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Lidocaine in the rostroventromedial medulla and the periaqueductal gray attenuates allodynia in neuropathic rats
ELSEVIER
Neuroscience Letters 218 (1996) 127-130
IItUROZlEIIC[ LEIT[II8
Lidocaine in the rostroventromedial medulla and the periaqueductal gray attenuates allodynia in neuropathic rats A n t t i P e r t o v a a r a a'b'*, H o n g W e i a, M i n n a M . H/im~il/iinen a aDepartment of Physiology, Institute of Biomedicine, POB 9, University of Helsinki, F1N-O0014 Helsinki, Finland bDepartment of Physiology, Institute of Biomedicine, University of Turku, Turku, Finland
Received 16 September 1996; accepted 30 September 1996
Abstract
In the present study we attempted to find out if the rostroventromedial medulla (RVM) or the periaqueductal gray (PAG) might contribute to chronic allodynia induced by unilateral ligation of two spinal nerves in the rat. Lidocaine was microinjected in the RVM or PAG and allodynia was quantitatively determined by measuring the hindlimb withdrawal thresholds to mechanical stimulation of the paw. For comparison, lidocaine was also injected systemically (s.c.). Lidocaine in the RVM produced a dose-related (20 and 40/~g) antiallodynic effect. Lidocaine (20 #g) in the PAG produced identical antiallodynic effect as in the RVM. With systemic administrations of lidocaine, a considerably higher dose ( > > 40/xg) was needed to produce a significant antiallodynic effect. Naloxone, an opioidantagonist (1 mg/kg s.c.), did not attenuate the antiallodynic effect of lidocaine in the RVM. An antiallodynic dose of lidocaine (20 #g) in the RVM or the PAG did not influence the withdrawal response in the unoperated hindlimb nor the heat-induced tail-flick reflex. The results indicate that the RVM and the PAG have a facilitatory influence on the spinal segmental mechanisms underlying chronic allodynia. The selective attenuation of allodynia induced by lidocaine in the RVM and the PAG is independent of opiate receptors, and it can not be explained by a systemic spread of the drug. Keywords: Allodynia; Hyperalgesia; Lidocaine; Neuropathic pain; Mesencephalon; Medulla; Rat
Previous studies indicate that lidocaine, a local anesthetic, can alleviate various types of neuropathic pain in humans [2,3] and alleviate allodynia in animals [1,5], independent of its local anesthetic action. Lidocaine has induced a suppression of pathophysiological activity in peripheral nerve fibers at a low systemic dose that does not suppress the action potentials in intact nerve fibers [4,6,18]. This finding inclicates that the antiallodynic effect of lidocaine might be paa'tly due to action on the peripheral nerve or the dorsal root ganglion. On the other hand, a selective attenuation of the C-fiber evoked polysynaptic reflex by systemic lidocaine indicates that lidocaine might have selective antinociceptive effects in the central nervous system [22]. In neuropathic rats intravenous administration of lidocaine, unlike regional lidocaine block of the injured nerve or spinal administration, pro* Corresponding author. Fax: +358 9 1918681; e-mail: [email protected]
duced prolonged antiallodynic effects [5]. This finding raises the possibility that supraspinal structures might contribute to the lidocaine-induced antiallodynic effect. In the current investigation we attempted to study the involvement of two supraspinal sites, the rostroventromedial medulla (RVM) and the periaqueductal gray (PAG), in chronic allodynia induced by ligation of two spinal nerves in the rat. Following lidocaine microinjection into the RVM or the PAG, the effect on allodynia was evaluated by determining the hindlimb withdrawal response to mechanical stimulation. Since the PAG and the RVM have an important role in opioidergic antinociception [7], we also attempted to reverse the possible lidocaineinduced antiallodynic effects with naloxone, an opioid antagonist. The experiments were performed with adult male Hannover-Wistar rats (The Finnish National Laboratory Animal Center; weight range, 2 5 0 - 4 5 0 g). The experiments were approved by the Institutional Ethics Committee of
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940(96) 13136-0
128
A. Pertovaara et al. / Neuroscience Letters 218 (1996) 127-130
the University of Helsinki. The unilateral ligation of two spinal nerves (L5 and L6) w a s performed under pentobarbital anesthesia (40 mg/kg i.p.) as described in detail earlier [ 10]. For intracerebral injections, the anesthetized rats were placed in a stereotaxic apparatus according to the atlas of Paxinos and Watson [16] and they were implanted with a chronic guide cannula (26-gauge) made of stainless steel as described in detail earlier [12]. The desired injection site in the RVM was: posterior 2.00 mm, lateral 0.00 mm, dorsal -0.50 mm (reference, the ear bar). The desired injection site in the PAG was: anterior: 1.36 mm, lateral 0.70 mm, and dorsal 4.00 mm. The tip of the guide cannula was positioned 1 mm dorsal to the desired injection site. Before behavioral testing, the rats were allowed to recover from