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Descending modulation of central neural plasticity in the formalin pain test
BRAIN RESEARCH ELSEVIER
Brain Research 666 (1994) 104-108
Research report
Descending modulation of central neural plasticity in the formalin pain test Anthony L. Vaccarino *, Diane A. Chorney Department of Psychology, University of New Orleans, New Orleans, LA 70148, USA Accepted 13 September 1994
Abstract
Subcutaneous injection of formalin produces a biphasic profile of pain response: a transient early phase followed by a tonic late phase. A number of studies have indicated that the development of the late phase of formalin pain is dependent upon prolonged changes in central neural function produced by neural activity that is generated during the early phase (i.e. central sensitization). In support of this, the present study demonstrates that stimulation- or morphine-produced analgesia derived from the periaqueductal grey (PAG) during the early phase prevents the development of the late phase. These results suggest that descending mechanisms of pain inhibition, as reflected by PAG stimulation- and morphine-produced analgesia, can prevent the development of central neural plasticity following injury.
Keywords: Stimulation-produced analgesia; Morphine microinjection; Periaqueductal grey; Formalin pain; Central sensitization; Plasticity
I. Introduction
Peripheral tissue and nerve damage often leads to pathological pain syndromes such as phantom limb pain, spontaneous pain, hyperalgesia and allodynia. There is good evidence that the pain that develops after peripheral nerve or tissue damage is related to long-lasting changes in central nervous system function produced by the injury (i.e. central sensitization) [3]. Subcutaneous injection of dilute formalin produces a characteristic biphasic profile of pain response: an early phase of pain develops in the first 5 min after injection, followed by a period of little or no pain for about 10-15 min; the pain then rises to a stable plateau that lasts for about 1 h [7]. Recent evidence suggests that the pain in the late phase is, in part, dependent upon neural activity generated during the early phase [4,6,21,23]. For example, blocking neural activity selectively during the early phase with local anesthetics applied spinally [4] or in supraspinal structures [23]
* Corresponding author. Fax: (1) (504) 286-6049. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved
SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 1 1 3 1 - l
diminishes the expression of the tonic late phase. Conversely, locally anesthetizing the hind-paw only during the late phase does not completely abolish pain behavior [4], suggesting that the early phase produces persistent neural activity which can trigger pain behavior during the late phase independent of peripheral input. Different forms of stress can produce potent analgesia, a phenomenon known as stress-induced analgesia (SIA) [1]. We recently demonstrated that blocking the early phase of pain in the formalin test by SIA attenuates the development of the tonic late phase, whereas the same stressor given after the early phase does not [22]. SIA has been attributed, in part, to an activation of an endogenous descending pain-inhibitory system involving various supraspinal structures [2,20]. Electrical stimulation of and morphine microinjections into midbrain structures, such as the periaqueductal grey (PAG), have been shown to suppress nociceptive dorsal horn neurons and produce potent analgesia [9,10,13, 15,17,19,26]. In an effort to elucidate whether descending neural mechanisms may modulate central sensitization in the formalin test, we examined whether the late phase of the formalin test could also be suppressed by PAG stimulation-produced analgesia (SPA), or by mot-
A.L. Vaccarino, D.A. Chorney / Brain Research 666 (1994) 104-108 phine
microinjection
into the PAG,
during the early
phase.
2. Materials and methods 2.1. Subjects and housing Forty-eight male Long-Evans hooded rats (Harlan Laboratories, IN) weighing 400-500 g at the time of surgery served as subjects. The rats were housed individually with free access to food and water and were maintained on a 12-h light cycle (light onset at 07.00 h).
2.2. Formalin test The formalin test [7] was performed in a clear Plexiglas formalin test box, 32 × 32 × 16 cm in size. A mirror was positioned at a 45 ° angle below the floor of the test box, allowing an unobstructed view of the animal's paw. Following a l h habituation period, 50 /zl of 2.5% formalin was injected s.c. into the plantar surface of the hind-paw contralateral to the stimulating electrode or infusion cannula. The degree of pain was determined by measuring the amount of time the formalin-injected paw was elevated 0-5 rain after formalin injection (early phase) and 30-70 min after formalin injection (late phase).
105
beyond the tip of the outer cannula. The cannula was left in place for an additional minute to prevent the reflux of fluids. To examine the effects of morphine microinjection during the early phase on the late phase response, one group of rats were injected with morphine 5 min prior to formalin injection and with saline 25 min after formalin ('morphine before' group). To examine the effects of morphine microinjection after the early phase, a second group of rats received saline 5 min prior to formalin and morphine 25 min after formalin ('morphine after' group). A control group received saline 5 rain before and 25 min after formalin ('saline' group).
