Pain, 48 (1992) 421-427

0 1992 Elsevier Science Publishers B.V. All rights reserved 0304-3959/92/$05.00

421

PAIN 01958

Rat knee-joint carrageenin incapacitation test: an objective screen for central and peripheral analgesics

Carlos Rogtrio Tonussi and Shgio H. Ferreira Department of Pharmacology, Faculty of Medicine of Rib&Go Prefo, Ribeirgo Preto, SP, CEP 14049 (Brazil)

(Received 8 May 1991, revision received and accepted 8 July 1991)

Summary A new behavioral test is described in which quantitation is independent of the observer and is sensitive to all classes of analgesics, A computer-assisted device measures the period during which a rat hind paw fails to touch the surface of a rotating cylinder for 1 min (paw elevation time). Intra-articular injection of carrageenin induces a progressive and dose-dependent incapacitation of the limb. The maximum paw elevation time is attained 3-4 h after carrageenin challenge. The model showed dose-dependent sensitivity to (a> a central acting opiate (morphine, ID,, = 1.5 mg/kg, i.p.1, (b) cyclooxygenase inhibitors (indomethacin, ID,, = 0.8 mg/kg, i.p.; diclofenac, ID,,, = 0.22 mg/kg, i.p.1, and (cl peripheral analgesics which directly antagonize nociceptor hypersensi- tivity: dipyrone (ID,,, = 21 mg/kg, i.p.1, N-methyl-nalorphine (ID,, = 14 mg/kg, i.p.1 and BW443C (ID,, = 17.5 mg/kg, i.p.). The knee-joint carrageenin incapacitation was also blocked by the sympatholytics, propranolol and guanethidine. After the blockade by either indomethacin or guanethidine, intra-articular injections of prostaglandin E, or dopamine, respectively, reversed carrageenin-induced incapacitation. These results suggest that during inflammatory articular incapacitation cyclooxygenase and sympathomimetic mediators are involved, as has been suggested for the rat paw carrageenin hyperalgesia test and formalin test.

Key words: Peripheral analgesia; Prostaglandin E 2; Dopamine; Diclofenac; Tranquillizers

Introduction

The analgesic mechanism of action of non-steroidal anti-inflammatory drugs is thought to involve preven- tion of nociceptor sensitization, by the blockade of the generation of cyclooxygenase products (Ferreira 1972). In the absence of inflammatory nociceptor hypersensi- tivity, the previous mechanical or chemical algogenic stimulation ceases to cause pain. At present, in addi- tion to prostaglandins, there is growing experimental evidence indicating the involvement of the sympathetic system in inflammatory nociceptor hypersensitivity (Nakamura and Lice 1983; Nakamura and Ferreira 1987; Duarte et al. 1988). It is known that most non- steroidal anti-inflammatory drugs are unable to block ongoing nociceptor hypersensitivity. Nevertheless, some

Correspondence to: S.H. Ferreira, Dept. of Pharmacology, Faculty of Medicine of RibeirHo Preto, Ribeiriio Preto, SP, CEP 14049. Brazil.

drugs can directly down-regulate the nociceptors. An example is dipyrone (Lorenzetti and Ferreira 1985). A similar mode of action has also been described for peripherally acting opiates (Lorenzetti and Ferreira 1982, 1987; Rios and Jacob 1982, 1983; Smith et al. 1982; Follenfant et al. 1988) and some monoterpenes (Lorenzetti et al. 1991).

Notwithstanding the development of new experi- mental models of nociception, most of the parameters for quantitation remain subjective and dependent on the careful training of the observer (Pardo and Rod- riguez 1966; Van Arman et al. 1970; Moncada et al. 1975; Pircio et al. 1975; Nakamura and Lice 1983; Otsuki et al. 1986; Hargreaves et al. 1988). However, these methods are relatively expensive and in general useless for drug screening. Up to now, the develop- ment of central acting analgesics has been based on the use of several variations of the tail flick, hot plate and pinch tests (Haffner 1929; D’amour and Smith 1941; Woolfe and McDonald 1944). On the other hand, peripherally acting analgesics, such as non-steroidal