surgery for 3-5 days. Intracerebral microinjections were performed through a 33-gauge stainless steel injection cannula inserted through and protruding 1 mm beyond the tip of the guide cannula. The microinjection procedure is described in detail earlier [12]. The volume of intracerebral injection was 0.5 /zl, except in one group it was 1.0/zl. Lidocaine at the volume of 0.5 ~tl has an effect at 0.8-0.9 mm from the injection site, whereas lidocaine at a volume of 1.0/~1 should effectively suppress neuronal activity within a radius of 1.4-1.7 mm [13,17]. Before actual testing with drugs, the rats were habituated to the testing environment by allowing them to spend 1-2 h in the laboratory during three days preceding the testing. The determination of withdrawal thresholds of the hindlimb ipsilateral and contralateral to the nerve lesion was performed with a series of calibrated monofilaments (Stoelting, Wood Dale, IL, USA) as described in detail earlier [12]. Moreover, in some of the experiments the heat-induced tail-flick response was determined as described elsewhere [11 ]. There were nine experimental groups, (1) saline (1/zl) in the RVM, (2) lidocaine 20/zg (0.5 /zl; Astra, S6dert~ilje, Sweden) in the RVM, (3) lidocaine 40/xg (1.0/zl) in the RVM, (4) lidocaine 20/zg (0.5 /zl) in the PAG, (5) lidocaine 20 /zg in the RVM + naloxone (Sigma, St. Louis, MO, USA) 1 mg/kg systemically (s.c.), (6) naloxone 1 mg/kg s.c., (7) saline (50/xl) s.c., (8) lidocaine 40/zg (in 50/~1 saline) s.c., (9) lidocaine 200/~g (in 50/A saline) s.c. Withdrawal thresholds were bilaterally determined before the drug administrations (pre-drug control threshold), and 5, I0, 15, 30 and 60 min following the drug administrations. Furthermore, in groups 1, 2, and 4 the tail-flick latency was determined before the drug administration and 20 rain following the drug administration. Each rat participated in three to five experiments at an interval of 4 - 7 days. Following this interval the thresholds were recovered to the starting level of the previous experiment as verified in threshold measurements performed before each new test session. The order of testing various drug combinations in each rat was varied to avoid serial effects. At the end of experiments, the rats were sacrificed. The
injection sites in the brain were verified histologically and the extent of the area covered by lidocaine was estimated based on previous studies [13,17] (Fig. 1C,D). In statistical evaluation of the withdrawal threshold data one-way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparison test was applied to evaluate the significance of the drug-induced threshold change from the corresponding pre-drug value within each group. The tail-flick latencies before versus after drug administration were compared with a paired t-test. P < 0.05 was considered to represent a significant change. Only rats with a unilateral allodynia (hindlimb withdrawal thresholds in the operated side < 7 g) were selected for further studies. Following saline injection in the RVM, the hindlimb withdrawal threshold in the allodynic or the control paw was not changed. Lidocaine in the RVM produced a dose-related (20-40 /zg.) threshold elevation in the allodynic paw (Fig. IA and 2A). The antiallodynic effect started within 5 rain following the lidocaine injection and it lasted up to 30 rain (Fig. 1A). At the dose of 20 /~g of lidocaine in the RVM, that produced a marked threshold elevation in the allodynic paw (F(5,4|) = 3.895, P < .0.01; ANOVA), lidocaine did not change the threshold of the control paw (F(5,41) = 2.27, ns; ANOVA). Lidocaine at the dose of 20/zg in the PAG also produced a (A)
(B)
INTRACEREBRAL INJECTION 103 I • ctd paw polt 20.ug RVM o ol~r paw post 20 ;.,gRVM 1 02 ~ ~ °P" Paw po~ SAL RVM
/
S Y S T E M I C INJECTION 103 / . . . . . . • cta p a w post 40 UO ~ oper plw po=t 40 ug l~, 10 ;~~ ~ °Par Paw Post SAL
/
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10,1] -5
5 10 15 3 0 6 0
(C~)
TIME
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30
60
T I M E [min]
_
Fig. 1. (A) Hindlimb withdrawal thresholds at various time points following administration of lidocaine (20/~g) or saline (SAL) in the RVM. (B) Hindlimb withdrawal thresholds at various time points following systemic administration of lidocaine (40 #g) or saline. In (A,B) the drugs were administered at time point 0. The open symbols represent the threshold of the allodynic limb (oper paw) and the filled symbols the threshold of the intact hindlimb (ctrl paw). The error bars represent + SEM (n = 4-7). *P < 0.05 (Tukey-Kramer test; reference, the corresponding threshold before drug administration). (C,D) The estimated minimum extent of the area covered by lidocaine (20/zg) microinjected at a volume of 0.5/zl is shown by the dotted area in the rostral medulla (C) and in the mesencephalon (D). g7, Genu of the facial nerve; DPGi, dorsal paragigantocellular nucleus; Gi, gigantocellular nucleus; GiA, gigantocellular nucleus pars alpha; RMg, raphe magnus nucleus; PAG, periaqueductal gray. The calibration bars in (C,D) represent 1 mm.
