Intrathecal MK-801 and local nerve anesthesia synergistically reduce nociceptive behaviors in rats with experimental peripheral mononeuropathy

254

Brain Research, 576 (1992) 254-262 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00

BRES 17573

Intrathecal MK-801 and local nerve anesthesia synergistically reduce nociceptive behaviors in rats with experimental peripheral mononeuropathy J. Mao 1, D.D. Price 2, D.J. Mayer 1, J.

Lu I

and R.L. Hayes 3

Departments of ZPhysiology and 2Anesthesiology, Medical College of Virginia, Virginia Commonwealth University Richmond, VA 23298 (U.S.A.) and 3Division of Neurosurgery, University of Texas Health Science Center at Houston, Houston, TX 77030 (U.S.A.) (Accepted 19 November 1991) Key words: Hyperalgesia; Spontaneous pain; Neuropathic pain; MK-801; N-Methyl-D-aspartate; Hyperactivity

The hyperalgesia and spontaneous pain that occur following peripheral nerve injury may be related to abnormal peripheral input or altered central activity, or both. The present experiments investigated these possibilities by examining the effects of MK-801 (a non-competitive N-methyl-D-aspartate, NMDA, receptor antagonist) and bupivacaine (a local anesthetic agent) on thermal hyperalgesia and spontaneous nociceptive behaviors in rats with painful peripheral mononeuropathy. Peripheral mononeuropathy was produced by loosely ligating the rat's common sciatic nerve, a procedure which causes chronic constrictive injury (CCI) of the ligated nerve. The resulting hyperalgesia to radiant heat and spontaneous nociceptive behaviors was assessed by using a foot-withdrawal test and a spontaneous pain behavior rating method, respectively. CCI rats receiving 4 daily intraperitoneal (i.p.) MK-801 injections (0.03, 0.1, 0.3 mg/kg) beginning 15 min prior to nerve ligation exhibited less hyperalgesia (i.e., longer foot-withdrawal latencies) on days 3, 5, 7, 10, and 15 after nerve ligation as compared to those receiving saline injections. Thermal hyperalgesia also was reduced when a single MK-801 injection was given intrathecally (i.t.) onto the spinal cord lumbar segments on Day 3 after nerve ligation. This effect of postinjury MK-801 treatment was dose-dependent (2.5 - 20 nmol) and lasted for at least 48 h after injection. Moreover, i.t. injection of MK-801 (10 nmol) reliably lowered spontaneous pain behavior rating scores in CCI rats compared to those in the saline group. The spinal site of MK-801 action is situated within the caudal (probably lumbar) spinal cord, since i.t. injection of MK-801 (10 nmol) onto the spinal cord thoracic segments did not affect thermal hyperalgesia. Local anesthesia of the ligated sciatic nerve induced by a single perinerve bupivacaine injection on Day 3 after nerve ligation reliably reduced thermal hyperalgesia when tested 24 h after injection, indicating that abnormal peripheral input contributes to maintenance of neuropathic pain. While i.t. MK-801 (2.5 nmol) or local nerve anesthesia on Day 3 after nerve ligation attenuated thermal hyperalgesia for less than 48 h after injection, the combined application of MK-801 and bupivacaine extended the duration of the attenuation of thermal hyperalgesia for at least 4 days after injection. The data indicate that the central blockade of NMDA receptors and peripheral nerve anesthesia synergistically attenuate nociceptive behaviors associated with painful peripheral mononeuropathy. The results suggest that hyperalgesia and spontaneous pain behaviors following constrictive nerve injury may be related to both abnormal peripheral input and altered central activity involving NMDA receptor activation. These results also suggest novel approaches to the clinical management of postinjury neuropathic pain syndromes. INTRODUCTION H y p e r a l g e s i a a n d s p o n t a n e o u s p a i n are two salient clinical features of n e u r o p a t h i c pain s y n d r o m e s that occur following p e r i p h e r a l n e r v e injury 3°'3s. O t h e r characteristic features of n e u r o p a t h i c pain, which are distinguishable from acute pain, include allodynia, r a d i a t i o n of pain, a n d t e m p o r a l s u m m a t i o n of p a i n in response to r e p e a t e d s t i m u l a t i o n 7'3°. Most of these clinical s y m p t o m s have b e e n p r o d u c e d e x p e r i m e n t a l l y in rats by loosely ligating the c o m m o n sciatic n e r v e ~. This p r o c e d u r e results in chronic constrictive injury (CCI) of the ligated nerve a n d b e h a v i o r a l changes including hyperalgesia to r a d i a n t heat 4 or h i n d paw pressure 3, allodynia following w a r m 3 or cold 4 s t i m u l a t i o n , as well as distinctive g u a r d i n g po-

