KC, buprenorphine, meperidine and pentazocine inhibited the pain response at the first and second phases equally. The analgesic effects of morphine on formalin-induced pain responses are in agreement with the previous studies by Hunskaar et al. [5] and Vaccarino et al. [15]. On the other hand, peripherally acting drugs such as aspirin, oxyphenbutazone, dexamethasone and hydrocortisone inhibited the second phase only. Aminopyrine and mefenamic acid which had a central site of action as well as a peripheral one
[11,16] affected both phases, but the second phase response was inhibited by lower doses of these drugs than the first phase response. The second phase inhibition of aminopyrine was caused by a one-quarter dose of the first phase inhibition, and in the case of mefenamic acid it involved a onethird dose. Based on these findings, it is concluded that this method enables one to distinguish the site of action of analgesics: whether it is central, peripheral or both central and peripheral. Our results show approximate agreement with the classification of analgesics by the inflamed foot method of Randall and Selitto [lo] in rats. Capsaicin is a relatively selective neurotoxin for unmyelinated sensory neurons containing substance P [4,7,13]. In our study, capsaicin desensitization of the sciatic nerve produced analgesia in the first phase, but not in the second phase. This finding suggests that substance P may be related to the manifestation of the first phase response. Bradykinin inhibitor also inhibited the first phase response, and the second phase as well, and so bradykinin may be released in the formalin injury reaction. We reported previously that a combination of bradykinin with substance P exerted a synergistic effect on pain response in mouse paws [12]. It may be that formalin stimulation causes substance P release mediated by axon reflex, and substance P may play a role through cooperation with bradykinin in the first phase response. However, no action by 10 mg/kg of bromelain (which causes a specific depletion of high molecular weight kininogens [8]) was detected at the first phase response (Fig. 6b), suggesting that participation of bradykinin may not be very important for the manifestation of the first phase response. On the other hand, as shown in Fig. 6a, pretreatment with compound 48/80 caused an inhibition of the second phase response, and so histamine and serotonin were related to the second phase response as well as bradykinin. Prostaglandin synthetic inhibitors such as indomethacin, aspirin and dexamethasone only inhibited the second phase, and bromelain potentiated the indomethacin analgesia in the second phase, This indicates, therefore, that both prostaglandin and bradykinin cooperated to produce the second phase response.
352
Generally, hydrocortisone and dexamethasone are effective against the inflammatory pain, but show no activity against the non-inflammatory pain. Therefore, it is concluded that the first phase response is evoked by the direct formalin stimulation of the nerve endings followed by substance P release, and the second phase may be mainly due to subsequent inflammation. This conclusion agrees with those of Dubuisson and Dennis [3], Brown et al. [l] and Hunskaar and Hole [6]. These observations indicate that the first and second phase responses have obvious differential properties, and it is a very useful method for not only assessing the analgesics but also elucidating the mechanism of pain and analgesia.
6 Hunskaar.
7
8
References Brown, J.H., Kissel, J.W. and Lish, P.M., Studies on the acute inflammatory response. 1. Involvement of the central nervous system in certain models of inflammation, J. Pharmacol. Exp. Ther., 160 (1968) 231-242. Cohen, S.R., Abbott, F.V. and Melzack, R., Unilateral analgesia produced by intraventricular morphine, Brain Res., 303 (1984) 277-288. 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. Hinsey, J.C. and Gasser, H.S., The component of the dorsal root mediating vasodilation and the Sherrington contracture, Am. J. Physiol., 92 (1930) 679-689. Hunskaar, S., Fasmer, O.B. and Hole, K., Formalin test in mice, a useful technique for evaluating mild analgesics, J. Neurosci. Meth., 14 (1985) 69-76.