422

anti-inflammatory drugs, have been screened using var- ious modifications of either the writhing test in mice (Siegmund et al. 1957; Koster et al. 1959) or the rat paw pressure test introduced by Randall and Selitto (1957). However, it must be pointed out that the writhing test, apart from lacking specificity, exhibits great variability in response (Hendershot and Forsaith 1959; Emmele and Shanaman 1963). Results in the rat paw pressure test vary among laboratories (Gilfoil et al. 1963; Winter and Flataker 1965; Takesue et al. 1969; Swingle et al. 1971) so that difficulties arise when comparing them. At present, the formalin test is gain- ing popularity, probably due to the simplicity of the end point rather than due to its sensitivity to the various classes of analgesics (Dubuisson and Dennis 1977; Hunskaar et al. 1986; Hunskaar and Hole 1987; Bustamante et al. 1989; Shibata et al. 1989). In fact, the reliability of this test also depends on careful training of the observer.

Thus, there is a need for the development of noci- ceptive methods which (a) are relatively inexpensive, (b) allow the testing of a large number of animals per session, (c) discriminate between the different types of peripheral analgesics, and, more important, Cd) facili- tate measurements that must be automatic and inde- pendent of the observer subjectivity. The method de- scribed here fulfills all these criteria and is itself a modification of a test originally non-steroidal anti-inflammatory 1982).

Methods

Animals used

proposed for testing drugs (Bustamante

Male Wistar rats weighing 170-200 g were fasted overnight hut

had free access to water.

Drugs The drugs BW443C (Wellcome, UK), chlorpromazine (Sigma.

USA), carrageenin (Marine Colloids, USA), diazepam (Sigma, USA).

diclofenac (Voltaren ‘, Biogalinica, Brazil), dipyrone (Hoechst.

Brazil), dopamine (Sigma, USA), guanethidine (Ciba-Geygy, Basle).

indomethacin (Merck-Sharpe and Dohme, USA), morphine (Merck,

Germany), N-methyl-morphine, N-methyl-nalorphine (Wellcome,

UK), prostaglandin E, (Sigma, USA) and propranolol (Sigma. USA)

were used.

Rat incapacitation register system Fig. 1 shows the device, made of a non-oxidisable steel drum (30

cm wide x 30 cm in diameter) covered with a fine-mesh non-oxidisa-

ble wire screen, which rotates at 3 rpm. The drum is divided into 3

equal tracks and electrically earthed with a Apple Be-like microcom-

puter. A specially designed metal gaiter is wrapped around both hind

paws. One of them is directly connected via a simple circuit to

microcomputer data input/output port (Fig. 1). In our system up to

3 animals can be registered simultaneously.

Cylinder+ an _ I/+ 5 Vcc

Gro;nd

Fig. I. incapacitation registration system. Panel A: rats walking on

top of the activity cylinder. the right paw gaiters arc connected to a

microcomputer. Panels B and C: detail of the gaiter placed around

the hind paw and in relation to a scale in cm, respectively. Panel D:

circuit used to connect the gaiter to the microcomputer.

Experimental procedures Thirty minutes after the placement of the gaiters was allowed for

habituation. After the control measurement session (1 min). 20 ~1 of

carrageenin solution (300 pg) was injected into the right knee-joint.

The period for which the hind paw failed to touch the surface of the

cylinder during a 60 set session was investigated every hour (paw

elevation time, PET). All drugs were tested by intraperitoneal administration (Lp.). In

order to calculate the ID,,, for the various drugs, they were injected

1 h before the maximum incapacitation induced by carrageenin, as

indicated in pilot studies (2nd or 3rd hour after the challenge). All

calculations were made taking the maximum carrageenin hyperalge-

sia as control, i.e. 1 h after administration of the test drug. The

opioid antagonist (N-methyl-nalorphine) was given i.p. 30 min before

the agonist tested. The experiments on reversal of inflammatory

incapacitation by a specific mediator (PGE, or dopamine) were

carried out as follows: (a) the animals were pretreated with in-

domethacin (5 mg/kg, i.p.1 and immediately challenged with car-

rageenin and 2, 3 and 4 h later. PGE, (500 ng/O.l ml) was injected

intra-articularly; (b) guanethidine (30 mg/kg, s.c.) was administered

3 days consecutively before the carrageenin challenge and 2, 3 and 4

h later dopamine (300 &g/O.1 ml) was injected intra-articularly. The

same procedure was taken for the prostaglandin E, and dopamine control groups, but without carrageenin challenge.

