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Central nervous system plasticity in the tonic pain response to subcutaneous formalin injection
Brain Research, 535 (1990) 155-158 Elsevier
155
BRES2~14
Central nervous system plasticity in the tonic pain response to subcutaneous formalin injection Terence J. Coderre 1'2, A n t h o n y L. Vaccarino 1 and R o n a l d Melzack 1 1Department of Psychology, McGill University and 2pain Mechanisms Research Laboratory, Clinical Research Institute of Montreal, Montreal, Que. (Canada)
(Accepted 21 August 1990) Key words: Nociception; Inflammation; Hyperaigesia; Injury; Local Anesthesia; Neuroplasticity
Evidence is presented which suggests that central neural changes occur during the brief early phase after subcutaneous formalin injection that are essential for the expression of pain during the long-lasting (tonic) later phase. First, tonic pain responses to subcutaneous formalin injections are abolished only if the injected hindpaw is locally anesthetized at the time of injection as well as the time of testing (30-60 rain later). Second, tonic formalin pain is substantially reduced by brief spinal anesthesia given 5 min before, but not 5 min after the formalin injection. Subcutaneous injection of dilute formalin produces a biphasic pain response which has been observed in rats, cats s, mice 11 and primates 1. An earl~, brief phase of intense pain occurs in the first 5 min, pain then subsides for 10-15 min, and a later tonic phase of moderate pain is expressed from 20 to 60 min after injection. It has been suggested that the transient early phase is due to direct stimulation of nociceptors, while the tonic phase is due to an ensuing inflammatory response 8. Indeed, non-steroidal anti-inflammatory agents (aspirin, indomethacin) and steroids (hydrocortisone, dexamethasone) produce analgesia in the late phase, but have little or no effect on the early pain produced by formalin injection 11'12'18. The dissociation of the effects of anti-inflammatory agents on the early and late phases of the formalin response has suggested that the two phases represent two independent processes which rely on separate neural systems or hyperalgesic mediators. Although the above studies show clearly that analgesia can be produced in the late phase of the formalin test by agents that do not affect the early phase, the opposite has not been demonstrated - - that is, analgesia during the early phase only. Centrally acting narcotic analgesics, such as morphine, pentazocine and meperidine, which produce analgesia during the early phase, are also analgesic in the late phase 11'12"18"23. Furthermore, Dickenson and Sullivan 7 have demonstrated that intrathecal administration of the /~-opiate agonist D A M G O (TyrD-Ala,Gly,Me,Phe,Gly-ol) significantly inhibits forma-
lin-induced increases in dorsal horn activity. However, this inhibition occurs only if the drug is given before the formalin injection, and not if it is given 2 min after the formalin injection. These results imply that the dorsal horn activity associated with the late phase of formalin pain depends upon spinal activation during the early phase 7. Thus, in addition to a dissociation of early and late formalin pain based on separate mechanisms involving nociceptor stimulation and inflammation, there is also the possibility of a second dissociation in which the early phase is a response to the direct stimulation of nociceptors, while the late phase is dependent on changes in central nervous system (CNS) function induced by neural activity generated during the early phase. The present experiment examines the contribution of CNS plasticity to the tonic late phase of the formalin response by assessing the effects of anesthetic agents, given in a local subcutaneous injection or intrathecally, either before or after the early phase of the formalin response. In all studies, Long-Evans male rats (250-350 g) were given a subcutaneous injection of 50 pl of 2.5% formalin to the plantar surface of one hindpaw. Observations for the purpose of generating pain scores began 30 min after formalin injection and were continued for 30 min. A pain score was determined for each 5 min block by measuring the amount of time spent in each of 4 behavioural categories: 0, the injected paw is not favored; 1, the injected paw has little or no weight on it; 2, the injected paw is elevated and is not in contact with
Correspondence: T.J. Coderre, Pain Mechanisms Research Laboratory, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, Que., Canada H2W 1R7.
0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
156
3
t_
•
2
before
•
after
O control
.~ 2
O
1
,
*
t
1
"T--
!