14
15
16
S. and Hole. K.. 1 he formalm te,t in rn~ce. dissociation between inflammatory and non-inflammator\ pain, Pain. 30 (1987) 103-l 14. Jancsh, G.. Kiraly, E. and Jancsb-Gibor, A., Pharmacologrtally induced selective degeneration of chemosensrtrve primary sensory neurones. Nature. 270 (1977) 741.~743. Katori, M., Ikeda, K.. Harada, Y., U&da. Y.. Tanaka. K. and Oh-ishi. S.. A possible role of prostaglandins and bradykinin as a trigger of exudation in carrageenin-induced rat pleurisy, Agents Actions, 8 (1978) 108 -112 Lim. R.K.S.. Guzman. F.. Rogers, D.W., Goto. K.. Braun. C.. Dickerson, C.D. and Engle, R.J., Site of action of narcotic and non-narcotic analgesics determined by hlocking bradykinin-evoked visceral pain, Arch. Int. Pharmacodyn.. 152 (1964) 25-58. Randall, L.O. and Selitto, J.J.. A method for measurement of analgesic activity on inflamed tissue. Arch. fnt. Pharmacodyn.. 111 (1957) 409-419. Sasa. M.. Fukuda, N. and Takaori. S.. Effect\ ol aminopyrine and phenylbutasone on the activity of neurons in the trigeminal nuclei and hypothalamus, Jpn. J. Pharmacol., 19 (1969) 363 ,372. Shibata. M.. Ohkubo, T.. Takahashi, H. and Inokr, R.. Interaction of bradykinin with substance P on vascular permeability and pain response, Jpn. J. Pharmacol.. 41 (1986) 427-429. Szolcsanyi, J.. A pharmacological approach to elucrdation of the role of different nerve fibres and receptor endings in mediation of pain, J. Physiol. (Paris). 73 (1977) 251-259. Takahashi, H., Ohkubo, T., Shibata, M. and Narese, S.. A modified formalin test for measuring analgesia in mice. Jpn. J. Oral Biol., 26 (1984) 543-548. Vaccarino, A.L.. Tasker, R.A.R. and Melzack. R., Analgesia produced by normal doses of opioid antagonists alone and in combination with morphine, Pain, 36 (1989) 103-109. Whittle, B.A., The use of changes in capillary permeability in mice to distinguish between narcotic and nonnarcotic analgesics, Br. J. Pharmacol., 22 (1964) 246-253.
Pain, 38 (1989) 347-352 Elsevier
PAIN 01453
Modified formalin test: characteristic biphasic pain response Manabu
Shibata,
Tsuyako
Ohkubo,
Hiroshi
Takahashi
and Reizo Inoki
*
Department of Pharmacology, Fukuoka Dental College, 700 Ta, Sawara-ku, Fukuoka 814-01 (Japan), and * Department of Pharmacology, Faculty of Dentistry, Osaka University, Suita 565, Osuka (Japan) (Received
20 December
1988, revision
received 5 April 1989, accepted
11 April 1989)
Summary
A modified formalin test in mice was investigated. The pain response curve induced by 0.5% formalin was biphasic, having 2 peaks, from 0 to 5 min (first phase) and from 15 to 20 min (second phase). A low concentration of formalin was used, allowing the effects of weak analgesics to be detected. Centrally acting drugs such as narcotics inhibited both phases equally. Peripherally acting drugs such as aspirin, oxyphenbutazone, hydrocortisone and dexamethasone only inhibited the second phase. Aminopyrine and mefenamic acid which acted on both central and peripheral sites inhibited both phases, but the second phase was inhibited by lower doses. Thus, this method enables one to easily distinguish the site of action of analgesics. Furthermore, pain response in the first phase was inhibited by capsaicin-treated desensitization and Des-Arg9-(Leu*)-bradykinin (bradykinin inhibitor). The second phase was inhibited by compound 48/80 pretreatment, indomethacin and bradykinin inhibitor. Therefore, it is suggested that substance P and bradykinin participate in the manifestation of the first phase response, and histamine, serotonin, prostaglandin and bradykinin are involved in the second phase. These results indicate that the first and second phase responses induced by formalin have distinct characteristic properties, and it is a very useful method for examining pain, nociception and its modulation by pharmacological or other means. Key words:
Formalin
test; Analgesics;
Substance
P; Bradykinin;
Introduction The formalin test was introduced by Dubuisson and Dennis [3] and the subcutaneous injection of formalin produced a biphasic pain response in rats. They noted that this method required little or no restraint and the pain stimulus bore a resemblance to most clinical pain. Formalin is a useful tool for obtaining neurogenic inflammation [l] and continuous pain. We previously modified the formalin test in mice, using the forepaw as the site of formalin injection, and simplified the pain rating scale by means of using only licking behavior
Correspondence to: Tsuyako Ohkubo, Ph.D., Department of Pharmacology, Fukuoka Dental College, 700 Ta, Sawara-ku, Fukuoka 814-01, Japan. 0304-3959/89/$03.50
0 1989 Elsevier Science Publishers
Prostaglandin
of the injected paw as an indicator of pain response [14]. Consequently, pain assessment was very easily performed without the experimenter’s subjective assessment and skill. A low concentration of formalin was used, allowing detection of the analgesic effects of weak non-narcotic analgesics. Hunskaar et al. [5] also reported the formalin test which used the same pain rating scale and the hind paw of mice. As licking the forepaw as an indication of pain response may be confounded with the normal grooming response [2], in the present study we made use of the hind paw of mice. Formalin produced a distinct biphasic response, and it has been reported that the action of analgesics differs in the early phase and the late phase [5,6,14]. Although it is conceivable that this might be due to the differential properties of these
B.V. (Biomedical
Division)
34X
2 phase responses, little is known about the features of the formalin test as the screening method and the mechanism of manifestation of formalininduced pain. We examined the effects of many narcotic and non-narcotic analgesics on the biphasic pain response, and report here its usefulness for differentiating the site of action (central versus peripheral) of various analgesics. In addition, participation of various chemical mediators in the manifestation of formalin pain was investigated. In particular, capsaicin was used to determine the influence of C fibers and substance P in the production of formalin-induced pain response.