2.5. Histology Following testing, the rats were overdosed with sodium pentobarbital (100 mg/kg, i.p.) and perfused with physiological saline followed by 10% formalin. The brains were removed and stored in formalin for at least 3 days. Electrode and cannulae tip placements were verified from 30 ~ m coronal sections stained with Cresyl violet.
2.6. Data analysis The data during the early phase (0-5 min after formalin) and during the late phase (30-70 min after formalin) were analyzed separately as total time the formalin-injected paw was elevated using a one-way analysis of variance (ANOVA). Post-hoc comparisons of group means were made using Newman Keul's test.
2.3. Electrical stimulation Under sodium pentobarbital anesthesia (60 mg/kg, i.p.), 24 rats were implanted on one side with a bipolar stimulating electrode aimed at the PAG using the stereotaxic coordinates of Paxinos and Watson [18] ( - 0 . 6 5 AP; +0.05 ML; -0.55 DV). The electrode was anchored into place with dental cement poured around the electrode and the heads of three jeweler's screws which were placed in the skull. Following a 7-14 day recovery period, rats were placed individually in the formalin test box and an electrode lead was connected to the stimulating electrode. To examine the effects of PAG SPA during the early phase on the late phase response, one group of rats received electrical stimulation (60 s with 0.5 ms monophasic square wave pulses at a frequency of 50 Hz and a current intensity of 50 izA) starting 30 s prior to formalin ('stimulation before' group). To examine the effects of stimulation after the early phase, a second group of rats received PAG stimulation starting 5 rain after formalin ('stimulation after' group), A control group was implanted with a stimulating electrode and connected to the electrode lead, but no current was passed ('no stimulation' group). Stimulation was delivered by a Grass $88 stimulator via a constant current unit, and the electrode lead remained connected for the entire observation period. If any rats showed signs of aversion/escape during stimulation, the stimulation was terminated and the rat was excluded from the study.
2.4. Morphine microinjection Under sodium pentobarbital (60 mg/kg, i.p.), 24 rats were implanted on one side with a 23-ga outer cannula aimed at the PAG. The cannula was anchored into place with dental cement poured around the outer cannula and the heads of three jeweler's screws which were placed in the skull. A stainless-steel stylet (size 00), which extended 0.5 mm beyond the tip of the outer cannula, was inserted after surgery and left in place until the time of testing. Following a 7-14 day recovery period, the rats were placed individually in the formalin test box and received a localized infusion of 3/zg morphine sulphate (Malinerodt, MO) in 1 ~1 saline or saline during a 2-min period via a 30-ga inner cannula that extended 0.5 mm
3. Results
3.1. Histology O n e r a t in t h e ' s t i m u l a t i o n b e f o r e ' g r o u p s h o w e d signs of aversion during stimulation and was excluded from the study. Histological examination revealed that o n e r a t in t h e ' m o r p h i n e b e f o r e ' g r o u p a n d t w o r a t s in the 'morphine after' group had cannulae tips outside the area of the PAG and were therefore excluded from the data analysis. All remaining rats had tips located in areas of the PAG previously reported to support SPA a n d m o r p h i n e a n a l g e s i a [5,14,15,26] (Fig. 1).
3.2. Electrical stimulation Analysis of paw elevation during the early phase r e v e a l e d a s i g n i f i c a n t g r o u p e f f e c t (F2,a0 = 42.44, P < 0.01, Fig. 2). P A G ' s t i m u l a t i o n b e f o r e ' p r o d u c e d significant analgesia during the early phase as compared to the 'stimulation after' and 'no stimulation' groups (both P < 0.05). A n a l y s i s o f p a w e l e v a t i o n d u r i n g t h e l a t e p h a s e r e v e a l e d a s i g n i f i c a n t g r o u p e f f e c t (F2,z0 = 12.04, P < 0.01, s e e Fig. 2). R a t s e x p o s e d t o P A G ' s t i m u l a t i o n b e f o r e ' f o r m a l i n i n j e c t i o n s p e n t less t i m e e l e v a t i n g t h e formalin-injected paw as compared to both the 'stimulation after' and 'no stimulation' groups (both P < 0.05). N o s i g n i f i c a n t d i f f e r e n c e w a s f o u n d b e t w e e n ' n o stimulation' controls and rats that received 'stimulation after' the early phase.