30
0 Control
Early & Late
Late only
Fig. 1. Mean + S.E.M. formalin pain score averaged over the 30 min test period for rats treated with bupivicaine or saline in the paw according to the following schedule. Early and late phase group: bupivicaine given 5 min before and 25 min after formalin. Late phase only group: bupivicaine given 5 and 25 min after formalin. Control group: saline given 5 min before and 25 min after formalin injection. ANOVA reveals a significant main effect of group (F2.24 = 244.4, P < 0.001). Significant differences from the control group are indicated by asterisks (*P < 0.01, Scheffe). The mean formalin pain score for the late phase only group is significantly greater than the pain score for the early and late phase group (*P < 0.0l, Scheffe). any surface; 3, the injected paw is licked, bitten or shaken. In subcutaneous local anesthesia studies, 3 groups of 9 rats each were given, in addition to formalin injections, subcutaneous injections of 150/~1 of 0.5% bupivicaine (with epinephrine) or saline according to the following schedule. To anesthetize the hindpaw during both the early and late phase of the formalin response, the first group of rats received bupivicaine injections both 5 min before and 25 min after the formalin injection (early and late phase). To anesthetize the hindpaw during all but the early phase, a second group of rats received the same bupivicaine injections 5 and 25 min after the formalin injection (late phase only). A control group received saline 5 min before and 25 min after the formalin injection (control). Behavioural observations were performed between 30 and 60 min after formalin injection. This time course (beginning 5 min after the second injection of bupivicaine) occurs during a period when bupivicaine treatment produces an effective anesthetic block, as indicated by a complete insensitivity of the rats's hindpaw to a noxious thermal stimulus (water at 50 °C) (unpublished data). Compared to a mean pain score of 2.1 for the control group, the overall mean pain score was reduced 100% in the group given bupivicaine during both the early and late phases, and was reduced by only 72% for the group which received, bupivicaine only during the late phase
35
!
40 45 Time (min)
!
i
50
55
Fig. 2. Mean + S.E.M. formalin pain score over time for rats treated with intrathecal lidocaine or saline according to the following schedule. Before group: lidocaine given 5 min before formalin. After group: lidocaine given 5 min after formalin. Control group: saline given 5 min before formalin injection. ANOVA reveals a significant main effect of group (Fz.15 = 56.0, P < 0.001), and a significant group x time interaction (F10,75 = 3.4, P < 0.001), but a non-significant main effect for time (F5.75 = 0.5, P > 0.05). Significant differences from the control group are indicated by asterisks (*P < 0.01, Dunnett's t-test). (both P < 0.01, see Fig. 1). Thus, although the hindpaw was completely anesthetic, a significant degree of formalin pain behaviour was still exhibited by rats given bupivicaine only during the late phase. It required the presence of a local anesthetic during both the early and late phases of the formalin test to completely suppress pain behaviours (late phase only group had a significantly higher pain score than the zero pain score of the early and late phase group, P < 0.01). In spinal anesthesia studies, 3 groups of 6 rats each were given, in addition to subcutaneous formalin injections, intrathecal injections of 20 pl of 2.0% lidocaine or saline according to the schedule below, lntrathecal injections were given by lumbar puncture between the L4 and L 5 vertebrae while the rats were under brief ether anesthesia. The first group received an intrathecal injection of lidocaine 5 min before subcutaneous formalin, so that spinal anesthesia was present during the early phase (before). A second group received an intrathecal injection of lidocaine 5 min after formalin, so that the spinal anesthesia did not affect the early phase (after). A third group received an intrathecal injection of saline 5 min before formalin (control). Observations for pain scores were performed between 30 and 60 min after formalin injections. At this time, all effects of ether and spinal anesthesia had completely worn off, as indicated by the return of normal locomotor behaviour. As illustrated in Fig. 2, formalin pain scores were dramatically reduced, as compared with the control group, when rats were given spinal anesthesia 5 min before injection of formalin, but pain scores were