sitions indicative of s p o n t a n e o u s p a i n 3'4. H o w e v e r , as is the case with n e u r o p a t h i c pain s y n d r o m e s in m a n , mechanisms that subserve postinjury hyperalgesia a n d spont a n e o u s pain behaviors in this m o d e l r e m a i n poorly u n derstood. Hyperalgesia and s p o n t a n e o u s p a i n due to tissue injury m a y be related to a b n o r m a l p e r i p h e r a l i n p u t 23"39. T h e r e is evidence that sensitivity of p r i m a r y afferent nociceptors increases after injury with a r e s u l t a n t increase in p r i m a r y nociceptive afferent discharges in r e s p o n s e to noxious s t i m u l a t i o n (hyperalgesia) 5'8'35. O n g o i n g s p o n t a n e o u s discharges m a y also originate from the severed nerve in the a b s e n c e of overt p e r i p h e r a l s t i m u l a t i o n a n d t h e r e b y possibly c o n t r i b u t e to s p o n t a n e o u s p a i n 12'17'19'4°. R e c e n t studies indicate that p o s t i n j u r y nociceptive be-

Correspondence: J. Mao, Department of Physiology, Medical College of Virginia, Virginia Commonwealth University, P.O. Box 551, Richmond, VA 23298, U.S.A. Fax: (I) (804) 371 7382.

255 haviors (hyperalgesia, allodynia, or spontaneous pain) may be associated with a central hyperactive state ~5'45 which is, at least partly, mediated by central N-methylD-aspartate (NMDA) receptor activation 1°'36'47. Glutamate has been shown to play important roles in nociceptive processes at the spinal cord level 1'18'37. Electrophysiological studies have demonstrated involvement of NMDA receptor activation in central hyperactive states that occur following repeated noxious stimulation 11'14'47. Such studies have observed decreased thresholds of dorsal horn nociceptive neurons following repeated noxious stimulation 9 and temporal summation of C-fiber-evoked responses in nociceptive neurons, a phenomenon known a s windup 14'26'31'32'48. The phenomenon of windup is characterized by slow postsynaptic potentials in spinal cord dorsal horn neurons 32, and may be prevented by pretreatment with NMDA receptor antagonists 11'14'47 and attenuated by the antagonists after its establishment 11'47. Recently, the blockade of NMDA receptors has also been shown to reduce nociceptive behaviors in a peripheral neuropathy model 1° and in an autotomy model 36. Therefore, it is of interest to know whether and to what extent NMDA receptor activation and abnormal peripheral input are necessary for neuropathic pain-related behaviors (hyperalgesia and spontaneous pain) that occur following nerve injury. If NMDA receptor activation is the necessary prelude of postinjury nociceptive behaviors, then the application of NMDA receptor antagonists initiated prior to nerve injury should have protective effects on the development of these nociceptive behaviors. If postinjury nociceptive behaviors are maintained either by tonic abnormal peripheral input or altered central NMDA receptor activation, then local nerve anesthesia of the injured nerve or intrathecal administration of NMDA receptor antagonists after nerve injury should also attenuate nociceptive behaviors. However, if postinjury nociceptive behaviors are maintained by both peripheral and central mechanisms, the combined treatment of both local nerve anesthesia and intrathecal NMDA receptor antagonists would be expected to produce additive effects on postinjury nociceptive behaviors. In order to investigate these possibilities, we examined the effects of MK-801 (a non-competitive NMDA receptor antagonist) administration initiated before or after peripheral nerve injury on thermal hyperalgesia and spontaneous nociceptive behaviors resulting from sciatic nerve ligation 4. MK-801 was injected either intraperitoneally (i.p.) or intrathecally (i.t.) in order to determine the site of MK-801 action. Thermal hyperalgesia and spontaneous nociceptive behaviors were assessed by using a foot-withdrawal test and a spontane-