obtain obvious biphasic and reproducible ~c’spon~ ses. 25 1-11of 0.5% sterile formalin was administered into the right hind paw. Each aninlnl W:I\ then returned to the chamber and pain response was recorded for a period of 30 min. The summation of time (in seconds) spent in licking and biting responses of the injected paw during each 5 min block was measured as an indicator of pain response. Statistical analysis Significant differences dent’s I test.
were determined
by Stu-
Results Methods Groups of 7 male ddY mice weighing 20-25 g were used. Chemicals used were: morphine hydrochloride; meperidine hydrochloride (Tanabe, Japan), pentazocine; mefenamic acid (Sankyo, Japan), ethylketocyclazocine; ketocyclazocine (Sterling-Winthrop Res. Inst.), buprenorphine hydrochloride (Ootsuka, Japan), codeine phosphate (Shionogi, Japan), indomethacin (Sumitomo, Japan), bromelain; compound 48/80 (Sigma), aspirin (Ken-ei, Japan), hydrocortisone acetate; dexamethasone acetate (Merck-Banyu, Japan), arninopyrine (Ono, Japan), oxyphenbutazone (Fujisawa, Japan), capsaicin (Merck), Des-Arg’-(Leu’)-bradykinin (Peptide Institute, Japan). Aspirin, mefenamic acid and oxyphenbutazone were suspended in 0.5% tragacanth. Ketocyclazocine was dissolved in a small quantity of acetic acid and then diluted with saline. Capsaicin was dissolved in 50% dimethyl sulfoxide (DMSO). When not specified, other drugs were dissolved in saline and administered 30 min prior to the formalin injection. The observation chamber was a glass cylinder of diameter 20 cm on an acrylic transparent plate floor. Beneath the floor, a large mirror was mounted at a 45 o angle to allow clear observation of the paws of the animal. Each mouse was placed in the chamber more than 5 min prior to the formalin injection in order to allow it to acclimatize to the new environment; this is necessary to
Formalin-induced biphasic pain response and morphine inhibition As shown in Fig. 1 (see pain response curve of control), the subcutaneous injection of 0.5% formalin caused an acute pain response which lasted about 5 min (first phase). Subsequently, this response disappeared for about 5 min and then recurred and lasted about 20 min (second phase). The peak period of the second phase was from 15 to 20 min. When morphine (1, 3, 6 mg/kg) was administered subcutaneously 20 min prior to the formalin
-.-
‘formalin
intectlon
control morphine
(saline. s.c ) ( 1 mg/kg,
SC
min. Fig. 1. Time course of pain response induced by formalin and effect of morphine. Pain response was measured from immediately after intraplantar injection of 25 ~1 of 0.5% formalin. Ordinate (pain response): the time (in set) spent in paw kinking or biting response during each 5 min block. Each value represents the meanfS.E. (n = 7). * P < 0.05, *** P-C 0.005.
a
-o-control ( tragacanlh. -c-aspirin
-.-.-
set 120
s. ,.
( 100 mg/kg.
t ZOOmg/kg,
(300mg/kg.
PO I, ‘.
