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Influence of formalin concentration on the antinociceptive effects of anti-inflammatory drugs in the formalin test in rats: separate mechanisms underlying the nociceptive effects of low- and high-concentration formalin
European
Journal
of Pain ( 1998) 2: 63-68
Influence.of formalin concentration on the antinociceptive effects of anti-inflammatory drugs in the formalin test in rats: separate mechanisms underlying the nociceptive effects of low- and high-concentration formalin K. Yashpal” and T. J. CoderTea* “Pain Mechanisms Laboratory, Clinical Research Institute of Montrkal, Quebec,, Canada bCentre de recherche en sciences neurologiques et dbpartement de mhdecine, Universiki Mont&al, Montrkal, Quebec, Canada ‘Department of Psychology, McGill University, Montrkal, Quebec, Canada
de
The present study has assessed the relationship between formalin-induced nociception and formalin-induced inflammation by comparing the dose-related effects of anti-inflammatory treatments on both nociceptive scores and plasma extravasation in the rat hind paw in response to high and low concentrations of formalin. The degree of plasma extravasation produced by 1% formalin did not differ significantly from that produced by the same volume of saline, and was not significantly affected by either of the anti-inflammatory agents. The 5% formalin injection produced significant plasma extravasation that was dose-dependently reduced by both dexamethasone and ibuprofen. The early-phase nociceptive responses to either 1 or 5% formalin were not affected significantly by either of the anti-inflammatory agents. In contrast, the late-phase nociceptive responses to 5%, but not l%, formalin were dose-dependently reduced by both dexamethasone and ibuprofen. The present study suggests that there is a positive correlation between the nociceptive and inflammatory effects of formalin in the rat hind paw. However, only a high concentration of formalin, which produces significant plasma extravasation, is capable of demonstrating the antinociceptive effects of anti-inflammatory agents, and the effects are restricted to the late phase of the formalin test.
INTRODUCTION The formalin test is now used frequently as a nociceptive assay for modelling persistent pain in laboratory animals (Tjolsen et al., 1992). Although originally described as an animal model of tonic inflammatory pain (O’Keefe, 1964; Dubuisson & Dennis, 1977), the formalin test has also received much attention recently Paper received 11 November 1997 and accepted in revised form 15 February 1998. Correspondence to: T. J. Coderre, Director, Pain Mechanisms Laboratory, Clinical Research Institute of Montreal, 110 Pine Ave. West, Montreal, Quebec, H2W 1R7, Canada. 1090-3801/98/010063+06 $12.0010 0 1998 European Federation of Chapters
of the International
as a proposed model of persistent pain which depends on sensitization in spinal cord dorsal horn (Coderre et al., 1990, 1993b, 1994) and brain (Vaccarino et al., 1989, 1992; Coderre et al., 1993b). In particular, it has been demonstrated that nociceptive responsesin the late phaseof the formalin test are, in part, dependent on nociceptive inputs which impinge on the central nervous system during the early phase (Dickenson & Sullivan, 1987; Vaccarino et al., 1989, 1992; Coderre et al., 1990). While persistent nociceptive responses to formalin may, in part, be dependent on central sensitization, there is also, most likely, a significant role of peripheral inflammation (Winter et al., 1962; Association
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Yashpal et al., 1996). However, the relationship dependenceon central sensitization. The hypobetween the nociceptive and the inflammatory thesis that nociceptive responses to high-, opresponsesto formalin is not well understood. posed to low-, concentration formalin depend to Early reports suggestedthat the early phase of a greaterextent on peripheral inflammation could be further tested by comparing the effects of the formalin responseis due to the direct stimulation of nociceptors, while the late phase is anti-inflammatory treatments in animals given directly due to an ensuinginflammatory response different concentrations of formalin. Thus, the present study has assessedthe re(O’Keefe, 1964;Dubuisson & Dennis, 1977).Inlationship betweenformalin-induced nociception deed, steroids and non-steroidal anti-inflammatory drugs (NSAIDs) reduce nociception and formalin-induced inflammation by comin the late phase, but have little or no effect on paring the dose-related effects of antiinflammatory treatments (steroids and NSAIDs) the early phaseresponsesto formalin (Hunskaar et al., 1986; Hunskaar & Hole, 1987; Shibata et on both nociceptive scores and plasma extravasation in the rat hind paw in response to al., 1989).A significant contribution of peripheral varying concentrations of formalin. Since the inflammation to the formalin responseis further supported by the fact that injection of between authors’ previous data (Yasphal et al., 1996) 4 and 5% formalin into a rat’s hind paw increases suggeststhat nociceptive responsesto low conpaw volume by 30-35% within 1 h after the centrations of formalin dependto a greater extent injection (Brown et al., 1968; Wheeler-Aceto et on central sensitization, and nociceptive responses to higher concentrations depend to a al., 1991).It is important to note, however, that formalin-induced oedemadoesnot reach its peak greater extent on peripheral inflammation, the until 4-5 h after injection (Brown et al., 1968; authors expectthat anti-inflammatory treatments will more effectively alleviate nociceptive reWheeler-Aceto et al., 1990), while its peak nosponses in rats given high concentrations of formciceptive responseis exhibited between20 and 35 min post-injection (Dubuisson & Dennis, 1977; alin. Wheeler-Aceto et al., 1991). Furthermore, the authors’ recent findings indicate that a 2.5% METHODS concentration of formalin producesa high degree of nociceptive responses,but elicits inflammatory The dose-related antinociceptive and antiresponses(plasma extravasation) which do not inflammatory effectsof systemically administered differ significantly from that produced by hindsteroid (dexamethasone)and non-steroidal (ibupaw injection of saline (Yashpal et al., 1996). profen) anti-inflammatory agentswere examined Recent data suggestthere is a concentrationin rats that received a subcutaneous (s.c.) independent increase in nociceptive responsesto jection of formalin into the plantar surface of formalin in both mice (Rosland et al., 1990)and the hind paw. Fourteen groups of male Long rats (Coderre et al., 1993~). However, there is Evans rats (250-350g) were administered either also evidence suggesting that there are different dexamethasone [ 1.O-6.25mg/kg, intraperitoneal mechanismsunderlying the nociceptive responses (i.p.)], ibuprofen (40-250 mg/kg, i.p.) or vehicle to low and high concentrations of formalin. The (45% 2-hydroxypropyl-P-cyclodextrin in saline). authors’ recent data (Yasphal et al., 1996) inOne hour later, rats were given 50 1.11 of either dicates that although it is possible for an in1 or 5% formalin, placed in a 30 x 30 x 30 cm trathecal lignocaine treatment to produce a pre- Plexiglas@ box and observed for 60min. Earlyemptive blockade of late phase nociceptive re- phase(O-5 min) and late-phase(25-60 min) nocisponsesto low concentration formalin, this effect ceptive responsesto formalin were assessedusing is lost at higher formalin concentrations. The the weighted-scoresmethod of behavioural rating authors concluded that as the concentration of (Coderre et al., 1993~). Briefly, a nociceptive formalin is increased, there is a greater de- score was derived by measuring the amount of pendence of the nociceptive responseson peri- time spent in eachof four behavioural categories: pheral inflammatory changes, and less 0, the injected paw is not favoured; 1, the injected European
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paw has little or no weight on it; 2, the injected paw is elevated and is not in contact with any surface; 3, the injected paw is licked, bitten or shaken. A weighted average nociceptive score, ranging from 0 to 3, wascalculated by multiplying the time spent in each category by the category weight, summing these products, and then dividing by the total time for each period. Formalin-induced inflammatory responses were assessedafter the same treatments by examining the degree of plasma extravasation 60 min after injection of 50 ~1 of either 1 or 5% formalin, as indicated by Evans Blue dye leakage, into the tissue of the injured hind paw. Responseswere compared with those in untreated rats that received a hind-paw injection of 50 ,~l of saline. Plasma extravasation was quantified by spectrophotometric analysisof the absorbanceat 620 nm of Evans Blue dye extracted from the hind paw, and converted to &ml using a standard (linear) curve for absorbancevs concentration. Rats were given an intravenous (tail vein) injection of Evans Blue dye (50mg/kg in 2.5 ml/kg) 30 min prior to the formalin injection. After formalin testing (60 min post-formalin), the rats were anaesthetized with a high dose of chloral hydrate (300mg/kg, i.p.), and after intracardiac perfusion with 0.9% saline to flush blood from the circulation, they were killed