423

Statistics The significance of differences between control and treated

groups was determined from the PET values after 3 h using Student’s t test for unpaired samples, P 5 0.05 being considered significant. The ID,, was calculated according to Litchfield and Wilcoxon (1949)

using mean PET values obtained at 3 h minus the respective mean

PET obtained at zero time. Per cent incapacity reversal was calcu-

lated by assuming a PET of 10 set as 100% reversal.

Results

Intra-articular (iart) administration of carrageenin caused dose-dependent incapacitation reaching a maxi- mum within 3-4 h (Fig. 2). The state of incapacitation slowly receded, paw elevation time returning to normal within 24-48 h after the challenge. Dextran at a dose which causes intense edema or saline (not shown in Fig. 2) did not produce functional incapacitation.

Antinociceptive effect of peripherally acting analgesics Indomethacin and dipyrone injected i.p. 2 h after

carrageenin iart injection caused a dose-dependent re- duction in the paw elevation time (Fig. 3). The esti- mated ID,, for indomethacin was 0.8 mg/kg (confi- dence limits = 0.25-2.49) and the maximum dose used (5 mg/kg) reversed 87.8% of the incapacitation meas- ured 3 h after carrageenin challenge (Fig. 3A). Fig. 3B shows the dose-dependent effect of dipyrone. The esti- mated ID,, was 21.9 mg/kg (confidence limits = 12.7- 37.8) and the dose of 50 mg/kg was able to reduce incapacity by 84%. Note that there was no difference between the antinociceptive effect of indomethacin at 3 and 4 h, at which time the effect of dipyrone tended towards the control values. Diclofenac caused a dose- dependent reversal of the incapacitation with an ID,,, of 0.2 mg/kg, i.p. (confidence limits = 0.13-0.38).

The Fig. 4 shows that N-methyl-nalorphine and BW443C caused a dose-dependent reversal of the inca-

60

0 ca 30 UQ _

O-w 2 3 4 5 6 7 a24

Time (h)

Fig. 2. Time course of the development of dose-dependent car-

rageenin (Cg)-induced incapacitation and the effect of T-70 dextran

(Dex) injected intra-articularly (300 pg). The paw elevation time was

measured over a 60 set period and the points are the means of 6

ratsfS.E.M. Note that 60 set of suspension of the paw is the

maximum value possible to register during the period of observation.

60

20

10

0 Tris A

:

60 B

50

40

30

20

10

0-l I / i I I ! : : : : : co 0 1 2 3 4 0 1 2 3 4

Time after Cg challenge (h)

Fig. 3. Panel A: development of the carrageenin (Cg)-induced inca-

pacitation under treatment with indomethacin Undo). Panel B: de-

velopment of the carrageenin (Cg)-induced incapacitation under

treatment with dipyrone (Dip). The drugs were given i.p. 2 h after

carrageenin injection. All points plotted represent the means+

S.E.M. from 6 animals. The values on the graph indicate doses in

mg/kg. The asterisks indicate a significant difference (P < 0.05) in

relation to the saline (or Tris)-treated group.

pacitation. The estimated ID,,, for N-methyl- nalorphine was 14 mg/kg (confidence limits = 7.5-26.2) and the maximum dose led to a reversal of incapacita- tion of 72.9% (panel A). The estimated ID,, for BW443C was 17.5 mg/kg (confidence limits = 10.4- 29.5) and the maximum reversal observed was 75.7% at the dose of 40 mg/kg (panel B).

Blockade of the antinociceptive effect of N-methyl- morphine and BW443C by a sub-analgesic dose of N- methyl-nalorphine

Pre-treatment of the animals with a sub-analgesic dose of N-methyl-nalorphine (1 mg/kg) abolished the antinociceptive effect of N-methyl-morphine (32 mg/kg) or BW443C (40 mg/kg) (Fig. 5).

60

50

40

30

20

10 /

0 saline 7 A 0 saline

0 10 A 20 A 40

0 0 0 1 2 3 4 0 1 2 3 4

Time after Cg challenge (h)

Fig. 4. Panel A: development of the carrageenin (Cg)-induced inca-

pacitation under treatment with N-methyl-nalorphine (MNal). Panel

B: development of the carrageenin (Cg)-induced incapacitation un-

der treatment with BW443C (BW). The drugs were given i.p. 2 h

after carrageenin injection. All points plotted represent the means k

S.E.M. from 6 animals. The values on the graph indicate doses in

mg/kg. The asterisks indicate a significant difference (P < 0.05) in

relation to the saline-treated group.