A, Pertovaara et al. / Neuroscience Letters 218 (1996) 127-130
(B)
(A)
INTRACEREBRAL INJECT.
INTRACEREBRAL INJECT.
5O
50
40
40
:z
oAJ
3o z
201 lo
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SAL
RVM
20 pg RVM
SAL RVM
40 pg 2(1 pg RVM PAG
20 pg 20 pg Nx sc RVM RVM+ Nx sc
(D)
(C)
TAIL-FLICK TEST
SYSTEMIC INJECTION
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50
8;
40
;8
q 30
,,z
0,iol 2o
5"
~_
i- lO SAL
40//g
200 pg
SAL P,VM
20/Jg RVM
20 h'g PAG
LIDOCAINE DOSE
Fig. 2. (A) Withdrawal threshold of the allodynic hindlimb following administration of lidocaine or saline (SAL) in the RVM or the PAG. (B) Attempted reversal of the lido:aine effect by naloxone (Nx; s.c.; 1 mg/kg s.c.) (C) Withdrawal threshoht of the allodynic hindlimb following systemic administration of lidocaine or saline. (D) Latency of the heatinduced tail-flick response following microinjection of lidocaine or saline in the brain. In (A-C) the thresholds were measured 15 min following the drug administration (the maximum effect), and in (D) the latencies were measured 20 rain following drug administration. *P < 0. 05 (Tukey-Kramer test; reference, the corresponding threshold before drug administration). The error bars represent SEM (n = 4-7).
significant threshold elevation in the allodynic paw (F(5,35) = 4.32, P < 0.0l; ANOVA) and this antiaUodynic effect was identical as that produced by an equal dose (20 /~g) of lidocaine in the RVM (Fig. 2A). The antiallodynic effect of lidocaine (20 lzg) in the RVM was not attenuated by systemic administration of naloxone (1 mg/kg; Fig. 2B). Naloxone alone (1 mg/kg, s.c.) had no effect on allodynia. Following systemic administration of lidocaine at a dose that produced strong ant:[allodynic effect in the RVM (40 /~g) no significant effect on hindlimb withdrawal thresholds was observed in the allodynic paw (F(6,27) = 2.60, ns; ANOVA) or the control paw (/7(6,27) = 0.67, ns, ANOVA; Fig. 1B). Systemic administration of lidocaine at a dose of 200/~g was enough to produce a significant antiallodynic effect (F(6,34) = 3. 51, P "-< 0.02; ANOVA; Fig. 2C). Systemic administration of saline had no effect on hindlimb withdrawal thresholds. Tail-flick latency was not significantly increased by an antiallodynic dose of lidocaine (20 #g) in the RVM or the
129
PAG nor by saline (paired t-test; reference, the corresponding pre-drug latency; Fig. 2D). According to the present results lidocaine in the RVM and the PAG produced a selective antiallodynic effect in neuropathic rats. In line with the present results, also in other studies an antiallodynic/-hyperalgesic or analgesic effect has been reported following microinjection of lidocaine in the RVM [9,11,15,21] indicating that supraspinal influence may facilitate the spinal segmental mechanisms underlying hyperalgesia or allodynia. A subpopulation of RVM and PAG neurons, 'On-cells', have been shown to facilitate spinal nociceptive responses under physiological conditions [8,9,15 ]. If an increased activity in 'On-cells' of the RVM and the PAG were responsible for the facilitation of spinal nociception under pathophysiological conditions, then a block of the 'On-cells' would provide a plausible explanation for the antiallodynic effect of lidocaine following intracerebral injections. The PAG has a large projection to the RVM [7], and this might also explain the identical antiallodynic effects induced by lidocaine in the PAG and the RVM. Although the PAG and the RVM have an important role in opioidergic antinociception [7], systemically administered naloxone did not reverse the antiallodynic effect induced by lidocaine in the RVM. Thus, the lidocaine-induced antiallodynia is not due to a release of opioidergic inhibitory mechanisms. It should also be noted that the present results do not exclude the possibility that microinjection of lidocaine into some other central structure(s) than the RVM and the PAG had produced antiallodynic effects. Other possible sites where lidocaine might produce antiallodynic effects are the dentate gyrus, the fornix and the anterior cingulum bundle, since lidocaine in these sites has produced antinociceptive effects in earlier rat studies [14,19,20]. In line with the results of earlier studies [1,5], systemic administration of