100
A.L. Uaccarino, D.A. Chorney / Brain Research 666 (1994) 104-108 early phase
late phase
300 "
/ A
250 200 2 •>
~5o
100 "
morphine before (n=7) morphine after (n=6) saline (n=8)
50-
B
0
. ! 0-5 30"35 35~40 40'-45 45~50 50'-55 55'-60 60~65 65~70 Time after formalin (rain)
Fig, 3. The effects of PAG morphine on formalin pain. The data are expressed as mean time (s) the formalin-injected paw was elevated
-6.3
-6.8
-7.3
Fig. 1. Location of electrode/cannula tips in the PAG for rats that received (A) 'stimulation before' (closed circles) or 'stimulation after' (open circles), and (B) 'morphine before' (closed circles) or 'morphine after' (open circles). Drawings are adapted from Paxinos and Watson [18].
3.3. Morphine microinjection Analysis of paw elevation during the early phase revealed a significant group effect (F2,]s = 3.94, P <
early 3hase
late
phase
300 -
during the early phase (0-5 min after formalin) and during the late phase (30-70 min after formalin injection). Rats received PAG morphine over a 2-min period starting 5 min before formalin injection ('morphine before'), 25 min after formalin injection ('morphine after'), or 'saline'. PAG 'morphine before' formalin injection significantly reduced pain during both the early and late phases.
0.05, Fig. 3). PAG 'morphine before' produced significant analgesia during the early phase as compared to the 'morphine after' and 'saline' groups (both P < 0.05). Analysis of paw elevation during the late phase revealed a significant group effect (Fm~8 = 4.16, P < 0.05, see Fig. 3). Rats receiving PAG 'morphine before' formalin injection spent less time elevating the formalin-injected paw as compared to 'saline' control group (P < 0.05). No significant difference was found between 'saline' controls and rats that received 'morphine after' formalin injection.
250 -
4. Discussion 200150 " 100
" •
50-
stimulationbefore (n=7) stimulation after (n=8) no stimulation (n=8)
o'-5 130~3535:40 40[45 45Z50 50:55 55~50
60]65 65170
Time after formalin (rain) Fig. 2.
o f P A G stimulation on f o r m a l i n pain. The data are expressed as mean time (s) the formalin-injected paw was elevated during the early phase (0-5 min after formalin) and during the The
effects
late phase (30-70 min after formalin injection). Rats. received PAG stimulation for 60 s starting 30 s before formalin injection ('stimulation before'), 5 min after formalin injection ('stimulation after'), or 'no stimulation', PAG 'stimulation before' formalin injection significatly reduced pain during both the early and late phases.
The present results show that PAG stimulation or PAG morphine prior to formalin injection reduces pain behavior during both the early and late phase. The finding that PAG stimulation and morphine produced analgesia in the early phase is consistent with the role of the PAG in descending mechanisms of pain inhibition [9,10,13,15,17,19,26]. It is unlikely, however, that the reduction in pain during the late phase is due to residual analgesia, as the same treatments given after the early phase were not effective. Previous studies have demonstrated that the presence of the early phase is essential for the development of the late phase [4,6,21,22,23]. It has been hypothesized that the late phase of formalin pain is, in part, a result of long-lasting changes in central neural function produced by neural activity generated during the early phase
A.L. Vaccarino, D.A. Chorney / Brain Research 666 (1994) 104-108
[4,6,21-23]. The present results are consistent with this hypothesis and suggests that descending mechanisms of pain inhibition (PAG SPA, PAG morphine or SIA [22]) can prevent the development of the central neural plasticity thought to contribute to the late phase of formalin pain. Although not statistically significant, there was a clear trend towards analgesia in rats that received 'morphine after' formalin injection (see Fig. 3). It is likely, therefore, that a higher dose of morphine would have produced a significant effect in this group. However, a closer inspection of the profile of pain scores in Fig. 3 suggests that the nature of pain reduction in the 'morphine before' and 'morphine after' groups was different. In rats that received 'morphine after' formalin, the greatest reduction of pain occurred during the early part of the late phase which was followed by a recovery of pain, suggesting that the analgesia produced by the morphine decreased as a function of time. On the other hand, in rats that received 'morphine before' formalin, that the greatest reduction of pain occurred during the latter half of the late phase. Morphine applied before formalin would be expected to inhibit spinal activity during the early phase, but not during the late phase. It is possible, therefore, that the pain during the later half of the late phase may be most representative of pain due to central neural plasticity and thus is sensitive to analgesia applied during the early phase. In fact, a similar profile was found in rats that received 'stimulation before' formalin (see Fig. 2), and has also been observed following injections of local anesthetics into supraspinal structures (cingulum and fornix) during the early phase [23]. Previous studies have suggested that SPA derived from the PAG may be accompanied by aversive sideeffects that may contribute to the analgesia [9,10]. In particular, analgesia derived from dorsal PAG stimulation has been reported to be accompanied signs of aversion, whereas analgesia derived from