423

60

* *

3 50 --

,i 40-- @

6 ‘Z 30 --

: a, o 20-- 3 0

CL lo--

o- Sal BW MMor MNol MNal

tt BW mar

Time after Cg challenge (h) lndo

Fig. 5. Illustration of the antagonistic effect of N-methyl-nalorphine

(MNal: 1 mg/kg, i.p.) upon N-methyl-morphine (MMor: 32 mg/kg.

i.p.) and BW443C (BW: 40 mg/kg, i.p.) analgesia. The peripheral

analgesics were administered 2 h after carrageenin (Cg) challenge

and the antagonist (MNal) was given 30 min before these drugs. The

bars represents the mean paw elevation time at 3 h+S.E.M. from 6

animals. The asterisks indicate a significant difference (P < 0.05) in

relation to the respective peripheral analgesic treated group. PET* S.E.M. from 6 animals.

Fig. 7. Panel A: restoration by prostaglandin E, (A ) of carrageenin

(Cg)-induced incapacitation inhibited by indomethacin (Indo: 5

mg/kg i.p., just before Cg challenge). Prostaglandin E, (Sot) ng/iart)

was given IO min before the 2nd. 3rd and 4th hour after Cg. The

same sequence of prostaglandin El injections in non-Cg-challenged

animals did not evoke incapacitation (0). The o and u represent.

respectively. the incapacitation induced by Cg iart injection in con-

trol and indomethacin-treated group. Panel B: detailed results ob-

tained at 4 h in panel A. All points or bars represent the mean

Antinociceptive effect of centrally acting drugs Morphine was very efficient in reducing inflamma-

tory incapacitation as illustrated in Fig. 6A. The IDS,, for morphine was 1.5 mg/kg (confidence limits = 0.93- 2.41) and the maximum reversal observed was 97.3%.

Fig. 6B shows that diazepam (1 mg/kg) and chlor- promazine (1 mg/kg) had no effect upon carrageenin- induced incapacitation.

Rerlersal by prostaglandin E, of antinociceptice blockade of incapacitation

Three successive injections of prostaglandin E, (Fig. 7) in non-inflamed rats treated with indomethacin did

saline A 1

2 /"\

0 saline

Time after Cg challenge (h)

Fig. 6. Panel A: development of the carrageenin (Cg)-induced inca-

pacitation under treatment with morphine (Mar). Panel B: develop-

ment of the carrageenin tCg)-induced incapacitation under treat-

ment with diazepan (Dzp) and chlorpromazine (Chlor). The drugs were given i.p. 2 h after carrageenin injection. All points plotted

represent the meanfS.E.M. from 6 animals. The values on the

graph indicate doses in mg/kg. The asterisks indicate a significant

difference (I’ < 0.05) in relation to the saline-treated group.

-- --IT 6o

+I 50

40

30

+20

. . . . . , 0

CL7 Q cg+pg pg 0

not cause knee-joint functional incapacitation. How- ever, carrageenin-induced incapacitation, totally inhib- ited by indomethacin, was significantly reversed by previously ineffective iart injections of prostaglandin EJ.

Sympatholytic blockade of incapacitation and its rer*ersal by dopamine

Sympatholytics such as propranolol and guanethi- dine pretreatment significantly blocked the rat paw

60 A B[

60

_

I--

I cg c 30

20

.,,...,. . . . , 0

I 50

r-h 40

Cg Q+Dp DP 0

Time after C-g challenge (h) Gnt

Fig. 8. Panel A: restoration by dopamine (0) of carrageenin (Cg)-in-

duced incapacitation partially blocked by guanethidine (Gnt: 30

mg/kg S.C. given 3 days consecutively before Cg challenge). Dopamine

(Dp: 300 pg, iart) was given 10 min before the 2nd, 3rd and 4th hour

after Cg. The same sequence of dopamine injections in non-Cg-chal-

lenged animals did not evoke incapacitation (0). The 0 and A

represent, respectively, the incapacitation induced by Cg iart injec- tion in control and Gnt-treated group. Panel B: detailed results

obtained at 4 h in panel A. All points or bars represent the mean

PET + S.E.M. from 6 animals.