157 unaffected when the same spinal anesthesia was given 5 min after formalin injection. Thus, there was a dramatic reduction in the late phase of the formalin response if spinal anesthesia was present only during the early phase. In contrast, the same spinal anesthesia was completely without effect when given after the early phase was finished. It should be noted that the possibility of residual effects of the ether or spinal anesthesia lowering pain scores is discounted, since the 'after' group received these treatments later than the 'before' group, yet was unaffected by the treatment. The present results demonstrate two major findings that support a contribution of CNS plasticity to the tonic late phase of the formalin test. The first finding is that rats display pain behaviour in response to formalin injection, even if the injected hindpaw is locally anesthetized at the time of testing. For pain responses to subside completely, it is necessary to locally anesthetize the hindpaw not only at the time of testing during the late phase, but also during the transient early phase of the formalin test. It is suggested that if the rat is subjected to the early phase, central changes occur which will trigger pain responses during the late phase, even if the paw is anesthetized at the time of testing. The fact that pain scores for rats given the subcutaneous local anesthetic only during the late phase are significantly lower than controls, however, suggests that local inflammatory changes, and neural inputs they would generate, may also contribute to the late phase pain response. It is possible that low level inputs from the inflamed region may contribute to late phase pain responses by activating cells in the CNS which are now more sensitive, due to neural plasticity which resulted during the early phase. A contribution of such a peripheral trigger is suggested by the fact that increases in spinal cord dorsal horn activity during the late phase of the formalin response are blocked by treatment of the formalin injected paw with local anesthetics at that time 6. However, the fact that pain responses are not completely blocked by local anesthesia, suggests that central changes induce excitation which is independent of peripheral inputs. These central changes can either act alone to produce pain responses, or act by enhancing peripheral inputs. The results also demonstrate that few or no tonic pain responses are exhibited if rats do not experience the early phase formalin pain. Thus, if rats are given spinal anesthesia during the early phase of the formalin test, pain scores during the late phase are substantially reduced. In fact, of the rats in this group, 4 of 6 were completely pain free throughout the testing period. Thus, although there may still be local inflammation in the hindpaw which the spinal anesthesia did not influence, pain during the late phase of the formalin test is
significantly reduced or absent in rats that did not experience the early-phase formalin pain. This clearly suggests that neural plasticity plays a more important role in the development of the late phase of the formalin response than does inflammation. While there is a possibility that spinal anesthesia may reduce subsequent pain behaviour by reducing local inflammation, these effects should be minor. Peripheral neurogenic effects are likely to overshadow any central effects, as indicated by the finding that neurogenic inflammation is eliminated several days after nerve sections distal to the dorsal root ganglion, but is unaffected by nerve sections proximal to the dorsal root ganglion 3'16. There are several reasons to expect that local inflammation would play a relatively minor role in the late phase of the formalin response. First, formalin-induced edema does not reach its peak until 4-5 h after injection 2'24, while its peak pain response is exhibited between 20 and 35 min post-injection 8'24. Second, inflammatory compounds which produce a much greater degree of inflammation than formalin, such as yeast and carrageenan, produce hyperalgesia, but little or no spontaneous pain behaviour 24. It is noteworthy that formalin, but not yeast or carrageenan, causes an immediate and intense increase in the acz'vity of C-fiber afferents 1°. Perhaps this intense barrage is needed to initiate central changes. Third, although anti-inflammatory agents are effective in reducing pain during the late phase of the formalin test, NSAIAs such as indomethacin and phenylbutazone are noted for their particularly weak activity in the inhibition of formalin edema 2. Indeed, over