ventral PAG stimulation is aversion-free [9,10]. Ifi the present study, with the exception of one rat that was excluded from the study, no signs of aversion were observed. This is likely due to the general ventral location of the electrode sites within the PAG, and the relatively low level of stimulation intensity that was used (50/xA). Also, it is unlikely that the analgesia observed in the late phase was due to a non-specific effect of aversion, since no analgesia was observed in rats that received PAG stimulation after the early phase. A number of clinical studies have indicated that opiate premedication is effective in reducing post-operative pain [11,12,16]. The notion is that pre-emptive narcotic treatment prevents the occurrence of central sensitization that may occur from surgical procedures, and thus reduces post-operative pain [3]. This is based
107
largely on observations by Woolf and Wall [24] that in rats opioids are much more effective at reducing stimulus-induced increases in dorsal horn excitability if administered prior to, rather than after noxious stimulation. In the formalin test, Dickenson and Sullivan [6] demonstrated that intrathecal administration of the tz-opiate agonist, DAMGO, reduces formalin-induced dorsal horn activity in the late phase when administered 2 min prior to, but not 2 min after formalin injection. The results of the present study are in accordance with these findings. Opiates suppress spinal activity both directly [8,25], and indirectly via descending mechanisms of pain inhibition involving the PAG [26]. Stimulation of or morphine microinjection into the PAG at the time of the formalin injection, therefore, would be expected to suppress formalin-induced dorsal horn activity and thus prevent central sensitization from occurring. Furthermore, since PAG stimulation, PAG morphine, stress [22], and spinal opiates [6] and anesthetics [4] are less effective on the late phase response when administered after the early phase, these results suggest that once initiated the central changes presumed to be induced by formalin injection may be maintained supraspinally [23].
Acknowledgements This research was supported by a Louisiana State University Neuroscience Incentive Grant, a University of New Orleans Research Council Grant and a Louisiana Education Quality Support Fund (LEQSF) Grant to A.L.V.
References [1] Akil, H., Madden, J., Patrick, R.L. and Brachas, J.D., Stress-induced increases in endogenous opiate peptides: concurrent analgesia and its partial reversal by naioxone. In H.W. Kosterlitz (Ed.), Opiates and Endogenous Opioid Peptides, North Holland, Amsterdam, 1976, pp. 63-70. [2] Basbaum, A.I. and Fields, H.L., Endogenous pain control mechanisms: review and hypothesis, Ann. Neurol., 4 (1978) 451-462. [3] Coderre, T.J., Katz, J., Vaccarino, A.L. and Melzack, R., Contribution of central neural plasticity to pathological pain: review of clinical and experimental evidence, Pain, 52 (1993) 259-285. [4l Coderre, T.J., Vaccarino, A.L. and Melzack, R., Central nervous system plasticity in the tonic pain response to subcutaneous formalin, Brain Res., 535 (1990) 155-158. [5] Dennis, S.G., Choiniere, M. and Melzack, R., Stimulation-produced analgesia in rats: a s s e s s m e n t by two pain tests and correlation with self-stimulation, Exp. Neurol., 68 (1980) 295-309. [6] Dickenson, A.H. and Sullivan, A.F., Subcutaneous formalin-induced activity of dorsal horn neurons in the rat: differential response to an intrathecal opiate administered pre or post formalin, Pain, 30 (1987) 349-360. [7] 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.
I (/8
A.L. Vaccarino. D.A. Chornev / Brain Research O00 (19941 104- lO,h'
[8] Duggan, A.W., Hall, J.G. and Headley, P.M., Morphine, enkephalin and substantia gelatinosa, Nature, 264 (t976) 456 458. [9] Fardin, V., Oliveras, J.L. and Besson, J.M., A reinvestigation of the analgesic effects induced by stimulation of the periaqueductal gray matter in the rat. I. The production of behavioral side effects together with analgesia, Brain Res., 306 (1984) 105-123. [10] Fardin, V., Oliveras, J.L. and Besson, J.M., A reinvestigation of the analgesic effects induced by stimulation of the periaqueductill gray matter in the rat. II. Differential characteristics of the analgesia produced by ventral and dorsal stimulation, Brain Res., 306 (1984) 125-139. [11] Katz, J., Kavanagh, B.P., Sandler, A.N., Nierenberg, H., Boylan, J.F. and Shaw, B.F., Pre-emptive analgesia: clinical evidence of neuroplasticity contributing to postoperative pain, Anesthesiolo~,9', 77 (1992) 439-446. [12] Kiss, I.E. and Killian, M., Does opiate premedication influence postoperative analgesia? A prospective study, Pain, 48 (1992) 157-158. [13] Liebeskind, J.C., Guilbaud, C., Besson, J.M. and Oliveras, J.L., Analgesia from electrical stimulation of the periaqueductal gray matter of the cat: behavioural observations and inhibitory effects on spinal cord interneurons, Brain Res., 50 (1973) 441-446. [14] Mayer, D.J. and Liebeskind, J.C., Pain reduction by focal electrical stimulation of the brain: an anatomical and behavioral analysis, Brain Res., 68 (1974) 73-93. [15] Mayer, D.J., Wolfe, T.L., Akil, H., Carder, B. and Liebeskind, J.C., Analgesia from electrical stimulation of the brain-stem of the rat, Science, 174 (1971) 1351-1354. [16] McQuay, H.J., Carroll, D. and Moore, R.A., Postoperative orthopaedic pain: the effects of opiate premedication and local anaesthetic blocks, Pain, 33 (1988) 291-295.