lidocaine at a low dose produced antiallodynic effects also in this study. The antiallodynic effect produced by systemically administered lidocaine may be explained due to a direct action on primary afferent fibers or the dorsal root ganglion as indicated by previous results [4,6,18]. This peripheral mechanism obviously cannot explain the antiallodynic effect induced by 20/zg of lidocaine in the RVM or the PAG, since 40 #g of lidocaine bad no antiallodynic effect following systemic administration. In an earlier study, the ED50 for blocking ongoing neuroma discharge was 1.9 mg of lidocaine/rat and the EDs0 for blocking ectopic activity in the dorsal root ganglion was 370 #g of lidocaine/rat [6]. The latter value is in the dose range that proved antiallodynic following systemic administration in the present study. It is also possible that the antiallodynic effect induced by systemically administered lidocaine was partly due to its action on the RVM and the PAG, although the local tissue concentration of lidocaine in these supraspinal sites war considerably higher following intracerebral than systemic administrations. A prolonged (>7 days) antiallodynic effect has been
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A. Pertovaara et al. / Neuroscience Letters 218 (1996) 127-130
reported f o l l o w i n g intravenous lidocaine administration [5]. In the present study, the antiaUodynic effect was not systemically d e t e r m i n e d f o l l o w i n g the 60 m i n observation period. H o w e v e r , the rats were retested at an interval o f 4 7 days, and in all of t h e m the antiallodynic effect c o m p l e tely disappeared within this interval. The lack o f prol o n g e d (>7 days) antiallodynia f o l l o w i n g systemic administration in the present study m a y be e x p l a i n e d by too slow a rate o f lidocaine administration (s.c.) and too low a p l a s m a concentration o f lidocaine, since these parameters are critical for the d e v e l o p m e n t of a p r o l o n g e d antiallodynia by lidocaine [5]. T h e present results add to the a c c u m u l a t i n g e v i d e n c e indicating that lidocaine m a y be useful in treating s o m e neuropathic conditions, since it is possible to obtain a selective antiallodynic effect at low doses that l e a v e physiological pain m e c h a n i s m s intact.
[10]
[11]
[12]
[13]
[14] [15]
[16] [1] Abram, S.E. and Yaksh, T.L., Systemic lidocaine blocks nerve injury-induced hyperalgesia and nociceptor-driven spinal sensitization in the rat, Anesthesiology, 80 (1994) 383-391. [21 Bach, F.W. and Jensen, T.S., Kastrup., J., Stigsby, B. and Dejgard, A., The effect of intravenous lidocaine on nociceptive processing in diabetic neuropathy, Pain, 40 (1990) 29-34. [3] Boas, R.A., Covino, B.G. and Shahnarian, A., Analgesic responses to i.v. lignocaine, Br. J. Anaesthiol., 54 (1982) 501-505. [4] Chabal, C., Russell, L.C. and Burchiel, K.J., The effect of intravenous lidocaine, tocainide, and mexiletine on spontaneously active fibers originating in rat sciatic neuromas, Pain, 38 (1989) 333-338. [5] Chaplan, S.R, Bach, F.W., Sharer, S.L. and Yaksh, T.L., Prolonged alleviation of tactile allodynia by intravenous lidocaine in nettropathic rats, Anesthesiology, 83 (1995) 775-785. [6] Devor, M., Wall, P.D. and Catalan, N., Systemic lidocaine silences ectopic neuroma and DRG discharge without blocking nerve conduction, Pain, 48 (1992) 261-268. [7] Fields, H.L., Neural mechanisms of opiate analgesia, Adv. Pain Res. Ther., 9 (1985) 479-486. [8] Heinricher, M.M., Cheng, Z.F. and Fields, H.L., Evidence for two classes of nociceptive modulating neurons in the periaqueductal gray, J. Neurosci., 7 (1987) 271-278. [9] Kaplan, H. and Fields, H.L., Hyperalgesia during acute opioid
[17]
[18]
[19]
[20]
[21]
[22]