the time course of the typical formalin test, the edema induced by formalin is more significantly inhibited by centrally acting narcotic analgesics than by NSAIAs 2. Based on their weak effects at inhibiting formalin edema, it is surprising that NSAIAs produce analgesia in the formalin test at all. It is possible, however, that NSAIAs may produce some of their analgesic effects on formalin pain by an action within the CNS 13. Preliminary results in our laboratory indicate that formalin pain is reduced more than 50% by intrathecal injection of 100 /~g of indomethacin or zomepirac sodium (unpublished data). Since subcutaneous formalin injections produce both an intense activation of C-fiber afferents 1° and a significant increase in substance P within the dorsal horn 14, it is possible that activity in C-fiber afferents initiates the CNS changes that we believe to occur in response to formalin. C-fiber neuropeptides such as substance P, which are capable of producing prolonged slow depolarizations 21, may trigger alterations in membrane excitability through interactions with second messenger systems and protein kinases which phosphorylate membranebound proteins ~7. Recent evidence suggests that excit-
158 atory amino acids acting at N-methyi-o-aspartic acid ( N M D A ) receptors may also contribute to formalininduced pain responses. Formalin injections produce a release of glutamate and aspartate from the dorsal horn of the spinal cord 19. In addition, N M D A receptor antagonists reduce formalin-induced increases in both dorsal horn activity5, and the expression of the protein product of the c-fos-proto-oncogene 15. These observa-
and guanylate cyclases 2°. Thus it is possible that the formalin stimulus triggers long-term alterations in CNS neural functioning by an interaction with both intracellular second and third messengers (proteins such as Fos) following the stimulation of N M D A receptors. While there is strong evidence for noxious stimulusinduced changes in neural function in the spinal cord 25"26,
tions are of particular interest given the well established role of excitatory amino acids in long-term potentiation 4,
it is also possible that neuroplasticity occurs in supraspinal structures. Recent evidence indicates that microinjection of lidocaine into the cingulum bundle 22, during
and evidence that the Fos protein appears to regulate
the early phase of the formalin test, significantly reduces
nuclear events that are expected to result in long-term neural changes 9. Several studies also indicate that N M D A receptors are coupled to various second messen-
tonic pain in the late phase. It is expected that by selectively blocking neural activity in these areas it is possible to prevent the development of these central
gers, such as inositol triphosphate (IP3), and adenylate
changes.
1 Alreja, M., Mutalik, P.G., Nayar, U. and Manchanda, S.K., The formalin test: a tonic pain model in the primate, Pain, 20 (1984) 97-105. 2 Brown, J.H., Kissel, J.W. and Lish, P.M., Studies on the acute inflammatory response. I. Involvement of the central nervous system in certain models of inflammation, J. Pharrnacol. Exp. Ther., 160 (1968) 231-242. 3 Chapman, L.E, Ramos, A.O., Goodell, H. and Wolff, H.G., Neurohumoral features of afferent fibers in man, Arch. Neurol., 4 (1961) 617-650. 4 Collingridge, C.L. and Bliss, T.V.P., NMDA receptors - - their role in long-term potentiation, Trends Neurosci., 10 (1987) 288-293. 5 Dickenson, A.H., Magnuson, D.S.K., Sullivan, A.E and Haley, J.E., Spinal opioid mechanisms of antinociception and their modulation, Pain, Suppl. 5 (1990) S121. 6 Dickenson, A.H. and Sullivan, A.E, Peripheral origins and central modulations of subcutaneous formalin-induced activity of rat dorsal horn neurons, Neurosci. Lett., 83 (1987) 207-211. 7 Dickenson, A.H. and Sullivan, A.E, Subcutaneous formalininduced activity of dorsal horn neurons in the rat: differential response to an intrathecal opiate administered pre or post formalin, Pain, 30 (1987) 349-360. 8 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. 9 Greenberg, M.E., Greene, L.A. and Ziff, E.B., Nerve growth factor and epidermal growth factor induce rapid transient changes in protooncogene transcription in PC12 cells, J. Biol. Chem., 260 (1985) 14101-14110, 10 Heapy, C.G., Jamieson, A. and Russell, N.J.W., Afferent C-fibre and A-delta activity in models of inflammation, Br. J. Pharmacol., 90 (1987) 164P. 11 Hunskaar, S., Berge, O.-G. and Hole, K., Dissociation between antinociceptive and anti-inflammatory effects of acetyl salicytic acid and indomethacin in the formalin test, Pain, 25 (1986) 125-132. 12 Hunskaar, S. and Hole, K., The formalin test in mice: dissociation between inflammatory and non-inflammatory pain, Pain, 30 (1987) 103-114. 13 Jurna, I. and Brune, K., Central effect of the non-steroid