[17] Oliveras, J.L., Besson, J.M., Ouilbaud, G. and Liebeskind, J.C.. Behavioral and electrophysiological evidence ~f pain inhibition from midbrain stimulation in the cat, Exp. Brain Res., 20(1974) 32 44. [18] Paxinos, G. and Watson. C., The Rat Brain in Stereotaxic ('oordinares. Academic Press, New York, 1986. [19] Reynolds, D.V., Surgery in the rat during electrical analgesia induced by focal brain stimulation, Science, 164 (t969) 444-445. [20] Terman, G.W., Penner, E.R. and Liebeskind, J.C., Stimulationproduced analgesia and stress-induced analgesia: cross-tolerance between opioid forms, Brain Res.. 360 (1985) 374-378. [21] Vaccarino, A.L., Marek, P., Kest, B., Weber, E., Keana, J.F.W. and Liebeskind, J.C., NMDA receptor antagonists, MK-801 and ACEA-1011, prevent the development of tonic pain following subcutaneous formalin, Brain Res., 615 (1993) 331-334. [22] Vaccarino, A.L., Marek, P. and Liebeskind, J.C., Stress-induced analgesia prevents the development of the tonic, late phase of pain produced by subcutaneous formalin, Brain Res., 572 (1992) 25(/-252. [23] Vaccarino, A.L. and Melzack, R., Temporal processes of formalin pain: differential role of the cingulum bundle, fornix pathway and medial bulboreticular formation, Pain, 49 (1992) 257-271. [24] Woolf, C.J. and Wall, P.D., Morphine-sensitive and morphineinsensitive actions of C-fibre input on the Ial spinal cord, Neurosci. Lett., 64 (1986) 221-225. [25] Yaksh, T.L., Analgetic actions of intrathecal opiates in cat and primate, Brain Res., 153 (1978) 205-210. [26] Yaksh, T.L. and Rudy, T.A., Narcotic analgesics: CNS sites and mechanisms of action as revealed by intracerebral injection techniques, Pain, 4 (1978) 299-359,
Brain Research 666 (1994) 104-108
Research report
Descending modulation of central neural plasticity in the formalin pain test Anthony L. Vaccarino *, Diane A. Chorney Department of Psychology, University of New Orleans, New Orleans, LA 70148, USA Accepted 13 September 1994
Abstract
Subcutaneous injection of formalin produces a biphasic profile of pain response: a transient early phase followed by a tonic late phase. A number of studies have indicated that the development of the late phase of formalin pain is dependent upon prolonged changes in central neural function produced by neural activity that is generated during the early phase (i.e. central sensitization). In support of this, the present study demonstrates that stimulation- or morphine-produced analgesia derived from the periaqueductal grey (PAG) during the early phase prevents the development of the late phase. These results suggest that descending mechanisms of pain inhibition, as reflected by PAG stimulation- and morphine-produced analgesia, can prevent the development of central neural plasticity following injury.