abstinence: evidence for a nociceptive facilitating function of the rostral ventromedial medulla, J. Neurosci., 11 (1991) 1433-1439. Kim, S.H. and Chung, J.M., An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat, Pain, 50 (1992) 355-363. Mansikka, H. and Pertovaara, A., Supraspinal influence on hindlimb withdrawal thresholds and mustard oil-induced secondary allodynia in rats, Brain Res. Bull., (1996) in press. Mansikka, H., Idanp~i~n-Heikkil~i,J.-J. and Pertovaara, A., Different roles of alpha2-adrenoceptors of the medulla versus the spinal cord in modulation of mustard oil-induced central hyperalgesia in rats, Eur. J. Pharmacol., 297 (1996) 19-26. Martin, J.H., Autoradiographic estimation of the extent of reversible inactivation produced by microinjection of lidocaine and muscimol in the rat, Neurosci. Lett., 127 (1991) 160-164. McKenna, J.E. and Melzack, R., Analgesia produced by lidocaine microinjection into the dentate gyms, Pain, 49 (1992) 105-112. Morgan, M.M. and Fields, H.L., Pronounced changes in the activity of nociceptive modulatory neurons in the rostral ventromedial medulla in response to prolonged thermal noxious stimuli, J. Neurophysiol., 72 (1994) 1161-1170. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1986. Sandktihler, J., Maisch, B. and Zimmermann, M., The use of local anesthetic microinjections to identify central pathways: a quantitative evaluation of the time course and extent of the neuronal block, Exp. Brain Res., 68 (1987) 168-178. Tanelian, D.L. and Maclver, M.B., Analgesic concentrations of lidocaine suppress tonic A-delta and C-fiber discharges produced by acute injury, Anesthesiology, 74 (1991) 934-936. Vaccarino, A.L. and Melzack, R., Analgesia produced by injection of lidocaine into the anterior cingulum bundle of the rat, Pain, 39 (1989) 213-219. 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. Wiertelak, E.P., Furness, L.E., Horan, R., Martinez, J., Maier, S.F. and Watkins, L.R., Subcutaneous formalin produces centrifugal hyperalgesia at a non-injected site via the NMDA-nitric oxide cascade, Brain Res., 649 (1994) 19-26. Woolf, C.J. and Wiesenfeld-Hallin, Z., The systemic administration of local anesthetics produces a selective depression of C-afferent fibre evoked activity in the spinal cord, Pain, 23 (1985) 361374.
Neuroscience Letters 218 (1996) 127-130
IItUROZlEIIC[ LEIT[II8
Lidocaine in the rostroventromedial medulla and the periaqueductal gray attenuates allodynia in neuropathic rats A n t t i P e r t o v a a r a a'b'*, H o n g W e i a, M i n n a M . H/im~il/iinen a aDepartment of Physiology, Institute of Biomedicine, POB 9, University of Helsinki, F1N-O0014 Helsinki, Finland bDepartment of Physiology, Institute of Biomedicine, University of Turku, Turku, Finland
Received 16 September 1996; accepted 30 September 1996
Abstract
In the present study we attempted to find out if the rostroventromedial medulla (RVM) or the periaqueductal gray (PAG) might contribute to chronic allodynia induced by unilateral ligation of two spinal nerves in the rat. Lidocaine was microinjected in the RVM or PAG and allodynia was quantitatively determined by measuring the hindlimb withdrawal thresholds to mechanical stimulation of the paw. For comparison, lidocaine was also injected systemically (s.c.). Lidocaine in the RVM produced a dose-related (20 and 40/~g) antiallodynic effect. Lidocaine (20 #g) in the PAG produced identical antiallodynic effect as in the RVM. With systemic administrations of lidocaine, a considerably higher dose ( > > 40/xg) was needed to produce a significant antiallodynic effect. Naloxone, an opioidantagonist (1 mg/kg s.c.), did not attenuate the antiallodynic effect of lidocaine in the RVM. An antiallodynic dose of lidocaine (20 #g) in the RVM or the PAG did not influence the withdrawal response in the unoperated hindlimb nor the heat-induced tail-flick reflex. The results indicate that the RVM and the PAG have a facilitatory influence on the spinal segmental mechanisms underlying chronic allodynia. The selective attenuation of allodynia induced by lidocaine in the RVM and the PAG is independent of opiate receptors, and it can not be explained by a systemic spread of the drug. Keywords: Allodynia; Hyperalgesia; Lidocaine; Neuropathic pain; Mesencephalon; Medulla; Rat