anti-inflammatory agents, indomethacin, ibuprofen, and diclofenac, determined in C fibre evoked activity in single neurons of the rat thalamus, Pain, 41 (1990) 71-80. 14 Kantner, R.M., Kirby, M.L. and Goldstein, B.D., Increase in substance P in the dorsal horn during a chemogenic nociceptive stimulus, Brain Research, 290 (1985) 196-199. 15 Kehl, L.J., Basbaum, A.I., Pollock, C.H., Mayes, M. and Wilcox, G.L., The NMDA antagonist MK801 reduces noxious stimulus-evoked los expression in the spinal dorsal horn, Pain, Suppl. 5 (1990) S165. 16 Lewis, T., Harris, K.E. and Grant, R.T., Observations relating to the influences of the cutaneous nerves on various reactions of the cutaneous vessels, Heart, 14 (1927) 1-17. 17 Nestler, E.J. and Greengard, P., Protein phosphorylation in brain, Nature, 305 (1983) 583-588. 18 Shibata, M., Ohkubo, T., Takahashi, H. and Inoki, R., Modified formalin test: characteristic biphasic pain response, Pain, 38 (1989) 347-352. 19 Skilling, S.R., Smullin, D.H. and Larson, A.A., Extracellular amino acid concentrations in the dorsal spinal cord of freely moving rats following veratridine and nociceptive stimulation, J. Neurochem., 51 (1988) 127-132. 20 Smart, T.G., Excitatory amino acids: the involvement of second messengers in the signal transduction process, Cell. Mol. Neurobiol., 9 (1989) 193-206. 21 Urban, L. and Randic, M., Slow excitatory transmission in rat dorsal horn: possible mediation by peptides, Brain Research, 290 (1984) 336-341. 22 Vaccarino, A.L. and Melzack, R., An examination of the time course of analgesia produced by injection of lidocaine into the anterior cingulum, Soc. Neurosci. Abstr., 15 (1989) 150. 23 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. 24 Wheeler-Aceto, H., Porreca, F. and Cowan, A., The rat paw formalin test: comparison of noxious agents, Pain, 40 (1990) 229-238. 25 Woolf, C.J., Evidence for a central component of post-injury pain hypersensitivity, Nature, 306 (1983) 686-688. 26 Woolf, C.J. and MacMahon, S.B., Injury-induced plasticity of the flexor reflex in chronic decerebrate rats, Neuroscience, 16 (1985) 395-404.
155
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Central nervous system plasticity in the tonic pain response to subcutaneous formalin injection Terence J. Coderre 1'2, A n t h o n y L. Vaccarino 1 and R o n a l d Melzack 1 1Department of Psychology, McGill University and 2pain Mechanisms Research Laboratory, Clinical Research Institute of Montreal, Montreal, Que. (Canada)
(Accepted 21 August 1990) Key words: Nociception; Inflammation; Hyperaigesia; Injury; Local Anesthesia; Neuroplasticity
Evidence is presented which suggests that central neural changes occur during the brief early phase after subcutaneous formalin injection that are essential for the expression of pain during the long-lasting (tonic) later phase. First, tonic pain responses to subcutaneous formalin injections are abolished only if the injected hindpaw is locally anesthetized at the time of injection as well as the time of testing (30-60 rain later). Second, tonic formalin pain is substantially reduced by brief spinal anesthesia given 5 min before, but not 5 min after the formalin injection. Subcutaneous injection of dilute formalin produces a biphasic pain response which has been observed in rats, cats s, mice 11 and primates 1. An earl~, brief phase of intense pain occurs in the first 5 min, pain then subsides for 10-15 min, and a later tonic phase of moderate pain is expressed from 20 to 60 min after injection. It has been suggested that the transient early phase is due to direct stimulation of nociceptors, while the tonic phase is due to an ensuing inflammatory response 8. Indeed, non-steroidal anti-inflammatory agents (aspirin, indomethacin) and steroids (hydrocortisone, dexamethasone) produce analgesia in the late phase, but have little or no effect on the early pain produced by formalin injection 11'12'18. The dissociation of the effects of anti-inflammatory agents on the early and late phases of the formalin response has suggested that the two phases represent two independent processes which rely on separate neural systems or hyperalgesic mediators. Although the above studies show clearly that analgesia can be produced in the late phase of the formalin test by agents that do not affect the early phase, the opposite has not been demonstrated - - that is, analgesia during the early phase only. Centrally acting narcotic analgesics, such as morphine, pentazocine and meperidine, which produce analgesia during the early phase, are also analgesic in the late phase 11'12"18"23. Furthermore, Dickenson and Sullivan 7 have demonstrated that intrathecal administration of the /~-opiate agonist D A M G O (TyrD-Ala,Gly,Me,Phe,Gly-ol) significantly inhibits forma-