Keywords: Stimulation-produced analgesia; Morphine microinjection; Periaqueductal grey; Formalin pain; Central sensitization; Plasticity
I. Introduction
Peripheral tissue and nerve damage often leads to pathological pain syndromes such as phantom limb pain, spontaneous pain, hyperalgesia and allodynia. There is good evidence that the pain that develops after peripheral nerve or tissue damage is related to long-lasting changes in central nervous system function produced by the injury (i.e. central sensitization) [3]. Subcutaneous injection of dilute formalin produces a characteristic biphasic profile of pain response: an early phase of pain develops in the first 5 min after injection, followed by a period of little or no pain for about 10-15 min; the pain then rises to a stable plateau that lasts for about 1 h [7]. Recent evidence suggests that the pain in the late phase is, in part, dependent upon neural activity generated during the early phase [4,6,21,23]. For example, blocking neural activity selectively during the early phase with local anesthetics applied spinally [4] or in supraspinal structures [23]
* Corresponding author. Fax: (1) (504) 286-6049. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved
SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 1 1 3 1 - l
diminishes the expression of the tonic late phase. Conversely, locally anesthetizing the hind-paw only during the late phase does not completely abolish pain behavior [4], suggesting that the early phase produces persistent neural activity which can trigger pain behavior during the late phase independent of peripheral input. Different forms of stress can produce potent analgesia, a phenomenon known as stress-induced analgesia (SIA) [1]. We recently demonstrated that blocking the early phase of pain in the formalin test by SIA attenuates the development of the tonic late phase, whereas the same stressor given after the early phase does not [22]. SIA has been attributed, in part, to an activation of an endogenous descending pain-inhibitory system involving various supraspinal structures [2,20]. Electrical stimulation of and morphine microinjections into midbrain structures, such as the periaqueductal grey (PAG), have been shown to suppress nociceptive dorsal horn neurons and produce potent analgesia [9,10,13, 15,17,19,26]. In an effort to elucidate whether descending neural mechanisms may modulate central sensitization in the formalin test, we examined whether the late phase of the formalin test could also be suppressed by PAG stimulation-produced analgesia (SPA), or by mot-
A.L. Vaccarino, D.A. Chorney / Brain Research 666 (1994) 104-108 phine
microinjection
into the PAG,
during the early
phase.
2. Materials and methods 2.1. Subjects and housing Forty-eight male Long-Evans hooded rats (Harlan Laboratories, IN) weighing 400-500 g at the time of surgery served as subjects. The rats were housed individually with free access to food and water and were maintained on a 12-h light cycle (light onset at 07.00 h).
2.2. Formalin test The formalin test [7] was performed in a clear Plexiglas formalin test box, 32 × 32 × 16 cm in size. A mirror was positioned at a 45 ° angle below the floor of the test box, allowing an unobstructed view of the animal's paw. Following a l h habituation period, 50 /zl of 2.5% formalin was injected s.c. into the plantar surface of the hind-paw contralateral to the stimulating electrode or infusion cannula. The degree of pain was determined by measuring the amount of time the formalin-injected paw was elevated 0-5 rain after formalin injection (early phase) and 30-70 min after formalin injection (late phase).
105
beyond the tip of the outer cannula. The cannula was left in place for an additional minute to prevent the reflux of fluids. To examine the effects of morphine microinjection during the early phase on the late phase response, one group of rats were injected with morphine 5 min prior to formalin injection and with saline 25 min after formalin ('morphine before' group). To examine the effects of morphine microinjection after the early phase, a second group of rats received saline 5 min prior to formalin and morphine 25 min after formalin ('morphine after' group). A control group received saline 5 rain before and 25 min after formalin ('saline' group).
2.5. Histology Following testing, the rats were overdosed with sodium pentobarbital (100 mg/kg, i.p.) and perfused with physiological saline followed by 10% formalin. The brains were removed and stored in formalin for at least 3 days. Electrode and cannulae tip placements were verified from 30 ~ m coronal sections stained with Cresyl violet.
2.6. Data analysis The data during the early phase (0-5 min after formalin) and during the late phase (30-70 min after formalin) were analyzed separately as total time the formalin-injected paw was elevated using a one-way analysis of variance (ANOVA). Post-hoc comparisons of group means were made using Newman Keul's test.
2.3. Electrical stimulation Under sodium pentobarbital anesthesia (60 mg/kg, i.p.), 24 rats were implanted on one side with a bipolar stimulating electrode aimed at the PAG using the stereotaxic coordinates of Paxinos and Watson [18] ( - 0 . 6 5 AP; +0.05 ML; -0.55 DV). The electrode was anchored into place with dental cement poured around the electrode and the heads of three jeweler's screws which were placed in the skull. Following a 7-14 day recovery period, rats were placed individually in the formalin test box and an electrode lead was connected to the stimulating electrode. To examine the effects of PAG SPA during the early phase on the late phase response, one group of rats received electrical stimulation (60 s with 0.5 ms monophasic square wave pulses at a frequency of 50 Hz and a current intensity of 50 izA) starting 30 s prior to formalin ('stimulation before' group). To examine the effects of stimulation after the early phase, a second group of rats received PAG stimulation starting 5 rain after formalin ('stimulation after' group), A control group was implanted with a stimulating electrode and connected to the electrode lead, but no current was passed ('no stimulation' group). Stimulation was delivered by a Grass $88 stimulator via a constant current unit, and the electrode lead remained connected for the entire observation period. If any rats showed signs of aversion/escape during stimulation, the stimulation was terminated and the rat was excluded from the study.