Previous studies indicate that lidocaine, a local anesthetic, can alleviate various types of neuropathic pain in humans [2,3] and alleviate allodynia in animals [1,5], independent of its local anesthetic action. Lidocaine has induced a suppression of pathophysiological activity in peripheral nerve fibers at a low systemic dose that does not suppress the action potentials in intact nerve fibers [4,6,18]. This finding inclicates that the antiallodynic effect of lidocaine might be paa'tly due to action on the peripheral nerve or the dorsal root ganglion. On the other hand, a selective attenuation of the C-fiber evoked polysynaptic reflex by systemic lidocaine indicates that lidocaine might have selective antinociceptive effects in the central nervous system [22]. In neuropathic rats intravenous administration of lidocaine, unlike regional lidocaine block of the injured nerve or spinal administration, pro* Corresponding author. Fax: +358 9 1918681; e-mail: [email protected]
duced prolonged antiallodynic effects [5]. This finding raises the possibility that supraspinal structures might contribute to the lidocaine-induced antiallodynic effect. In the current investigation we attempted to study the involvement of two supraspinal sites, the rostroventromedial medulla (RVM) and the periaqueductal gray (PAG), in chronic allodynia induced by ligation of two spinal nerves in the rat. Following lidocaine microinjection into the RVM or the PAG, the effect on allodynia was evaluated by determining the hindlimb withdrawal response to mechanical stimulation. Since the PAG and the RVM have an important role in opioidergic antinociception [7], we also attempted to reverse the possible lidocaineinduced antiallodynic effects with naloxone, an opioid antagonist. The experiments were performed with adult male Hannover-Wistar rats (The Finnish National Laboratory Animal Center; weight range, 2 5 0 - 4 5 0 g). The experiments were approved by the Institutional Ethics Committee of
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940(96) 13136-0
128
A. Pertovaara et al. / Neuroscience Letters 218 (1996) 127-130
the University of Helsinki. The unilateral ligation of two spinal nerves (L5 and L6) w a s performed under pentobarbital anesthesia (40 mg/kg i.p.) as described in detail earlier [ 10]. For intracerebral injections, the anesthetized rats were placed in a stereotaxic apparatus according to the atlas of Paxinos and Watson [16] and they were implanted with a chronic guide cannula (26-gauge) made of stainless steel as described in detail earlier [12]. The desired injection site in the RVM was: posterior 2.00 mm, lateral 0.00 mm, dorsal -0.50 mm (reference, the ear bar). The desired injection site in the PAG was: anterior: 1.36 mm, lateral 0.70 mm, and dorsal 4.00 mm. The tip of the guide cannula was positioned 1 mm dorsal to the desired injection site. Before behavioral testing, the rats were allowed to recover from surgery for 3-5 days. Intracerebral microinjections were performed through a 33-gauge stainless steel injection cannula inserted through and protruding 1 mm beyond the tip of the guide cannula. The microinjection procedure is described in detail earlier [12]. The volume of intracerebral injection was 0.5 /zl, except in one group it was 1.0/zl. Lidocaine at the volume of 0.5 ~tl has an effect at 0.8-0.9 mm from the injection site, whereas lidocaine at a volume of 1.0/~1 should effectively suppress neuronal activity within a radius of 1.4-1.7 mm [13,17]. Before actual testing with drugs, the rats were habituated to the testing environment by allowing them to spend 1-2 h in the laboratory during three days preceding the testing. The determination of withdrawal thresholds of the hindlimb ipsilateral and contralateral to the nerve lesion was performed with a series of calibrated monofilaments (Stoelting, Wood Dale, IL, USA) as described in detail earlier [12]. Moreover, in some of the experiments the heat-induced tail-flick response was determined as described elsewhere [11 ]. There were nine experimental groups, (1) saline (1/zl) in the RVM, (2) lidocaine 20/zg (0.5 /zl; Astra, S6dert~ilje, Sweden) in the RVM, (3) lidocaine 40/xg (1.0/zl) in the RVM, (4) lidocaine 20/zg (0.5 /zl) in the PAG, (5) lidocaine 20 /zg in the RVM + naloxone (Sigma, St. Louis, MO, USA) 1 mg/kg systemically (s.c.), (6) naloxone 1 mg/kg s.c., (7) saline (50/xl) s.c., (8) lidocaine 40/zg (in 50/~1 saline) s.c., (9) lidocaine 200/~g (in 50/A saline) s.c. Withdrawal thresholds were bilaterally determined before the drug administrations (pre-drug control threshold), and 5, I0, 15, 30 and 60 min following the drug administrations. Furthermore, in groups 1, 2, and 4 the tail-flick latency was determined before the drug administration and 20 rain following the drug administration. Each rat participated in three to five experiments at an interval of 4 - 7 days. Following this interval the thresholds were recovered to the starting level of the previous experiment as verified in threshold measurements performed before each new test session. The order of testing various drug combinations in each rat was varied to avoid serial effects. At the end of experiments, the rats were sacrificed. The