ous pain behavior rating method, respectively. Bupivacaine, a local anesthetic agent, was used to block the conduction of the injured nerve. MK-801 and bupivacaine were given alone or simultaneously to CCI rats in order to test whether the elimination of both peripheral and central factors would potentiate the reduction of hyperalgesia beyond that seen after application of either one of them. The data derived from the present experiments indicate that both abnormal peripheral input and central NMDA receptor activation contribute to hyperalgesia and spontaneous pain that occur after constrictive nerve injury. MATERIALS AND METHODS

Subjects Adult male Sprague-Dawley rats (Hilltop) weighing 340-360 g at the time of surgery were used. Animals were individually housed in cages with water and food pellets available ad libitum. The animal room was artificially illuminated from 07.00 to 19.00 h. All experimental procedures were approved by our Institutional Animal Care and Use Committee.

Surgical preparation of neuropathic rats Rats were anesthetized with i.p. 50 mg/kg sodium pentobarbital. Neuropathic pain behaviors were produced according to the method of Bennett and Xie 4. Briefly, the rat's right sciatic nerve was exposed and a 5-7 mm long nerve segment was then dissected. Four loose ligatures (4-0 chromic gut) were made around the dissected nerve with a 1.0-1.5 mm interval between each of them. The ligation was carefully manipulated such that the nerve was barely constricted, but no arrest of blood circulation through the superficial epineural vasculature was viewed under a microscope. The skin incision was closed with 4-0 silk sutures. All animals received one postoperative injection of potassium penicillin (30,000 IU/rat) intramuscularly in order to prevent infection.

Behavioral assessments 1. The foot-withdrawal test. Thermal hyperalgesia to radiant heat was assessed by using a foot-withdrawal test. The foot-withdrawal latency to radiant heat stimulation was defined as the time from onset of radiant heat to withdrawal of the rat's hind paw. The radiant heat source was adjusted to result in baseline latencies of approximately 12 s. Three test trials were made for each of the rat's hind paws, and scores from each hind paw were averaged to provide a mean withdrawal latency (MWL). The latency difference score (MWL contralateral to the side of nerve ligation minus M W L ipsilateral to the side of nerve ligation) was used to indicate the degree of thermal hyperalgesia. In order to determine the relationship between hind paw skin temperature and foot-withdrawal latency, hind paw skin temperature was measured in separate groups of both normal rats and CCI rats 3 days after nerve ligation (n = 6/group). A hypodermic thermoprobe (24 gauge, 0.1 s time constant), connected to an electronic temperature monitor (made by Yellow Stone Inc.), was inserted intracutaneously into the plantar region of the ipsilateral hind paw under pentobarbital anesthesia. The time course of skin temperature increase was then plotted on an oscilloscope screen as a function of exposure time to radiant heat produced by the same device used for the foot-withdrawal test. Temperature values measured from photos of these recordings along with foot-withdrawal latencies taken from separate groups of 48 CCI rats provided a graph of the relationship between hind paw skin temperature ( ° C ) and foot-withdrawal latencies (Fig. 1). 2. Spontaneous behavior ratings. In order to quantitatively analyze spontaneous nociceptive behaviors in CCI rats, a spontaneous

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Fig. 1. Relationship of the rat's hind paw skin temperature and the foot-withdrawal latency• The rat's hind paw skin temperature was measured under pentobarbital anesthesia by using a hypodermic thermoprobe (24 gauge, 0.1 s time constant) in both normal rats (dotted line) and CCI rats (solid line) 3 days after nerve ligation. Arrows above the X-axis indicate the average baseline latency (10.39 s) and postsurgical latency (7.36 s) on Day 3 after nerve ligation taken from 48 CCI rats, while arrows on the right of the Y-axis indicate the corresponding temperature shifts inferred from temperature plots from anesthetized rats.

pain behavior rating method modified from the formalin test rating system 16 was used. Each rat was allowed to freely move without intervention within an open-top plexiglas cylinder (diameter 19 cm x height 30 cm) and was habituated for 5 min before the observation. Within a 5 min observation period, the total duration over which a rat presented one or more of the following behaviors was timed with a stopwatch: (1) the placement of only the medial edge or the heel of the ligated hind paw on the ground, and (2) elevation or licking of the ligated hind paw. In addition, a count of the ligated hind paw elevations was made and recorded. Each rat underwent 3 observation trials, and was graded on days 0, 4, 5, and 7 after nerve ligation. The average score for the 3 trials was calculated and denoted as the spontaneous behavior rating score (s). Numbers of ligated hind paw elevations also were averaged from 3 trials for statistical analysis.