2.4. Morphine microinjection Under sodium pentobarbital (60 mg/kg, i.p.), 24 rats were implanted on one side with a 23-ga outer cannula aimed at the PAG. The cannula was anchored into place with dental cement poured around the outer cannula and the heads of three jeweler's screws which were placed in the skull. A stainless-steel stylet (size 00), which extended 0.5 mm beyond the tip of the outer cannula, was inserted after surgery and left in place until the time of testing. Following a 7-14 day recovery period, the rats were placed individually in the formalin test box and received a localized infusion of 3/zg morphine sulphate (Malinerodt, MO) in 1 ~1 saline or saline during a 2-min period via a 30-ga inner cannula that extended 0.5 mm
3. Results
3.1. Histology O n e r a t in t h e ' s t i m u l a t i o n b e f o r e ' g r o u p s h o w e d signs of aversion during stimulation and was excluded from the study. Histological examination revealed that o n e r a t in t h e ' m o r p h i n e b e f o r e ' g r o u p a n d t w o r a t s in the 'morphine after' group had cannulae tips outside the area of the PAG and were therefore excluded from the data analysis. All remaining rats had tips located in areas of the PAG previously reported to support SPA a n d m o r p h i n e a n a l g e s i a [5,14,15,26] (Fig. 1).
3.2. Electrical stimulation Analysis of paw elevation during the early phase r e v e a l e d a s i g n i f i c a n t g r o u p e f f e c t (F2,a0 = 42.44, P < 0.01, Fig. 2). P A G ' s t i m u l a t i o n b e f o r e ' p r o d u c e d significant analgesia during the early phase as compared to the 'stimulation after' and 'no stimulation' groups (both P < 0.05). A n a l y s i s o f p a w e l e v a t i o n d u r i n g t h e l a t e p h a s e r e v e a l e d a s i g n i f i c a n t g r o u p e f f e c t (F2,z0 = 12.04, P < 0.01, s e e Fig. 2). R a t s e x p o s e d t o P A G ' s t i m u l a t i o n b e f o r e ' f o r m a l i n i n j e c t i o n s p e n t less t i m e e l e v a t i n g t h e formalin-injected paw as compared to both the 'stimulation after' and 'no stimulation' groups (both P < 0.05). N o s i g n i f i c a n t d i f f e r e n c e w a s f o u n d b e t w e e n ' n o stimulation' controls and rats that received 'stimulation after' the early phase.
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A.L. Uaccarino, D.A. Chorney / Brain Research 666 (1994) 104-108 early phase
late phase
300 "
/ A
250 200 2 •>
~5o
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morphine before (n=7) morphine after (n=6) saline (n=8)
50-
B
0
. ! 0-5 30"35 35~40 40'-45 45~50 50'-55 55'-60 60~65 65~70 Time after formalin (rain)
Fig, 3. The effects of PAG morphine on formalin pain. The data are expressed as mean time (s) the formalin-injected paw was elevated
-6.3
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Fig. 1. Location of electrode/cannula tips in the PAG for rats that received (A) 'stimulation before' (closed circles) or 'stimulation after' (open circles), and (B) 'morphine before' (closed circles) or 'morphine after' (open circles). Drawings are adapted from Paxinos and Watson [18].
3.3. Morphine microinjection Analysis of paw elevation during the early phase revealed a significant group effect (F2,]s = 3.94, P <
early 3hase
late
phase
300 -
during the early phase (0-5 min after formalin) and during the late phase (30-70 min after formalin injection). Rats received PAG morphine over a 2-min period starting 5 min before formalin injection ('morphine before'), 25 min after formalin injection ('morphine after'), or 'saline'. PAG 'morphine before' formalin injection significantly reduced pain during both the early and late phases.
0.05, Fig. 3). PAG 'morphine before' produced significant analgesia during the early phase as compared to the 'morphine after' and 'saline' groups (both P < 0.05). Analysis of paw elevation during the late phase revealed a significant group effect (Fm~8 = 4.16, P < 0.05, see Fig. 3). Rats receiving PAG 'morphine before' formalin injection spent less time elevating the formalin-injected paw as compared to 'saline' control group (P < 0.05). No significant difference was found between 'saline' controls and rats that received 'morphine after' formalin injection.
250 -
4. Discussion 200150 " 100
" •
50-
stimulationbefore (n=7) stimulation after (n=8) no stimulation (n=8)
o'-5 130~3535:40 40[45 45Z50 50:55 55~50
60]65 65170
Time after formalin (rain) Fig. 2.
o f P A G stimulation on f o r m a l i n pain. The data are expressed as mean time (s) the formalin-injected paw was elevated during the early phase (0-5 min after formalin) and during the The
effects
late phase (30-70 min after formalin injection). Rats. received PAG stimulation for 60 s starting 30 s before formalin injection ('stimulation before'), 5 min after formalin injection ('stimulation after'), or 'no stimulation', PAG 'stimulation before' formalin injection significatly reduced pain during both the early and late phases.