by decapitation and their hind paws were removed by amputation at the ankle joint. The hind paws were then incubated in 4 ml of formamide at 60°C for 24 h to extract Evans Blue dye from the tissue. After cooling to room temperature, plasma extravasation was recorded as the absorbance of the resulting supernate in a spectrophotometer at a wavelength of 620 nm. RESULTS The effects of ibuprofen and dexamethasone on formalin-induced plasma extravasation are shown in Fig. 1. The degree of plasma extravasation produced by 1% formalin did not differ significantly from that produced by the same volume of saline (No Form), and was not affected significantly by any dose of either of the antiinflammatory agents[Fig. 1(a)]. The 5% formalin injection produced significant plasma ex-
g (b) t
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FIG. 1. Plasma extravasation indicated by Evans Blue dye (pg/ml) extracted from the injected hind paw of rats given a hind paw injection of 50~1 1% (a) or 5% (b) formalin preceded by treatment with systemic (intraperitoneal)vehicle(n=lOforl%andn=6for5%), ibuprofen (IBU, 40-250 mg/kg, n=6,6,7 for 1% and n= 6,6,6 for 5%) or dexamethasone (DEX,l.O-6.25 mg/kg, n=7,6,6for 1% and n=8,6,5for 5%), ascompared with rats that received only a hind-paw injection of the same volume of saline (No Form, n=4). ANOVAfor groups of rats receiving 1% formalin (a) reveals a non-significant effect of treatment group [F(7,44) = 1.78, p>O.O51. ANOVA for groups of rats receiving 5% formalin (b) reveals a significant effect oftreatment group [F(7,39) = 6.6, p
travasation in vehicle-treated rats that was dosedependentlyreducedby both dexamethasoneand ibuprofen [Fig. l(b)]. The effectsof ibuprofen and dexamethasoneon formalin-induced nociceptive European
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2.5 c (a)
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FIG. 2. Early (a) and late-phase (b) phase nociceptive scores (+SEM) of rats given a hind-paw injection of 50 ~1 1% formalin preceded by treatment with systemic (intraperitoneal) vehicle (n=8), ibuprofen (IBU, 40-250 mg/kg, n=6,6,9) or dexamethasone (DEX, 1.0-6.25 mg/kg, n=7,7,6). ANOVAs for both (a) earlyphase [F(6,42)=0.11, ~0.051 and (b) late-phase [F(6, 42 =0.37, p10.051 nociceptive scores reveal nonsignificant effects of treatment group.
behaviours are shown in Fig. 2 (1% formalin) and Fig. 3 (5% formalin). The early-phasenociceptive responsesto either 1% [Fig. 2(a)] or 5% [Fig. 3(a)] formalin were not affected significantly by either of the anti-inflammatory agents. In contrast, the late phase nociceptive responsesto 5% [Fig. 3(b)], but not 1% [Fig. 2(b)], formalin were dose-dependently reduced by both dexamethasone and ibuprofen. Significant reductions of late-phase nociceptive responsesto 5% formalin occurred with the 100 and 250mg/kg doses European
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FIG. 3. Early-phase (a) and late-phase(b) nociceptive scores (*SEMI of rats given a hind-paw injection of 50~1 5% formalin preceded by treatment with systemic (intraperitoneal) vehicle (n=6), ibuprofen (IBU, 40-250 mg/kg, n=6,6,5) or dexamethasone (DEX,1.0-6.25 mg/kg,n=7,5,6).ANOVAfortheearlyphase (a) nociceptive scores reveals a nonsignificant effect of treatment group [F(6,401=0.46, ~0.05). ANOVA for the late phase (b) nociceptive scores reveals a significant main effect of treatment Significant [F(6,40) =8.35, pcO.01). group differences from the vehicle control group are indicated by asterisks (* pcO.05, ** pcO.01).
of ibuprofen and the 2.5 and 6.25mg/kg doses of dexamethasone. DISCUSSION The present study suggeststhat there is a positive correlation between the nociceptive and in-
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flammatory effects of formalin in the rat hind paw. However, this relationship is dependent on the concentration of formalin injected into the rat’s hind paw. Thus, only a high concentration of formalin, which produces significant plasma extravasation, is capable of demonstrating the antinociceptive effects of anti-inflammatory agents. Furthermore, the antinociceptive effects of dexamethasone and ibuprofen parallel their anti-inflammatory actions, but are observed exclusively in the late phaseof a high-concentration formalin test. To date, there are few studies of the varying effects of anti-inflammatory treatments on nociceptive responsesto different concentrations of formalin; these have been performed only in mice, and are contradictory. Thus, Hunskaar et al. (1985) found that indomethacin reduced late phase responsesto both 1 and 5% formalin, while Rosland et al. (1990) showed that indomethacin reduced late phase nociceptive