1
) )
)
90
60
a lormalln
inp?cllon
min.
0
Ob3
0.3
0.1
1
10
3b
Dose
lormalln
l”,eCm”
mm.
Fig. 2. Effects of aspirin and dexamethasone on formalin-induced pain response. Aspirin and dexamethasone were administered 30 min prior to the formalin. Each value represents the meanf S.E. (n = 7). * P < 0.05. * * P -L0.01, * * * P c 0.005.
mglkg
Fig. 3. Dose-response curves of analgesic action of morphine, pentazocine. EKC, KC, codeine, buprenorphine and meperidine in the first phase (F) and the second phase (S) of formalin-induced pain. Percentage of inhibition = [l -duration time (drug-treated)/duration time (vehicle-treated)] x 100. Each point is the mean value (n = 7).
injection, it inhibited the formalin-induced biphasic pain response dose-dependently. Six mg/kg of morphine produced almost complete analgesia. Effects of aspirin and dexamethasone on formalininduced pain Aspirin (100, 200, 300 mg/kg, p.o) inhibited the second phase response dose-dependently (Fig. 2a), but not the first phase. Similarly, subcutaneous injection of dexamethasone (0.25, 0.5, 1 mg/kg) caused an inhibition of the second phase dose-dependently (Fig. 2b). Dose-response curves of various analgesics and anti-inflammatory drugs In order to obtain dose-response curves of analgesics in both the first and second phase, percent inhibition was calculated from the values of saline-treated and drug-treated animals for each
0
i 0.3
-*--.
4--.-,_, 1
3
‘---+. 10
.&r,
F
30
KJo DOS-2
C?Qo mg,kg
Fig. 4. Dose-response curves of aminopyrine, mefenamic acid, dexamethasone, hydrocortisone, oxyphenbutasone and aspirin in the first phase (F) and the second phase (S) of formalin-induced pain. See Fig. 3 for details.
350
period of O-5 min (F) and 15-20 min (S) after formalin injection. Dose-response relationships of morphine, ethylketocyclazocine (EKC), ketocyclazocine (KC), pentazocine, buprenorphine, meperidine and codeine on the first and second phases were observed (Fig. 3). All of these drugs inhibited both phases equally and dose-dependently. Dose-response curves of aminopyrine, mefenamic acid, aspirin, oxyphenbutazone, dexamethasone and hydrocortisone are shown in Fig. 4. Aminopyrine and mefenamic acid inhibited both phases, but the second phase was inhibited by lower doses than the first phase. In the case of aspirin, oxyphenbutazone, dexamethasone and hydrocortisone, dose-response relationship was obtained only on the inhibition of the second phase, and they had no effect on the first phase pain response.
a 120.
-o-ca~sac,n-treated
go’
sc~ahc
O-5
formaIm
5-10
10-15
15-20
~n,ecf~on
20-25
25-30
min.
Fig. 6. Effects of compound 48/80 pretreatment and combination of indomethacin and bromelain on formalin-induced pain response. Bromelain and indomethacin were administered 90 and 30 min respectively before the formalin. Each value represents themean+SE.(n=7). * P~0.05, ** PiO.01, *** P
-.-solvent-treated SClatlCnerve (Stklrnoperaho” )
SK
T
nerve
i\
< 0.005.
b
(salme,
-.-control -o-
Des.Arg%(
I
PI
)
Leus )-bradykmm
-I-
5-10
IO-15
15-20
20-25
25-30
(150~mol.
!Pl )
(3OOPmol.
”
1
mm.
Fig. 5. Effects of pretreatment with capsaicin and Des-Arg’(Leu’)-bradykinin on formalin-induced pain response. Each value represents the mean& S.E. (n = 7). * * P < 0.01, * * * P
-=c 0.005.