lin-induced increases in dorsal horn activity. However, this inhibition occurs only if the drug is given before the formalin injection, and not if it is given 2 min after the formalin injection. These results imply that the dorsal horn activity associated with the late phase of formalin pain depends upon spinal activation during the early phase 7. Thus, in addition to a dissociation of early and late formalin pain based on separate mechanisms involving nociceptor stimulation and inflammation, there is also the possibility of a second dissociation in which the early phase is a response to the direct stimulation of nociceptors, while the late phase is dependent on changes in central nervous system (CNS) function induced by neural activity generated during the early phase. The present experiment examines the contribution of CNS plasticity to the tonic late phase of the formalin response by assessing the effects of anesthetic agents, given in a local subcutaneous injection or intrathecally, either before or after the early phase of the formalin response. In all studies, Long-Evans male rats (250-350 g) were given a subcutaneous injection of 50 pl of 2.5% formalin to the plantar surface of one hindpaw. Observations for the purpose of generating pain scores began 30 min after formalin injection and were continued for 30 min. A pain score was determined for each 5 min block by measuring the amount of time spent in each of 4 behavioural categories: 0, the injected paw is not favored; 1, the injected paw has little or no weight on it; 2, the injected paw is elevated and is not in contact with
Correspondence: T.J. Coderre, Pain Mechanisms Research Laboratory, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, Que., Canada H2W 1R7.
0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
156
3
t_
•
2
before
•
after
O control
.~ 2
O
1
,
*
t
1
"T--
!
30
0 Control
Early & Late
Late only
Fig. 1. Mean + S.E.M. formalin pain score averaged over the 30 min test period for rats treated with bupivicaine or saline in the paw according to the following schedule. Early and late phase group: bupivicaine given 5 min before and 25 min after formalin. Late phase only group: bupivicaine given 5 and 25 min after formalin. Control group: saline given 5 min before and 25 min after formalin injection. ANOVA reveals a significant main effect of group (F2.24 = 244.4, P < 0.001). Significant differences from the control group are indicated by asterisks (*P < 0.01, Scheffe). The mean formalin pain score for the late phase only group is significantly greater than the pain score for the early and late phase group (*P < 0.0l, Scheffe). any surface; 3, the injected paw is licked, bitten or shaken. In subcutaneous local anesthesia studies, 3 groups of 9 rats each were given, in addition to formalin injections, subcutaneous injections of 150/~1 of 0.5% bupivicaine (with epinephrine) or saline according to the following schedule. To anesthetize the hindpaw during both the early and late phase of the formalin response, the first group of rats received bupivicaine injections both 5 min before and 25 min after the formalin injection (early and late phase). To anesthetize the hindpaw during all but the early phase, a second group of rats received the same bupivicaine injections 5 and 25 min after the formalin injection (late phase only). A control group received saline 5 min before and 25 min after the formalin injection (control). Behavioural observations were performed between 30 and 60 min after formalin injection. This time course (beginning 5 min after the second injection of bupivicaine) occurs during a period when bupivicaine treatment produces an effective anesthetic block, as indicated by a complete insensitivity of the rats's hindpaw to a noxious thermal stimulus (water at 50 °C) (unpublished data). Compared to a mean pain score of 2.1 for the control group, the overall mean pain score was reduced 100% in the group given bupivicaine during both the early and late phases, and was reduced by only 72% for the group which received, bupivicaine only during the late phase
35
!