responsesto 1 but not 5% formalin. Until now, there has beenno detailed assessment of differencesin the antinociceptiveeffectsof antiinflammatory agents given to rats with varying concentrations of formalin. However, it is known that mice exhibit a relatively greater inflammatory responseto the same concentration of formalin given to rats (Tjolsen et al., 1992). Although there is no explanation for the discrepancy between the results of Hunskaar et al. (1985) and Rosland et al. (1990) in mice, it is likely that the 1% concentration of formalin produces a large enough degree of peripheral inflammation that it is sensitive to antiinflammatory treatments, while in rats, antiinflammatory treatments only reduce formalin responsesto higher concentrations of formalin. The data support the hypothesis that the nociceptive responsesto low- and high-concentration formalin rely on different mechanisms. Together with the authors’ previous data (Yashpal et al., 1996), there is evidenceto suggestthat, in rats, nociceptive responsesto low concentrations of formalin rely very little on peripheral inflammatory responses.It is likely that persistent nociceptive responsesto low concentrations of formalin rely partly on central sensitization, since late-phasenociceptive responsesare inhibited by pre-emptive treatments given during the early
phase. Persistent nociceptive responses to low concentration formalin may also depend on a direct effect of formalin on nociceptors, since several investigators have found that formalin produces a biphasic activation of C-fibres, although late-phaseC-fibre activity is considerably less with low concentration formalin (Abram et al., 1996; McCall et al., 1996) than for higher concentrations (Taylor et al., 1995; Puig & Sorkin, 1996). On the other hand, persistent nociceptive responses to high-concentration formalin rely to a greater extent on peripheral inflammation, since they are not influenced by pre-emptive treatment with spinal lignocaine, but are dose-dependently reduced by anti-inflammatory agents in a manner consistent with their anti-inflammatory effects.It should be noted that the inflammatory and nociceptive responses to formalin were only affected by relatively high dosesof the anti-inflammatory agents.This likely reflects the long known finding that formalin oedemais not particularly sensitiveto many antiinflammatory agents (Winter et al., 1962).As a consequence,it is possible that some of the effects of the systemic anti-inflammatory treatments may rely on central actions of theseagents,since the central administration of NSAIDs and steroids has been found to produce significant analgesia in the formalin test (Malmberg & Yaksh, 1992; Coderre & Yashpal, 1994). However, this possibility is unlikely sinceeven the highest doses of the anti-inflammatory agents did not reduce late-phase nociceptive responses to low-concentration formalin, or early-phaseresponsesto low or high concentration formalin. ACKNOWLEDGEMENTS The authors wish to thank Jennifer Ritchie for technical assistance.This work was supported by an MRC grant (MT-13236) to T.J.C. REFERENCES Abram SE, Dean C, O’Connor TC. Peroneal afferent nerve discharges underlying the behavioral response to the formalin test. Regional Anesth 1996; 21: 226-233. Brown JH, Kissel JW, Lish PM. Studies on the acute inflammatory response. I. Involvement of the central European
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68 nervous system in certain models of inflammation. J pharmacol Exp Ther 1968; 160: 231-242. Coderre TJ, Fundytus MF, McKenna J, Dahl S, Melzack R. The formalin test: a validation of the weighted-scores method of behavioural pain rating. Pain 1993a; 54: 43350. Coderre TJ, Katz J, Vaccarino AL, Melzack R. Contribution of central neuroplasticity to pathological pain: Review of clinical and experimental evidence. Pain 1993b; 52: 259-285. Coderre TJ, Vaccarino AL, Melzack R. Central nervous system plasticity in the tonic pain response to subcutaneous formalin injection. Brain Res 1990; 535: 155158. Coderre TJ, Yashpal K. Intracellular messengers contributing to persistent nociception and hyperalgesia induced by L-glutamate and substance P in the rat formalin pain model. Eur J Neurosci 1994; 6: 1328-1334. Coderre TJ, Yashpal K, Henry JL. Specific contribution of lumbar spinal mechanisms to persistent nociceptive responses in the formalin test. NeuroReport, 1994; 5: 133771340. Dickenson AH, Sullivan AF. Subcutaneous formalininduced activity of dorsal horn neurons in the rat: differential response to an intrathecal opiate administration pre or post formalin. Pain 1987; 30: 349-360. Dubuisson D, Dennis SG, The formalin test: a quantitative