injection sites in the brain were verified histologically and the extent of the area covered by lidocaine was estimated based on previous studies [13,17] (Fig. 1C,D). In statistical evaluation of the withdrawal threshold data one-way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparison test was applied to evaluate the significance of the drug-induced threshold change from the corresponding pre-drug value within each group. The tail-flick latencies before versus after drug administration were compared with a paired t-test. P < 0.05 was considered to represent a significant change. Only rats with a unilateral allodynia (hindlimb withdrawal thresholds in the operated side < 7 g) were selected for further studies. Following saline injection in the RVM, the hindlimb withdrawal threshold in the allodynic or the control paw was not changed. Lidocaine in the RVM produced a dose-related (20-40 /zg.) threshold elevation in the allodynic paw (Fig. IA and 2A). The antiallodynic effect started within 5 rain following the lidocaine injection and it lasted up to 30 rain (Fig. 1A). At the dose of 20 /~g of lidocaine in the RVM, that produced a marked threshold elevation in the allodynic paw (F(5,4|) = 3.895, P < .0.01; ANOVA), lidocaine did not change the threshold of the control paw (F(5,41) = 2.27, ns; ANOVA). Lidocaine at the dose of 20/zg in the PAG also produced a (A)
(B)
INTRACEREBRAL INJECTION 103 I • ctd paw polt 20.ug RVM o ol~r paw post 20 ;.,gRVM 1 02 ~ ~ °P" Paw po~ SAL RVM
/
S Y S T E M I C INJECTION 103 / . . . . . . • cta p a w post 40 UO ~ oper plw po=t 40 ug l~, 10 ;~~ ~ °Par Paw Post SAL
/
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Fig. 1. (A) Hindlimb withdrawal thresholds at various time points following administration of lidocaine (20/~g) or saline (SAL) in the RVM. (B) Hindlimb withdrawal thresholds at various time points following systemic administration of lidocaine (40 #g) or saline. In (A,B) the drugs were administered at time point 0. The open symbols represent the threshold of the allodynic limb (oper paw) and the filled symbols the threshold of the intact hindlimb (ctrl paw). The error bars represent + SEM (n = 4-7). *P < 0.05 (Tukey-Kramer test; reference, the corresponding threshold before drug administration). (C,D) The estimated minimum extent of the area covered by lidocaine (20/zg) microinjected at a volume of 0.5/zl is shown by the dotted area in the rostral medulla (C) and in the mesencephalon (D). g7, Genu of the facial nerve; DPGi, dorsal paragigantocellular nucleus; Gi, gigantocellular nucleus; GiA, gigantocellular nucleus pars alpha; RMg, raphe magnus nucleus; PAG, periaqueductal gray. The calibration bars in (C,D) represent 1 mm.
A, Pertovaara et al. / Neuroscience Letters 218 (1996) 127-130
(B)
(A)
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INTRACEREBRAL INJECT.
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TAIL-FLICK TEST
SYSTEMIC INJECTION
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LIDOCAINE DOSE
Fig. 2. (A) Withdrawal threshold of the allodynic hindlimb following administration of lidocaine or saline (SAL) in the RVM or the PAG. (B) Attempted reversal of the lido:aine effect by naloxone (Nx; s.c.; 1 mg/kg s.c.) (C) Withdrawal threshoht of the allodynic hindlimb following systemic administration of lidocaine or saline. (D) Latency of the heatinduced tail-flick response following microinjection of lidocaine or saline in the brain. In (A-C) the thresholds were measured 15 min following the drug administration (the maximum effect), and in (D) the latencies were measured 20 rain following drug administration. *P < 0. 05 (Tukey-Kramer test; reference, the corresponding threshold before drug administration). The error bars represent SEM (n = 4-7).