40 45 Time (min)
!
i
50
55
Fig. 2. Mean + S.E.M. formalin pain score over time for rats treated with intrathecal lidocaine or saline according to the following schedule. Before group: lidocaine given 5 min before formalin. After group: lidocaine given 5 min after formalin. Control group: saline given 5 min before formalin injection. ANOVA reveals a significant main effect of group (Fz.15 = 56.0, P < 0.001), and a significant group x time interaction (F10,75 = 3.4, P < 0.001), but a non-significant main effect for time (F5.75 = 0.5, P > 0.05). Significant differences from the control group are indicated by asterisks (*P < 0.01, Dunnett's t-test). (both P < 0.01, see Fig. 1). Thus, although the hindpaw was completely anesthetic, a significant degree of formalin pain behaviour was still exhibited by rats given bupivicaine only during the late phase. It required the presence of a local anesthetic during both the early and late phases of the formalin test to completely suppress pain behaviours (late phase only group had a significantly higher pain score than the zero pain score of the early and late phase group, P < 0.01). In spinal anesthesia studies, 3 groups of 6 rats each were given, in addition to subcutaneous formalin injections, intrathecal injections of 20 pl of 2.0% lidocaine or saline according to the schedule below, lntrathecal injections were given by lumbar puncture between the L4 and L 5 vertebrae while the rats were under brief ether anesthesia. The first group received an intrathecal injection of lidocaine 5 min before subcutaneous formalin, so that spinal anesthesia was present during the early phase (before). A second group received an intrathecal injection of lidocaine 5 min after formalin, so that the spinal anesthesia did not affect the early phase (after). A third group received an intrathecal injection of saline 5 min before formalin (control). Observations for pain scores were performed between 30 and 60 min after formalin injections. At this time, all effects of ether and spinal anesthesia had completely worn off, as indicated by the return of normal locomotor behaviour. As illustrated in Fig. 2, formalin pain scores were dramatically reduced, as compared with the control group, when rats were given spinal anesthesia 5 min before injection of formalin, but pain scores were
157 unaffected when the same spinal anesthesia was given 5 min after formalin injection. Thus, there was a dramatic reduction in the late phase of the formalin response if spinal anesthesia was present only during the early phase. In contrast, the same spinal anesthesia was completely without effect when given after the early phase was finished. It should be noted that the possibility of residual effects of the ether or spinal anesthesia lowering pain scores is discounted, since the 'after' group received these treatments later than the 'before' group, yet was unaffected by the treatment. The present results demonstrate two major findings that support a contribution of CNS plasticity to the tonic late phase of the formalin test. The first finding is that rats display pain behaviour in response to formalin injection, even if the injected hindpaw is locally anesthetized at the time of testing. For pain responses to subside completely, it is necessary to locally anesthetize the hindpaw not only at the time of testing during the late phase, but also during the transient early phase of the formalin test. It is suggested that if the rat is subjected to the early phase, central changes occur which will trigger pain responses during the late phase, even if the paw is anesthetized at the time of testing. The fact that pain scores for rats given the subcutaneous local anesthetic only during the late phase are significantly lower than controls, however, suggests that local inflammatory changes, and neural inputs they would generate, may also contribute to the late phase pain response. It is possible that low level inputs from the inflamed region may contribute to late phase pain responses by activating cells in the CNS which are now more sensitive, due to neural plasticity which resulted during the early phase. A contribution of such a peripheral trigger is suggested by the fact that increases in spinal cord dorsal horn activity during the late phase of the formalin response are blocked by treatment of the formalin injected paw with local anesthetics at that time 6. However, the fact that pain responses are not completely blocked by local anesthesia, suggests that central changes induce excitation which is independent of peripheral inputs. These central changes can either act alone to produce pain responses, or act by enhancing peripheral inputs. The results also demonstrate that few or no tonic pain responses are exhibited if rats do not experience the early phase formalin pain. Thus, if rats are given spinal anesthesia during the early phase of the formalin test, pain scores during the late phase are substantially reduced. In fact, of the rats in this group, 4 of 6 were completely pain free throughout the testing period. Thus, although there may still be local inflammation in the hindpaw which the spinal anesthesia did not influence, pain during the late phase of the formalin test is