the analgesic effects of morphine, study of meperidine, and brain stimulation in rats and cats. Pain 1977; 4:161-74. Hunskaar S, Fasmer OB, Hole K. Formalin test in mice, a useful technique for evaluating mild analgesics. J Neurosci Methods 1985; 14: 69-76. Hunskaar S, Berge O-G, Hole K. Dissociation between antinociceptive and anti-inflammatory effects of acetylsalicylic acid and indomethacin in the formalin test. Pain 1986; 25: 125-132. Hunskaar S, Hole K. The formalin test in mice: dissociation between inflammatory and non-inflammatory pain. Pain 1987; 30: 103-114. O’Keefe J. Spinal Cord Mechanisms Subserving Pain Perception, Unpublished M.Sc. Thesis, McGill University, Canada, 1964. Malmberg AB, Yaksh TL, Antinociceptive actions of spinal
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nonsteroidal anti-inflammatory agents on the formalin test in the rat. J Pharmacol Exp Ther 1992; 263: 136- 146. McCall WD, Tanner KD, Levine JD. Formalin induces biphasic activity in c-fibers in the rat. Neurosci Lett 1996; 208: 45-48. Puig S, Sorkin LS. Formalin-evoked activity in identified primary afferent fibers-systemic lidocaine suppresses phase-2 activity. Pain 1996; 64: 345-355. Rosland JH, Tjolsen A, Mahle B, Hole K. The formalin test in mice---effect of formalin concentration. Pain 1990; 42: 235-242. Shibata M, Ohkubo T, Takahashi H, Inoki R. Modified formalin test: Characteristic biphasic pain response. Pain 1989; 38:347-352. Taylor BK, Peterson MA, Basbaum AI. Persistent cardiovascular and behavioral nociceptive responses to subcutaneous formalin require peripheral nerve input. J Neurosci 1995; 15: 7575-7584. Tjolsen A, Berge O-G, Hunskaar S, Rosland JH, Hole K. The formalin test: an evaluation of the method. Pain 1992; 51: 5-17. Vaccarino AL, Melzack R. Analgesia produced by injection of lidocaine into the anterior cingulum bundle of the rat. Pain 1989; 39: 2133219. Vaccarino AL, Melzack R. Temporal processes of formalin pain: differential role of the cingulum bundle, fornix pathway and medial bulboreticular formation. Pain 1992; 49: 257-271. Wheeler-Aceto H, Porreca F, Cowan A. The rat paw formalin test: comparison of noxious agents. Pain 1990; 40: 229-238. Wheeler-Aceto H, Cowan A. Neurogenic and tissuemediated components of formalin-induced edema: Evidence for supraspinal regulation, Agents Actions. 1991; 34: 264269. Winter CA, Risley EA, Nuss GW. Carrageenin-induced edema in hind paw of rat as an assay for antiinflammatory drugs. Proc Sot exp Biol (N. E) 1962; 111: 544. Yashpal K, Katz J, Coderre TJ. Effects of preemptive or postinjury intrathecal local anesthesia on persistent nociceptive responses in rats: Confounding influences of peripheral inflammation and the general anesthetic regimen. Anesthesiology 1996; 84: 1119-1128.
Journal
of Pain ( 1998) 2: 63-68
Influence.of formalin concentration on the antinociceptive effects of anti-inflammatory drugs in the formalin test in rats: separate mechanisms underlying the nociceptive effects of low- and high-concentration formalin K. Yashpal” and T. J. CoderTea* “Pain Mechanisms Laboratory, Clinical Research Institute of Montrkal, Quebec,, Canada bCentre de recherche en sciences neurologiques et dbpartement de mhdecine, Universiki Mont&al, Montrkal, Quebec, Canada ‘Department of Psychology, McGill University, Montrkal, Quebec, Canada
de
The present study has assessed the relationship between formalin-induced nociception and formalin-induced inflammation by comparing the dose-related effects of anti-inflammatory treatments on both nociceptive scores and plasma extravasation in the rat hind paw in response to high and low concentrations of formalin. The degree of plasma extravasation produced by 1% formalin did not differ significantly from that produced by the same volume of saline, and was not significantly affected by either of the anti-inflammatory agents. The 5% formalin injection produced significant plasma extravasation that was dose-dependently reduced by both dexamethasone and ibuprofen. The early-phase nociceptive responses to either 1 or 5% formalin were not affected significantly by either of the anti-inflammatory agents. In contrast, the late-phase nociceptive responses to 5%, but not l%, formalin were dose-dependently reduced by both dexamethasone and ibuprofen. The present study suggests that there is a positive correlation between the nociceptive and inflammatory effects of formalin in the rat hind paw. However, only a high concentration of formalin, which produces significant plasma extravasation, is capable of demonstrating the antinociceptive effects of anti-inflammatory agents, and the effects are restricted to the late phase of the formalin test.