425

elevation time induced by an intra-articular injection of carrageenin. The administration of propranolol (100 pg/kg, i.p.1 given 2 h after Cg challenge reduced significantly the PET from 49.3 + 3.04 set to 19.34 * 1.3 sec. Fig. 8 shows that previous treatment with guan- ethidine (30 mg/kg s.c., for 3 days) partially blocked carrageenin-induced incapacitation and it was restored by 3 consecutive intra-articular doses of dopamine (300 pg). As for .PGE,, dopamine injected into normal joints did not cause incapacitation.

Discussion

In the present study, using a gaiter applied to the rat paw, an objective screening test for analgesics was developed. The incapacitation of the rat knee joint induced by carrageenin was quantitated by the time of ‘no contact’ during a 60 set registration period. It is intriguing that when the gaiters were applied to car- rageenin-inflamed paws, even in the presence of in- tense edema, there was no sign of incapacitation. The knee-joint test was able to detect in a dose-dependent fashion the antinociceptive activity of cyclooxygenase inhibitors (indomethacin and diclofenac), sympatholyt- its (the beta-blocker propranolol and the adrenergic terminal blocker guanethidine), as well as peripherally direct-acting analgesics such as dipyrone, methyl- nalorphine, methyl-morphine and BW443C. This method was also able to detect centrally acting anal- gesics (morphine) but was insensitive to the action of minor and major tranquillizers (diazepam and chlor- promazine, respectively).

Incapacitation of the rat knee joint has been ob- served previously with urate crystals using a system similar to that used in the present study (Bustamante 1982). However, the carrageenin-induced incapacita- tion is more sensitive to peripherally and centrally acting analgesics. When the knee joint was inflamed with urate crystals (Bustamante 1982) dipyrone only caused analgesia at doses (> 50 mg/kg) which had a clear anti-edematogenic effect, probably due to inhibi- tion of cyclooxygenase (Lorenzetti and Ferreira 1985). It must be pointed out, however, that is not the edema which is responsible for the incapacitation since an edematogenic substance such as dextran (Rowley and Benditt 1956; Rocha and Ferreira 1986) did not cause incapacitation. In fact, carrageenin-induced inflamma- tion involves a more complex tissue reaction than that evoked by T-70 dextran, since it involves many inflam- matory mediators as well as cellular infiltration (Van Arman et al. 1965; Vinegar et al. 1976; Lo et al. 1982).

The complex etiology of the rat articular incapacita- tion induced by carrageenin is illustrated by the fact that intra-articular injection of prostaglandin E, in doses up to 1 pg did not evoke incapacitation in

normal joints. This lack of effect contrasts with the direct enhancing action of prostaglandin E, on the rat paw hyperalgesia test (Ferreira et al. 1978) and dog knee-joint incapacitation test (Rosenthale et al. 19721. However, as shown here, if the joint was previously inflamed with carrageenin in animals pretreated with indomethacin (5 mg/kg, i.p.1 the successive (thereby mimicking continuous release by the inflamed tissues) intra-articular injection of prostaglandin E, caused functional incapacitation of the same order of magni- tude as the control. This result suggests that, in this test, for prostaglandin E, to induce knee-joint incapac- itation local release of other inflammatory mediators is required. Thus, the inhibition of carrageenin-induced rat knee-joint incapacitation by indomethacin is sup- ported by the assumption that its action is associated with blockade of cyclooxygenase activated during the inflammatory process.

It is now being recognized that in several experi- mental models the sympathetic nervous system con- tributes to the nociceptive response to different stimuli (Coderre et al. 1984; Nakamura and Lice 1986; Naka- mura and Ferreira 1987; Duarte et al. 1988). We have shown here that the carrageenin-induced rat knee-joint incapacitation was antagonized by propranolol and guanethidine and restored by successive injections of an adrenergic hyperalgesic stimulus, dopamine (Nakamura and Ferreira 1987). The sympathetic ner- vous system also seems to contribute to inflammatory sensibilization in this model.