significantly reduced or absent in rats that did not experience the early-phase formalin pain. This clearly suggests that neural plasticity plays a more important role in the development of the late phase of the formalin response than does inflammation. While there is a possibility that spinal anesthesia may reduce subsequent pain behaviour by reducing local inflammation, these effects should be minor. Peripheral neurogenic effects are likely to overshadow any central effects, as indicated by the finding that neurogenic inflammation is eliminated several days after nerve sections distal to the dorsal root ganglion, but is unaffected by nerve sections proximal to the dorsal root ganglion 3'16. There are several reasons to expect that local inflammation would play a relatively minor role in the late phase of the formalin response. First, formalin-induced edema does not reach its peak until 4-5 h after injection 2'24, while its peak pain response is exhibited between 20 and 35 min post-injection 8'24. Second, inflammatory compounds which produce a much greater degree of inflammation than formalin, such as yeast and carrageenan, produce hyperalgesia, but little or no spontaneous pain behaviour 24. It is noteworthy that formalin, but not yeast or carrageenan, causes an immediate and intense increase in the acz'vity of C-fiber afferents 1°. Perhaps this intense barrage is needed to initiate central changes. Third, although anti-inflammatory agents are effective in reducing pain during the late phase of the formalin test, NSAIAs such as indomethacin and phenylbutazone are noted for their particularly weak activity in the inhibition of formalin edema 2. Indeed, over the time course of the typical formalin test, the edema induced by formalin is more significantly inhibited by centrally acting narcotic analgesics than by NSAIAs 2. Based on their weak effects at inhibiting formalin edema, it is surprising that NSAIAs produce analgesia in the formalin test at all. It is possible, however, that NSAIAs may produce some of their analgesic effects on formalin pain by an action within the CNS 13. Preliminary results in our laboratory indicate that formalin pain is reduced more than 50% by intrathecal injection of 100 /~g of indomethacin or zomepirac sodium (unpublished data). Since subcutaneous formalin injections produce both an intense activation of C-fiber afferents 1° and a significant increase in substance P within the dorsal horn 14, it is possible that activity in C-fiber afferents initiates the CNS changes that we believe to occur in response to formalin. C-fiber neuropeptides such as substance P, which are capable of producing prolonged slow depolarizations 21, may trigger alterations in membrane excitability through interactions with second messenger systems and protein kinases which phosphorylate membranebound proteins ~7. Recent evidence suggests that excit-
158 atory amino acids acting at N-methyi-o-aspartic acid ( N M D A ) receptors may also contribute to formalininduced pain responses. Formalin injections produce a release of glutamate and aspartate from the dorsal horn of the spinal cord 19. In addition, N M D A receptor antagonists reduce formalin-induced increases in both dorsal horn activity5, and the expression of the protein product of the c-fos-proto-oncogene 15. These observa-
and guanylate cyclases 2°. Thus it is possible that the formalin stimulus triggers long-term alterations in CNS neural functioning by an interaction with both intracellular second and third messengers (proteins such as Fos) following the stimulation of N M D A receptors. While there is strong evidence for noxious stimulusinduced changes in neural function in the spinal cord 25"26,
tions are of particular interest given the well established role of excitatory amino acids in long-term potentiation 4,
it is also possible that neuroplasticity occurs in supraspinal structures. Recent evidence indicates that microinjection of lidocaine into the cingulum bundle 22, during
and evidence that the Fos protein appears to regulate
the early phase of the formalin test, significantly reduces
nuclear events that are expected to result in long-term neural changes 9. Several studies also indicate that N M D A receptors are coupled to various second messen-
tonic pain in the late phase. It is expected that by selectively blocking neural activity in these areas it is possible to prevent the development of these central
gers, such as inositol triphosphate (IP3), and adenylate
changes.
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