The present results show that PAG stimulation or PAG morphine prior to formalin injection reduces pain behavior during both the early and late phase. The finding that PAG stimulation and morphine produced analgesia in the early phase is consistent with the role of the PAG in descending mechanisms of pain inhibition [9,10,13,15,17,19,26]. It is unlikely, however, that the reduction in pain during the late phase is due to residual analgesia, as the same treatments given after the early phase were not effective. Previous studies have demonstrated that the presence of the early phase is essential for the development of the late phase [4,6,21,22,23]. It has been hypothesized that the late phase of formalin pain is, in part, a result of long-lasting changes in central neural function produced by neural activity generated during the early phase
A.L. Vaccarino, D.A. Chorney / Brain Research 666 (1994) 104-108
[4,6,21-23]. The present results are consistent with this hypothesis and suggests that descending mechanisms of pain inhibition (PAG SPA, PAG morphine or SIA [22]) can prevent the development of the central neural plasticity thought to contribute to the late phase of formalin pain. Although not statistically significant, there was a clear trend towards analgesia in rats that received 'morphine after' formalin injection (see Fig. 3). It is likely, therefore, that a higher dose of morphine would have produced a significant effect in this group. However, a closer inspection of the profile of pain scores in Fig. 3 suggests that the nature of pain reduction in the 'morphine before' and 'morphine after' groups was different. In rats that received 'morphine after' formalin, the greatest reduction of pain occurred during the early part of the late phase which was followed by a recovery of pain, suggesting that the analgesia produced by the morphine decreased as a function of time. On the other hand, in rats that received 'morphine before' formalin, that the greatest reduction of pain occurred during the latter half of the late phase. Morphine applied before formalin would be expected to inhibit spinal activity during the early phase, but not during the late phase. It is possible, therefore, that the pain during the later half of the late phase may be most representative of pain due to central neural plasticity and thus is sensitive to analgesia applied during the early phase. In fact, a similar profile was found in rats that received 'stimulation before' formalin (see Fig. 2), and has also been observed following injections of local anesthetics into supraspinal structures (cingulum and fornix) during the early phase [23]. Previous studies have suggested that SPA derived from the PAG may be accompanied by aversive sideeffects that may contribute to the analgesia [9,10]. In particular, analgesia derived from dorsal PAG stimulation has been reported to be accompanied signs of aversion, whereas analgesia derived from ventral PAG stimulation is aversion-free [9,10]. Ifi the present study, with the exception of one rat that was excluded from the study, no signs of aversion were observed. This is likely due to the general ventral location of the electrode sites within the PAG, and the relatively low level of stimulation intensity that was used (50/xA). Also, it is unlikely that the analgesia observed in the late phase was due to a non-specific effect of aversion, since no analgesia was observed in rats that received PAG stimulation after the early phase. A number of clinical studies have indicated that opiate premedication is effective in reducing post-operative pain [11,12,16]. The notion is that pre-emptive narcotic treatment prevents the occurrence of central sensitization that may occur from surgical procedures, and thus reduces post-operative pain [3]. This is based
107
largely on observations by Woolf and Wall [24] that in rats opioids are much more effective at reducing stimulus-induced increases in dorsal horn excitability if administered prior to, rather than after noxious stimulation. In the formalin test, Dickenson and Sullivan [6] demonstrated that intrathecal administration of the tz-opiate agonist, DAMGO, reduces formalin-induced dorsal horn activity in the late phase when administered 2 min prior to, but not 2 min after formalin injection. The results of the present study are in accordance with these findings. Opiates suppress spinal activity both directly [8,25], and indirectly via descending mechanisms of pain inhibition involving the PAG [26]. Stimulation of or morphine microinjection into the PAG at the time of the formalin injection, therefore, would be expected to suppress formalin-induced dorsal horn activity and thus prevent central sensitization from occurring. Furthermore, since PAG stimulation, PAG morphine, stress [22], and spinal opiates [6] and anesthetics [4] are less effective on the late phase response when administered after the early phase, these results suggest that once initiated the central changes presumed to be induced by formalin injection may be maintained supraspinally [23].
Acknowledgements This research was supported by a Louisiana State University Neuroscience Incentive Grant, a University of New Orleans Research Council Grant and a Louisiana Education Quality Support Fund (LEQSF) Grant to A.L.V.
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