Systemic MK-801 experiments In order to determine whether i.p. MK-801 given prior to nerve ligation had effects on thermal hyperalgesia, rats were divided into 4 groups (n = 8/group). Four daily i.p. MK-801 (0.03, 0.1, 0.3 mg/ kg) or saline injections were initiated 15 min prior to nerve ligation and then on days 1, 2, and 3 after nerve ligation. The daily systemic injection of 1 mg/kg MK-801 initiated immediately before surgery for 7 days after surgery has been shown to produce protective effects on the development of neuropathic pain behaviors in this same model 1°. In an additional 5 groups (n = 7/group), a single MK-801 (0.01, 0.03, 0.1, 0.3 mk/kg) or saline injection was given i.p. on Day 3 after nerve ligation in order to examine whether postinjury MK-801 treatment would attenuate thermal hyperalgesia. The rationale of giving a single MK-801 injection on Day 3 after nerve ligation was based on our behavioral observation that CCI rats exhibited the most pronounced thermal hyperalgesia approximately 3 days after nerve ligation. Animals in all groups (including preinjury treatment and postinjury treatment groups) were first tested before surgery to obtain baseline latencies. Rats in preinjury treatment groups were then tested on days 1, 3, 5, 7, 10, and 15 after nerve ligation. Rats in postinjury treatment groups were then tested on days 4 and 5 postsurgery. All experiments including those described below were made with the tester blind as to treatment conditions.

lntrathecal MK-801 experiments The possible direct spinal cord effects of MK-801 on hyperalgesia were examined by i.t. injection of MK-801. Rats were implanted with i.t. catheters immediately following the nerve ligation procedure. A gentamicin sulfate-flushed polyethylene (PE-10) tube was inserted into the subarachnoid space through an incision at the cisterna magna. The caudal end of the catheter was gently threaded to the lumbar enlargement or thoracic segments. The rostral end was then secured with dental cement to a screw embedded in the skull• The skin wound was closed with wound clips. Those rats exhibiting postsurgical motor deficits (e.g., limb paralysis) were excluded from the experiment. Two groups of CCI rats (n = 6/group) with or without i.t. catheter implantation were assessed during the first 5 days after surgery for hyperalgesic responses and spontaneous behaviors to examine possible effects of the i.t. catheter implantation procedure itself on neuropathic pain behaviors. Five groups of CCI rats (n = 7/group) received a single i.t. injection of MK-801 (1.25, 2.5, 5, 10, or 20 nmol) at the spinal cord lumbar segment level, and one group of CCI rats (n = 7) received an equal vol. of saline. All injections were given on Day 3 after nerve ligation. MK-801 or saline (in 10 #1) was delivered slowly (within 1 min) through the i.t. catheter followed by 10 #1 saline to flush the catheter• This dose regimen was employed to examine the dose-dependent effects of i.t. MK-801 on thermal hyperalgesia. In addition, MK-801 (10 nmol) was injected onto the thoracic spinal cord (n = 7) in order to determine the spinal cord site of MK-801 action. Thermal hyperalgesia was assessed on days 0 (baseline), 3. 4, 5, and 7 after nerve ligation in all groups. Two separate groups of CCI rats (n = 8/group) were used to examine the effects of i.t. 10 nmol MK-801 on spontaneous pain behaviors. These rats (n = 8/group) received either i.t. MK-801 (10 nmol) or saline on Day 3 after nerve ligation and were graded by using the spontaneous pain rating method on days 0 (baseline), 3, 4, 5, and 7 after nerve ligation. Local nerve anesthesia The effect of peripheral nerve anesthesia on hyperalgesia was examined by the injection of bupivacaine, a local anesthetic agent, into the perinerve space on Day 3 after nerve ligation in 8 CCI rats. The peripheral nerve block was performed by carefully inserting a needle into the perinerve space. The insertion angle and depth of the needle were guided by a mark made on the skin surface during the surgery for nerve ligation such that the needle was close to the perinerve space proximal to the site of nerve ligation. Contact with the nerve, as indicated by twitches of the affected hind paw, was avoided. The choice of guided injection avoided reopening the incision made during the surgery for nerve ligation thereby minimizing the confounding nociceptive input from the incision• Bupivacaine (0.5%, 0.6 ml) was delivered slowly into the perinerve space proximal to the site of nerve ligation. Effective anesthesia of the sciatic nerve trunk was inferred by (1) lack of responses of the affected hind paw to intense mechanical stimulation delivered by clamping the skin with a forceps, and (2) paralysis of the affected hind paw. The initial recovery from local anesthesia beginning approximately 1 h after injection was inferred by a slight response to mechanical stimuli (e.g., touching or a light clamping of the skin with a forceps). Full recovery from local nerve anesthesia (i.e., normal responses to mechanical stimuli and no sign of paralysis) occurred at about 3 h after injection. In addition, CCI rats in 3 control groups (n = 6/group) received equal vol. of either (1) ipsilateral perinerve saline injection, (2) contralateral perinerve bupivacaine injection, or (3) contralateral intramuscular bupivacaine injection. In two separate groups (n = 8/group) , one group received the lowest effective i.t. dose of MK801 (2.5 nmol), and the other received simultaneously i.t. MK-801 (2.5 nmol) and ipsilateral perinerve bupivacaine (0.6 ml) on Day 3 after nerve ligation. These two groups allowed examination of possible interactive effects of MK-801 treatment and peripheral nerve anesthesia on hyperalgesia. Foot-withdrawal latencies were assessed on days 0, 3, 4, 5, and 7 after nerve ligation in all groups.