Another important feature of the method is its ability to detect the peripheral antinociceptive effect of opiates (Ferreira and Nakamura 1979b; Bentley et al. 1981). Since peripheral opiates are not cyclooxygenase inhibitors, their antinociceptive effect cannot be at- tributed to the prevention of sensitization of the pain receptors by released prostaglandins. It has been sug- gested, however, that their analgesic action is mediated by a direct effect upon the molecular events which control nociceptor hypersensitivity. It was assumed that these drugs act by blocking the activation of adenylate cyclase (Ferreira and Nakamura 1979a1, but we re- cently showed that opiates may cause peripheral anal- gesia by stimulation of the cGMP pathway, via the release of nitric oxide (Duarte et al. 1990; Ferreira et al. 1991). It is interesting that at the peripheral noci- ceptors, partial antagonists such as nalorphine and its quaternized derivative, N-methyl-nalorphine, cause analgesia (Ferreira and Nakamura 1979b; Ferreira et al. 1984). In the present study N-methyl-nalorphine, N-methyl-morphine and the peripherally acting modi- fied enkephalin, BW443C, blocked carrageenin-in- duced incapacitation in a dose-dependent manner. We have further confirmed that the effect was peripheral by antagonizing the antinociceptive effect with a sub- analgesic dose of N-methyl-nalorphine.

426

Finally we have shown that this method is able to detect centrally actin’g analgesics (morphine) but is insensitive to the action of minor (diazepam) and major (chlorpromazine) tranquillizers, at doses which are in accordance with their anxiolytic activity (Davidson and Weidley 1976; Sepinwall and Cook 1978). This indi- cates that the observed effect of morphine was related to antinociception rather than to its proposed anxi- olytic action.

In conclusion, the carrageenin-induced hind paw incapacitation test, described in the present paper, constitutes a useful screen for all classes of analgesics, including those which act by directly blocking ongoing hypersensitivity of the nociceptors.

Acknowledgements

This work was supported by FAPESP, FINEP and CNPq (Brazil).

References

Bentley, G.A., Newton, S.H. and Starr, .I., Evidence for an action of

morphine and the enkephalins on sensory nerve endings in the

mouse peritoneum. Br. J. Pharmacol., 73 (1981) 325-332.

Bustamante, A.M.F., Caracterizacion de un Modelo para la Evalua-

cion de la Analgesia en Ratas: Medici& de la Revercion de la

Limitation Funcional Producida por Administration Intra-articu-

lar de Acido Urico, BSc thesis presented to Universidad Au-

tonoma del MCxico, 1982.

Bustamante, D., Miranda, H.F.. Pelisser. T. and Paeile, C., Analgesic

action of clonixin. nifedipine and morphine using the formalin

test, Gen. Pharmacol. 20 (1989) 319-322.

Coderre. T.J., Abbott, F.V. and Melzack, R., Effects of peripheral

antisympathetic treatments in the tail-flick, formalin and auto-

tomy tests. Pain 18 (1984) 13-23

D’amour, F.E. and Smith, D., A method for determining loss of pain

sensation. J. Pharmacol. Exp. Ther., 72 (1941) 74-79.

Davidson. A.B. and Weidley, E., Differential effects of neuroleptics

and other psychotropic agents on acquisition of avoidance in rats,

Life Sci., 18 (1976) 1279-1284.

Duarte, I.D.G., Nakamura, M. and Ferreira, S.H., Participation of

the sympathetic system in acetic acid-induced writhing in mice.

Braz. J. Med. Biol. Res., 21 (1988) 341-343.

Duarte, I.D.G., Lorenzetti, B.B. and Ferreira, S.H., Peripheral anal-

gesia and activation of the nitric oxide cyclic-GMP pathway. Eur.

J. Pharmacol., 186 (1990) 289-293. 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.

Emmele. J.F. and Shanaman, J., Bradykinin writhing: a method for

measuring analgesia, Proc. Sot. Exp. Biol. Med.. 114 (1963)

680-681. Ferreira, S.H., Prostaglandins, aspirin-like drugs and analgesia. Na-

ture (New Biol.), 240 (1972) 200-203.

Ferreira, S.H. and Nakamura, M., 1 prostaglandin hyperalgesia, a

cAMP/Ca’+ dependent process, Prostaglandins. 18 (1979a)

179-190. Ferreira, S.H. and Nakamura, M.. II prostaglandin hyperalgesia, the

peripheral analgesic activity of morphine. enkephalins and opioid

antagonists. Prostaglandins, IX (lY7Yb) 141-200.

Ferreira, S.H.. Lorenzetti, B.B. and Correa, F.M.A.. (‘entral and

peripheral antialgesic action of aspirin-like drugs. Eur. J. Phar-

mucol.. 53 (1978) 39-4X.

Ferreira, S.H.. Lorenzetti. B.B. and Rae, G.A., is methylnalor-

phiniun the prototype of an ideal peripheral analgesic‘?. Cur. J.