Capsaicin treatment of sciatic nerve and effect of bradykinin inhibitor Sciatic nerves were pretreated with 1.5% capsaicin solution 12 days prior to the experiment. Control mice were treated with sham operation and vehicle (50% DMSO). In mice treated with capsaicin, only the first phase response was inhibited significantly (Fig. 5a) Des-Arg9-(Leu’)-bradykinin, a specific bradykinin inhibitor, was injected intraplantarly 2 min before formalin injection at doses of 150 and 300 pmol. This drug caused an inhibition of both phases of formalin pain (Fig. 5b). Compound 48/80 treatment and combination effect of indomethacin and bromelain Compound 48/80, a histamine and serotonin depletor, was administered by intraplantar injec-
351
tion at a dose of 1.25 pg/paw twice a day for 2 days. Twelve hours after final administration, formalin was injected into the treated paw. Compound 48/80 reduced the second phase response only (Fig. 6a). Effects of indomethacin (5 mg/kg, p.o.) and bromelain (10 mg/kg, i.v.) and a combination of these 2 drugs were observed. To study the effect of such a combination, lower doses of the 2 drugs were chosen. When given alone, indomethacin and bromelain inhibited only the second phase. The effects of combination of these drugs were greater than mere summation on the second phase pain response, especially for the periods of 10-15 min and 20-25 min.
Discussion Among the screening methods of analgesics, there are many which are available to assess analgesic action of narcotics but are unsuitable for weak analgesics such as antipyretics. This modified formalin test can be used to evaluate the analgesic effects regardless of narcotics or nonnarcotics. Antinociceptive drugs are generally classified into central or peripheral according to the site of action. Typical of the former is morphine, and that of the latter is aspirin. Lim et al. [9] reported the method which determined the site of action by means of nociception caused by intra-arterial injection of bradykinin in dogs. This method is superior in estimating the drug’s action sites; however, the technique is difficult. Assessing the effects of various analgesics using the formalin-induced biphasic response, narcotic drugs such as morphine, codeine, EKC, KC, buprenorphine, meperidine and pentazocine inhibited the pain response at the first and second phases equally. The analgesic effects of morphine on formalin-induced pain responses are in agreement with the previous studies by Hunskaar et al. [5] and Vaccarino et al. [15]. On the other hand, peripherally acting drugs such as aspirin, oxyphenbutazone, dexamethasone and hydrocortisone inhibited the second phase only. Aminopyrine and mefenamic acid which had a central site of action as well as a peripheral one
[11,16] affected both phases, but the second phase response was inhibited by lower doses of these drugs than the first phase response. The second phase inhibition of aminopyrine was caused by a one-quarter dose of the first phase inhibition, and in the case of mefenamic acid it involved a onethird dose. Based on these findings, it is concluded that this method enables one to distinguish the site of action of analgesics: whether it is central, peripheral or both central and peripheral. Our results show approximate agreement with the classification of analgesics by the inflamed foot method of Randall and Selitto [lo] in rats. Capsaicin is a relatively selective neurotoxin for unmyelinated sensory neurons containing substance P [4,7,13]. In our study, capsaicin desensitization of the sciatic nerve produced analgesia in the first phase, but not in the second phase. This finding suggests that substance P may be related to the manifestation of the first phase response. Bradykinin inhibitor also inhibited the first phase response, and the second phase as well, and so bradykinin may be released in the formalin injury reaction. We reported previously that a combination of bradykinin with substance P exerted a synergistic effect on pain response in mouse paws [12]. It may be that formalin stimulation causes substance P release mediated by axon reflex, and substance P may play a role through cooperation with bradykinin in the first phase response. However, no action by 10 mg/kg of bromelain (which causes a specific depletion of high molecular weight kininogens [8]) was detected at the first phase response (Fig. 6b), suggesting that participation of bradykinin may not be very important for the manifestation of the first phase response. On the other hand, as shown in Fig. 6a, pretreatment with compound 48/80 caused an inhibition of the second phase response, and so histamine and serotonin were related to the second phase response as well as bradykinin. Prostaglandin synthetic inhibitors such as indomethacin, aspirin and dexamethasone only inhibited the second phase, and bromelain potentiated the indomethacin analgesia in the second phase, This indicates, therefore, that both prostaglandin and bradykinin cooperated to produce the second phase response.
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Generally, hydrocortisone and dexamethasone are effective against the inflammatory pain, but show no activity against the non-inflammatory pain. Therefore, it is concluded that the first phase response is evoked by the direct formalin stimulation of the nerve endings followed by substance P release, and the second phase may be mainly due to subsequent inflammation. This conclusion agrees with those of Dubuisson and Dennis [3], Brown et al. [l] and Hunskaar and Hole [6]. These observations indicate that the first and second phase responses have obvious differential properties, and it is a very useful method for not only assessing the analgesics but also elucidating the mechanism of pain and analgesia.
6 Hunskaar.
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