257

Data analysis Data derived from the foot-withdrawal test and the spontaneous pain behavior rating were first analyzed by using a two-way analysis of variance (ANOVA) repeated across test days to detect overall differences among treatment groups. Subsequently, the WallerDuncan K-ratio t-test (WD) was performed to determine sources of differences. The same statistical procedures were applied to detect the difference among test days repeated across treatment groups. Data on frequency counts of hind paw elevations were analyzed using a non-parametric Mann-Whitney U test (MW). RESULTS

Effect of systemic MK-801 on thermal hyperalgesia The average foot-withdrawal latency taken from 48 CCI rats on Day 3 after nerve ligation shifted from a baseline of 10.38 s to a postsurgical latency of 7.36 s. Corresponding skin temperature readings from anesthetized CCI rats were 47.88 and 45.45°C (Fig. 1), respectively, indicating that the procedure of sciatic nerve ligation produced thermal hyperalgesia in CCI rats. That is, noxious skin temperatures were still required to elicit withdrawal in CCI rats. Also shown in Fig. 1, skin temperature readings did not differ between normal rats and CCI rats (ANOVA, P > 0.05). As shown in Fig. 2, those rats receiving saline injection manifested hyperalgesia over the 15 day experimental period. In contrast, CCI rats receiving 4 daily i.p.

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MK-801 treatments (0.03, 0.1, 0.3 mg/kg) initiated 15 min prior to nerve ligation exhibited reliably lower difference scores (i.e., less hyperalgesia) on Day 3 after nerve ligation as compared to those in the saline group (ANOVA, P < 0.01). This reduction of thermal hyperalgesia following preinjury treatment with MK-801 lasted over the experimental period (days 5, 7, 10, 15) even after the withdrawal of MK-801 treatment prior to Day 4 after nerve ligation (ANOVA, P < 0.01). Within the dose range used in systemic MK-801 experiments, the attenuation of hyperalgesia produced by 0.03 mg/kg MK801 treatment was not quantitatively different from that obtained after 0.3 mg/kg MK-801 treatment (WD, P > 0.05). In addition, neither ipsilateral sciatic nerve ligation nor systemic MK-801 (0.03-0.3 mg/kg) treatments changed absolute foot-withdrawal latencies of the hind paw contralateral to the side of nerve ligation on each day of postinjury behavioral assessment (ANOVA, P > 0.05). Thus, MK-801 had no direct effect on baseline foot-withdrawal latencies. A single injection of MK-801 (0.3 mg/kg) on Day 3 after nerve ligation reduced thermal hyperalgesia when tested 24 h after injection compared to CCI rats receiving the saline injection (2.17 + 0.35 s vs. 4.37 + 0.35 s, WD, P < 0.05). However, unlike the results employing multiple injections of MK-801, single injection of MK801 reduced thermal hyperalgesia in a dose-dependent manner, i.e., a single injection of MK-801 at doses lower