Pharmacol., 99 (1984) 23-2’).

Ferreira. S.H., Duarte. I.D.G.. Lorenzetti. B.B.. Molecular base ol

acetylcholine and morphine analgesia. In: M.J. Parnham, MA

Bray and W.B. Van den Berg (Eds.), Drugs in Inflammation, Vol.

32. Birkhauser. 1901. pp. 101~106.

Follcnfant. R.L.. Hardy. G.W.. Lowe, L.A., Schneider, C. and Smith.

T.W.. Antinociceptivc cffectz of the novel opioid peptide BW443C

compared with classical opiates: peripheral versus central actionr,

Br. J. Pharmacol.. 43 (IYXX) X5-Y?.

Gilfoil. T.M.. Klavins. I. and Grumhach. L.. Effects of acetylsalicylic

acid on the oedema and hyperesthesia of experimentally inflamed

rat‘\ paw. J. Pharmacol. Exp. Ther.. 142 (1963) l--5.

llaffner. F., Experimentelle prufung schmerzstellender mittel.

Deutsch. Me. Worchenschr.. 55 (1929) 731-733.

Hargreaves. H.. Dubner, R., Brown. F.. Flares, C. and Joris, J.. A

new and sensitive method for measuring thermal nociception in

cutaneous hyperalgesia, Pain. 32 (IYXX) 77-7X

Hendershot, L.C. and Forsaith, J.. Antagonism of the frequency of

phenylquinone-induced writhing in the mouse by weak analgesics

and nonanalgesics, J. Pharmacol. Exp. Ther.. I25 (1959) 237-240.

Hunskaar, S. and Hole, K, The formalin test in mice dissociation

hetween inflammatory and non-inflammatory pain. Pain. 30 (19X7)

103-l 14.

Hunskaar. S., Berge, O.G. and Hole. K., Dissociation between

antinociceptive and anti-inflammatory effects of acetylsalicylic

acid and indomethacin in the formalin test, Pain, 25 (1986)

125-132.

Koster, R.. Anderson, M. and De Beer. J., Acetic acid for analgesic

screening, Fed. Proc.. IX (1959) 412-417

Litchfield. Jr.. J.T. and Wilcoxon, F.. A simplified method of evaluat-

ing dose-effect experiments. J. Pharmacol. Exp. Ther.. Y6 (1949) ‘)‘I- I 13.

Lo. T.N., Almeida, A.P. and Beaven. M.A., Dextran and carrageenin

evoke different inflammatory responses in rat with respect to

composition of infiltrates and effect of indomethacin, J. Pharma-

col. Exp. Ther., 221 (1982) 261-267.

Lorenzetti. B.B. and Ferreira, Sri.. The analgesic effect of quater-

nary analogues of morphine and nalorphine, Braz. J. Med. Biol.

Res., IS (1982) 285-290.

Lorenzetti, B.B. and Ferreira. S.H.. Mode of analgesic action of

dipyrone direct antagonism of inflammatory hyperalgesia. Eur. J.

Pharmacol., 114 (1985) 375-381.

Lorenzetti, B.B. and Ferreira, S.H., On the mode of analgesic action

of BW443C. Br. J. Pharmacol., 90 (19X7) 69.

Lorenzetti, B.B.. Souza, G.E.P.. Sarti, S.J., Santosfilho, D. and Ferreira. S.H.. Myrcene mimics the peripheral analgesic activity

of lemongrass tea, J. Ethopharmacol.. 34 (1991) 43-4X.

Moncada, S., Ferreira, S.H. and Vane, J.R.. Inhibition of

prostaglandin biosynthesis as the mechanism of analgesia of

aspirin-like drugs in the dog knee-joint, Eur. J. Pharmacol.. 31

(197% 250-260

Nakamura, M. and Ferreira, S.H., A peripheral sympathetic compo- nent in inflammatory hyperalgesia. Eur. J. Pharmacol., 135 (1987)

145-153. Nakamura, M. and Lice. M.C.,‘ Peripheral effect of cholinergic

agents on the response to noxious stimulation in the conscious

guinea-pig, Braz. J. Med. Biol. Res.. 16 (1983) 473.

Nakamura. M. and Lice, M.C.. Mechanism of peripheral pain in the

conscious guinea-pig: effect of propranolol, Braz. J. Med. Biol.

Res.. I9 (1986) 451-453.