INTRODUCTION The formalin test is now used frequently as a nociceptive assay for modelling persistent pain in laboratory animals (Tjolsen et al., 1992). Although originally described as an animal model of tonic inflammatory pain (O’Keefe, 1964; Dubuisson & Dennis, 1977), the formalin test has also received much attention recently Paper received 11 November 1997 and accepted in revised form 15 February 1998. Correspondence to: T. J. Coderre, Director, Pain Mechanisms Laboratory, Clinical Research Institute of Montreal, 110 Pine Ave. West, Montreal, Quebec, H2W 1R7, Canada. 1090-3801/98/010063+06 $12.0010 0 1998 European Federation of Chapters
of the International
as a proposed model of persistent pain which depends on sensitization in spinal cord dorsal horn (Coderre et al., 1990, 1993b, 1994) and brain (Vaccarino et al., 1989, 1992; Coderre et al., 1993b). In particular, it has been demonstrated that nociceptive responsesin the late phaseof the formalin test are, in part, dependent on nociceptive inputs which impinge on the central nervous system during the early phase (Dickenson & Sullivan, 1987; Vaccarino et al., 1989, 1992; Coderre et al., 1990). While persistent nociceptive responses to formalin may, in part, be dependent on central sensitization, there is also, most likely, a significant role of peripheral inflammation (Winter et al., 1962; Association
for the Study
of Pain
64
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AND
T. J. CODERRE
Yashpal et al., 1996). However, the relationship dependenceon central sensitization. The hypobetween the nociceptive and the inflammatory thesis that nociceptive responses to high-, opresponsesto formalin is not well understood. posed to low-, concentration formalin depend to Early reports suggestedthat the early phase of a greaterextent on peripheral inflammation could be further tested by comparing the effects of the formalin responseis due to the direct stimulation of nociceptors, while the late phase is anti-inflammatory treatments in animals given directly due to an ensuinginflammatory response different concentrations of formalin. Thus, the present study has assessedthe re(O’Keefe, 1964;Dubuisson & Dennis, 1977).Inlationship betweenformalin-induced nociception deed, steroids and non-steroidal anti-inflammatory drugs (NSAIDs) reduce nociception and formalin-induced inflammation by comin the late phase, but have little or no effect on paring the dose-related effects of antiinflammatory treatments (steroids and NSAIDs) the early phaseresponsesto formalin (Hunskaar et al., 1986; Hunskaar & Hole, 1987; Shibata et on both nociceptive scores and plasma extravasation in the rat hind paw in response to al., 1989).A significant contribution of peripheral varying concentrations of formalin. Since the inflammation to the formalin responseis further supported by the fact that injection of between authors’ previous data (Yasphal et al., 1996) 4 and 5% formalin into a rat’s hind paw increases suggeststhat nociceptive responsesto low conpaw volume by 30-35% within 1 h after the centrations of formalin dependto a greater extent injection (Brown et al., 1968; Wheeler-Aceto et on central sensitization, and nociceptive responses to higher concentrations depend to a al., 1991).It is important to note, however, that formalin-induced oedemadoesnot reach its peak greater extent on peripheral inflammation, the until 4-5 h after injection (Brown et al., 1968; authors expectthat anti-inflammatory treatments will more effectively alleviate nociceptive reWheeler-Aceto et al., 1990), while its peak nosponses in rats given high concentrations of formciceptive responseis exhibited between20 and 35 min post-injection (Dubuisson & Dennis, 1977; alin. Wheeler-Aceto et al., 1991). Furthermore, the authors’ recent findings indicate that a 2.5% METHODS concentration of formalin producesa high degree of nociceptive responses,but elicits inflammatory The dose-related antinociceptive and antiresponses(plasma extravasation) which do not inflammatory effectsof systemically administered differ significantly from that produced by hindsteroid (dexamethasone)and non-steroidal (ibupaw injection of saline (Yashpal et al., 1996). profen) anti-inflammatory agentswere examined Recent data suggestthere is a concentrationin rats that received a subcutaneous (s.c.) independent increase in nociceptive responsesto jection of formalin into the plantar surface of formalin in both mice (Rosland et al., 1990)and the hind paw. Fourteen groups of male Long rats (Coderre et al., 1993~). However, there is Evans rats (250-350g) were administered either also evidence suggesting that there are different dexamethasone [ 1.O-6.25mg/kg, intraperitoneal mechanismsunderlying the nociceptive responses (i.p.)], ibuprofen (40-250 mg/kg, i.p.) or vehicle to low and high concentrations of formalin. The (45% 2-hydroxypropyl-P-cyclodextrin in saline). authors’ recent data (Yasphal et al., 1996) inOne hour later, rats were given 50 1.11 of either dicates that although it is possible for an in1 or 5% formalin, placed in a 30 x 30 x 30 cm trathecal lignocaine treatment to produce a pre- Plexiglas@ box and observed for 60min. Earlyemptive blockade of late phase nociceptive re- phase(O-5 min) and late-phase(25-60 min) nocisponsesto low concentration formalin, this effect ceptive responsesto formalin were assessedusing is lost at higher formalin concentrations. The the weighted-scoresmethod of behavioural rating authors concluded that as the concentration of (Coderre et al., 1993~). Briefly, a nociceptive formalin is increased, there is a greater de- score was derived by measuring the amount of pendence of the nociceptive responseson peri- time spent in eachof four behavioural categories: pheral inflammatory changes, and less 0, the injected paw is not favoured; 1, the injected European