significant threshold elevation in the allodynic paw (F(5,35) = 4.32, P < 0.0l; ANOVA) and this antiaUodynic effect was identical as that produced by an equal dose (20 /~g) of lidocaine in the RVM (Fig. 2A). The antiallodynic effect of lidocaine (20 lzg) in the RVM was not attenuated by systemic administration of naloxone (1 mg/kg; Fig. 2B). Naloxone alone (1 mg/kg, s.c.) had no effect on allodynia. Following systemic administration of lidocaine at a dose that produced strong ant:[allodynic effect in the RVM (40 /~g) no significant effect on hindlimb withdrawal thresholds was observed in the allodynic paw (F(6,27) = 2.60, ns; ANOVA) or the control paw (/7(6,27) = 0.67, ns, ANOVA; Fig. 1B). Systemic administration of lidocaine at a dose of 200/~g was enough to produce a significant antiallodynic effect (F(6,34) = 3. 51, P "-< 0.02; ANOVA; Fig. 2C). Systemic administration of saline had no effect on hindlimb withdrawal thresholds. Tail-flick latency was not significantly increased by an antiallodynic dose of lidocaine (20 #g) in the RVM or the
129
PAG nor by saline (paired t-test; reference, the corresponding pre-drug latency; Fig. 2D). According to the present results lidocaine in the RVM and the PAG produced a selective antiallodynic effect in neuropathic rats. In line with the present results, also in other studies an antiallodynic/-hyperalgesic or analgesic effect has been reported following microinjection of lidocaine in the RVM [9,11,15,21] indicating that supraspinal influence may facilitate the spinal segmental mechanisms underlying hyperalgesia or allodynia. A subpopulation of RVM and PAG neurons, 'On-cells', have been shown to facilitate spinal nociceptive responses under physiological conditions [8,9,15 ]. If an increased activity in 'On-cells' of the RVM and the PAG were responsible for the facilitation of spinal nociception under pathophysiological conditions, then a block of the 'On-cells' would provide a plausible explanation for the antiallodynic effect of lidocaine following intracerebral injections. The PAG has a large projection to the RVM [7], and this might also explain the identical antiallodynic effects induced by lidocaine in the PAG and the RVM. Although the PAG and the RVM have an important role in opioidergic antinociception [7], systemically administered naloxone did not reverse the antiallodynic effect induced by lidocaine in the RVM. Thus, the lidocaine-induced antiallodynia is not due to a release of opioidergic inhibitory mechanisms. It should also be noted that the present results do not exclude the possibility that microinjection of lidocaine into some other central structure(s) than the RVM and the PAG had produced antiallodynic effects. Other possible sites where lidocaine might produce antiallodynic effects are the dentate gyrus, the fornix and the anterior cingulum bundle, since lidocaine in these sites has produced antinociceptive effects in earlier rat studies [14,19,20]. In line with the results of earlier studies [1,5], systemic administration of lidocaine at a low dose produced antiallodynic effects also in this study. The antiallodynic effect produced by systemically administered lidocaine may be explained due to a direct action on primary afferent fibers or the dorsal root ganglion as indicated by previous results [4,6,18]. This peripheral mechanism obviously cannot explain the antiallodynic effect induced by 20/zg of lidocaine in the RVM or the PAG, since 40 #g of lidocaine bad no antiallodynic effect following systemic administration. In an earlier study, the ED50 for blocking ongoing neuroma discharge was 1.9 mg of lidocaine/rat and the EDs0 for blocking ectopic activity in the dorsal root ganglion was 370 #g of lidocaine/rat [6]. The latter value is in the dose range that proved antiallodynic following systemic administration in the present study. It is also possible that the antiallodynic effect induced by systemically administered lidocaine was partly due to its action on the RVM and the PAG, although the local tissue concentration of lidocaine in these supraspinal sites war considerably higher following intracerebral than systemic administrations. A prolonged (>7 days) antiallodynic effect has been
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A. Pertovaara et al. / Neuroscience Letters 218 (1996) 127-130
reported f o l l o w i n g intravenous lidocaine administration [5]. In the present study, the antiaUodynic effect was not systemically d e t e r m i n e d f o l l o w i n g the 60 m i n observation period. H o w e v e r , the rats were retested at an interval o f 4 7 days, and in all of t h e m the antiallodynic effect c o m p l e tely disappeared within this interval. The lack o f prol o n g e d (>7 days) antiallodynia f o l l o w i n g systemic administration in the present study m a y be e x p l a i n e d by too slow a rate o f lidocaine administration (s.c.) and too low a p l a s m a concentration o f lidocaine, since these parameters are critical for the d e v e l o p m e n t of a p r o l o n g e d antiallodynia by lidocaine [5]. T h e present results add to the a c c u m u l a t i n g e v i d e n c e indicating that lidocaine m a y be useful in treating s o m e neuropathic conditions, since it is possible to obtain a selective antiallodynic effect at low doses that l e a v e physiological pain m e c h a n i s m s intact.
[10]
[11]
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[14] [15]
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