427

Otsuki, T., Nakahama, H., Niizuma, H. and Suzuki, J., Evaluation of

the analgesic effects of capsaicin using a new rat model for tonic

pain, Brain Res., 365 (1986) 235-240.

Pardo, E.G. and Rodriguez, R., Reversal by acetylsalicylic acid of

pain induced functional impairment, Life Sci., 5 (1966) 775-781.

Pircio, A.W., Fedele, C.T. and Bierwagen, M.E., A new method for

the evaluation of analgesic activity using adjuvant-induced arthri-

tis in the rat, Eur. J. Pharmacol., 31 (1975) 207-215.

Randall, L.O. and Sellito, J.J., A method for measurement of anal-

gesic activity on inflamed tissues, Arch. Int. Pharmacodyn., 111

(1957) 409-419.

Rios, L. and Jacob, J.J.C., Inhibition of inflammatory pain by nalox-

one and its N-methyl quaternary analogue, Life Sci., 31 (1982)

1209-1212.

Rios, L. and Jacob, J.J.C., Local inhibition of inflammatory pain by

naloxone and its N-methyl quaternary analogue, Eur. J. Pharma-

col., 96 (1983) 277-283.

Rocha, N.P. and Ferreira, S.H. Restoration by levamizole of endo-

toxin-inhibited neutrophil migration, oedema and increased vas-

cular permeability induced by carrageenin. Eur. J. Pharmacol.

122 (1986) 87-92

Rosenthale, M.E., Dervinis, A., Kassarich, J. and Singer, S.,

Prostaglandins and anti-inflammatory drugs in the dog knee-joint,

J. Pharm. Pharmacol., 24 (1972) 149-150.

Rowley, D.A. and Benditt, E.P., 5-Hydroxytryptamine and histamine

as mediators of the vascular injury produced by agents which

damage mast cells in rats, J. Exp. Med., 103 (1956) 399-415.

Sepinwall, J. and Cook, L., Behavioral pharmacology of antianxiety

drugs. In: L.L. Iversen, S.D. Iversen and S.H. Snyder (Eds.),

Handbook of Psychopharmacology-Biology of Mood and An-

tianxiety Drugs, Vol. 13, Raven Press, New York, 1978, pp. 345-393.

Shibata, M., Ohkubo, T., Takahashi, H. and Inoki, R., Modified

formalin test characteristic biphasic pain response, Pain, 38 (1989)

347-352.

Siegmund, E., Cadmus, R. and Lu, G., A method for evaluating both

non-narcotic and narcotic analgesics, Proc. Sot. Exp. Biol. Med.,

95 (1957) 729-731.

Smith, T.W., Buchan, P., Parsons, D.N. and Wilkinson, S., Periph-

eral antinociceptive effects of N-methyl morphine, Life Sci., 31

(1982) 1205-1208.

Swingle, K.F., Grant, T.J. and Kvam, D.C., Quanta1 responses in the

Randall-Sellito assay, Proc. Sot. Exp. Biol. Med., 137 (1971)

536-538.

Takesue, J.E., Schaeffer, W. and Jukniewicz, E., Modification of the

Randall-Sellito analgesic apparatus, J. Pharm., 21 (1969) 788-789.

Van Arman, G.G., Begany, A.J., Miller. L.M. and Pless, H.H., Some

details of the inflammations caused by yeast and carrageenin, J.

Pharmacol. Exp. Ther., 150 (1965) 328-334.

Van Arman, G.G., Carlson, R.P., Risley, E.A., Thomas, R.H. and

Nuss, G.W., Inhibitory effects of indomethacin, aspirin and cer-

tain other drugs on inflammations induced in rat and dog by

carrageenin. sodium urate and ellagic acid, J. Pharmacol. Exp.

Ther., 175 (1970) 459-468.

Vinegar, R., Truax, J.F and Selph, J.L., Quantitative studies of the

pathway to acute carrageenin inflammation, Fed. Proc., 35 (1976)

2447-2456.

Winter, C.A. and Flataker, L., Nociceptive threshold as affected by

parenteral administration of irritants and of various anti-nocicep-

tive drugs, J. Pharmacol. Exp. Ther., 148 (1965) 373-379.

Woolfe, G. and McDonald, A.D., The evaluation of the analgesic

action of pethidin hydrochloride (Demerol), J. Pharmacol. Exp. Ther., 80 (1944) 300-3b7.