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paw has little or no weight on it; 2, the injected paw is elevated and is not in contact with any surface; 3, the injected paw is licked, bitten or shaken. A weighted average nociceptive score, ranging from 0 to 3, wascalculated by multiplying the time spent in each category by the category weight, summing these products, and then dividing by the total time for each period. Formalin-induced inflammatory responses were assessedafter the same treatments by examining the degree of plasma extravasation 60 min after injection of 50 ~1 of either 1 or 5% formalin, as indicated by Evans Blue dye leakage, into the tissue of the injured hind paw. Responseswere compared with those in untreated rats that received a hind-paw injection of 50 ,~l of saline. Plasma extravasation was quantified by spectrophotometric analysisof the absorbanceat 620 nm of Evans Blue dye extracted from the hind paw, and converted to &ml using a standard (linear) curve for absorbancevs concentration. Rats were given an intravenous (tail vein) injection of Evans Blue dye (50mg/kg in 2.5 ml/kg) 30 min prior to the formalin injection. After formalin testing (60 min post-formalin), the rats were anaesthetized with a high dose of chloral hydrate (300mg/kg, i.p.), and after intracardiac perfusion with 0.9% saline to flush blood from the circulation, they were killed by decapitation and their hind paws were removed by amputation at the ankle joint. The hind paws were then incubated in 4 ml of formamide at 60°C for 24 h to extract Evans Blue dye from the tissue. After cooling to room temperature, plasma extravasation was recorded as the absorbance of the resulting supernate in a spectrophotometer at a wavelength of 620 nm. RESULTS The effects of ibuprofen and dexamethasone on formalin-induced plasma extravasation are shown in Fig. 1. The degree of plasma extravasation produced by 1% formalin did not differ significantly from that produced by the same volume of saline (No Form), and was not affected significantly by any dose of either of the antiinflammatory agents[Fig. 1(a)]. The 5% formalin injection produced significant plasma ex-
g (b) t
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FIG. 1. Plasma extravasation indicated by Evans Blue dye (pg/ml) extracted from the injected hind paw of rats given a hind paw injection of 50~1 1% (a) or 5% (b) formalin preceded by treatment with systemic (intraperitoneal)vehicle(n=lOforl%andn=6for5%), ibuprofen (IBU, 40-250 mg/kg, n=6,6,7 for 1% and n= 6,6,6 for 5%) or dexamethasone (DEX,l.O-6.25 mg/kg, n=7,6,6for 1% and n=8,6,5for 5%), ascompared with rats that received only a hind-paw injection of the same volume of saline (No Form, n=4). ANOVAfor groups of rats receiving 1% formalin (a) reveals a non-significant effect of treatment group [F(7,44) = 1.78, p>O.O51. ANOVA for groups of rats receiving 5% formalin (b) reveals a significant effect oftreatment group [F(7,39) = 6.6, p
travasation in vehicle-treated rats that was dosedependentlyreducedby both dexamethasoneand ibuprofen [Fig. l(b)]. The effectsof ibuprofen and dexamethasoneon formalin-induced nociceptive European
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2.5 c (a)
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FIG. 2. Early (a) and late-phase (b) phase nociceptive scores (+SEM) of rats given a hind-paw injection of 50 ~1 1% formalin preceded by treatment with systemic (intraperitoneal) vehicle (n=8), ibuprofen (IBU, 40-250 mg/kg, n=6,6,9) or dexamethasone (DEX, 1.0-6.25 mg/kg, n=7,7,6). ANOVAs for both (a) earlyphase [F(6,42)=0.11, ~0.051 and (b) late-phase [F(6, 42 =0.37, p10.051 nociceptive scores reveal nonsignificant effects of treatment group.
behaviours are shown in Fig. 2 (1% formalin) and Fig. 3 (5% formalin). The early-phasenociceptive responsesto either 1% [Fig. 2(a)] or 5% [Fig. 3(a)] formalin were not affected significantly by either of the anti-inflammatory agents. In contrast, the late phase nociceptive responsesto 5% [Fig. 3(b)], but not 1% [Fig. 2(b)], formalin were dose-dependently reduced by both dexamethasone and ibuprofen. Significant reductions of late-phase nociceptive responsesto 5% formalin occurred with the 100 and 250mg/kg doses European
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FIG. 3. Early-phase (a) and late-phase(b) nociceptive scores (*SEMI of rats given a hind-paw injection of 50~1 5% formalin preceded by treatment with systemic (intraperitoneal) vehicle (n=6), ibuprofen (IBU, 40-250 mg/kg, n=6,6,5) or dexamethasone (DEX,1.0-6.25 mg/kg,n=7,5,6).ANOVAfortheearlyphase (a) nociceptive scores reveals a nonsignificant effect of treatment group [F(6,401=0.46, ~0.05). ANOVA for the late phase (b) nociceptive scores reveals a significant main effect of treatment Significant [F(6,40) =8.35, pcO.01). group differences from the vehicle control group are indicated by asterisks (* pcO.05, ** pcO.01).
of ibuprofen and the 2.5 and 6.25mg/kg doses of dexamethasone. DISCUSSION The present study suggeststhat there is a positive correlation between the nociceptive and in-
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flammatory effects of formalin in the rat hind paw. However, this relationship is dependent on the concentration of formalin injected into the rat’s hind paw. Thus, only a high concentration of formalin, which produces significant plasma extravasation, is capable of demonstrating the antinociceptive effects of anti-inflammatory agents. Furthermore, the antinociceptive effects of dexamethasone and ibuprofen parallel their anti-inflammatory actions, but are observed exclusively in the late phaseof a high-concentration formalin test. To date, there are few studies of the varying effects of anti-inflammatory treatments on nociceptive responsesto different concentrations of formalin; these have been performed only in mice, and are contradictory. Thus, Hunskaar et al. (1985) found that indomethacin reduced late phase responsesto both 1 and 5% formalin, while Rosland et al. (1990) showed that indomethacin reduced late phase nociceptive responsesto 1 but not 5% formalin. Until now, there has beenno detailed assessment of differencesin the antinociceptiveeffectsof antiinflammatory agents given to rats with varying concentrations of formalin. However, it is known that mice exhibit a relatively greater inflammatory responseto the same concentration of formalin given to rats (Tjolsen et al., 1992). Although there is no explanation for the discrepancy between the results of Hunskaar et al. (1985) and Rosland et al. (1990) in mice, it is likely that the 1% concentration of formalin produces a large enough degree of peripheral inflammation that it is sensitive to antiinflammatory treatments, while in rats, antiinflammatory treatments only reduce formalin responsesto higher concentrations of formalin. The data support the hypothesis that the nociceptive responsesto low- and high-concentration formalin rely on different mechanisms. Together with the authors’ previous data (Yashpal et al., 1996), there is evidenceto suggestthat, in rats, nociceptive responsesto low concentrations of formalin rely very little on peripheral inflammatory responses.It is likely that persistent nociceptive responsesto low concentrations of formalin rely partly on central sensitization, since late-phasenociceptive responsesare inhibited by pre-emptive treatments given during the early
phase. Persistent nociceptive responses to low concentration formalin may also depend on a direct effect of formalin on nociceptors, since several investigators have found that formalin produces a biphasic activation of C-fibres, although late-phaseC-fibre activity is considerably less with low concentration formalin (Abram et al., 1996; McCall et al., 1996) than for higher concentrations (Taylor et al., 1995; Puig & Sorkin, 1996). On the other hand, persistent nociceptive responses to high-concentration formalin rely to a greater extent on peripheral inflammation, since they are not influenced by pre-emptive treatment with spinal lignocaine, but are dose-dependently reduced by anti-inflammatory agents in a manner consistent with their anti-inflammatory effects.It should be noted that the inflammatory and nociceptive responses to formalin were only affected by relatively high dosesof the anti-inflammatory agents.This likely reflects the long known finding that formalin oedemais not particularly sensitiveto many antiinflammatory agents (Winter et al., 1962).As a consequence,it is possible that some of the effects of the systemic anti-inflammatory treatments may rely on central actions of theseagents,since the central administration of NSAIDs and steroids has been found to produce significant analgesia in the formalin test (Malmberg & Yaksh, 1992; Coderre & Yashpal, 1994). However, this possibility is unlikely sinceeven the highest doses of the anti-inflammatory agents did not reduce late-phase nociceptive responses to low-concentration formalin, or early-phaseresponsesto low or high concentration formalin. ACKNOWLEDGEMENTS The authors wish to thank Jennifer Ritchie for technical assistance.This work was supported by an MRC grant (MT-13236) to T.J.C. REFERENCES Abram SE, Dean C, O’Connor TC. Peroneal afferent nerve discharges underlying the behavioral response to the formalin test. Regional Anesth 1996; 21: 226-233. Brown JH, Kissel JW, Lish PM. Studies on the acute inflammatory response. I. Involvement of the central European
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