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Fig. 2. MK-801 pretreatment reduces thermal hyperalgesia following sciatic nerve ligation. Four daily i.p. injections of MK-801 (0.03, 0.1, 0.3 mg/kg) initiated 15 rain prior to nerve ligation reliably reduced thermal hyperalgesia on Day 3 after nerve ligation. This reduction of thermal hyperalgesia lasted over the experimental period (up to day 15 after nerve ligation) even after the withdrawal of MK-801 treatment prior to Day 4 after nerve ligation. The latency difference score shown on the Y-axis was obtained by subtracting ipsilateral foot-withdrawal latencies from contralateral footwithdrawal latencies (also in Figs. 3 and 5). x, p < 0.05; x~, p < 0.01 (ANOVA), as compared among different groups. *, P < 0.05 (WD), as compared to the saline group.

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Fig. 3. Dose-dependent relationship of i.t. MK-801 effects on thermal hyperalgesia. A single i.t. injection of MK-801 on Day 3 after nerve ligation at doses higher than 2.5 nmol (5, 10, and 20 nmol) attenuated thermal hyperalgesia for at least 24 h after injection. Thermal hyperalgesia that was attenuated by 2.5 nmol MK-801 returned to the preinjection level within 48 h after injection. In contrast, the attenuation of hyperalgesia by higher doses of MK-801 (5, 10, 20 nmol) subsided gradually within 4 days after injection, x, P < 0.05; xx, p < 0.01 (ANOVA), as compared among different groups. *, P < 0.05 (WD), as compared to the saline group.

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Fig. 4. Intrathecal MK-801 reduces spontaneous pain-related behaviors. A single i.t. injection of 10 nmol MK-801 on Day 3 after nerve ligation reliably lowered spontaneous pain behavior rating scores as compared to the saline group. The spontaneous pain behavior rating score was defined as the total duration over which a CCI rat presented one or more of following spontaneous pain-related behaviors within a 5 min observation period: (1) placement of only the medial edge or the heel of the ligated hind paw onto the ground, and (2) the elevation or licking of the ligated hind paw. Standard errors are presented as vertical lines, x~, p < 0.01 (ANOVA), as compared to the saline group.

than 0.3 mg/kg (0.01, 0.03, 0.1 mg/kg) had no effect on thermal hyperalgesia. Effect o f intrathecal MK-801 on thermal hyperalgesia Before MK-801 administration on D a y 3 after nerve

ligation, CCI rats in all groups exhibited larger difference scores c o m p a r e d to baseline scores ( W D , P < 0.05). A single injection of MK-801 onto spinal cord lumbar segments at doses ranging from 2.5 to 20 nmol reliably reduced thermal hyperalgesia when tested 24 h after injection ( W D , P < 0.05, Fig. 3). As also shown in Fig. 3, the attenuation of hyperalgesia p r o d u c e d by higher effective doses of MK-801 (5-20 nmol) subsided gradually through D a y 7 after nerve ligation. Thus, difference scores in higher dose groups (5, 10, 20 nmol) remained lower 48 h after injection as c o m p a r e d both to

TABLE I Median numbers of ligated hind paw elevations Day

Saline (n = 8)

MK-801 P-value (10 nmol, n = 8) (MW)

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Fig. 5. Intrathecal MK-801 and local nerve anesthesia synergistically reduce thermal hyperalgesia. A single i.t. injection of 2.5 nmol MK-801 or a single perinerve injection of bupivacaine (0.5%, 0.6 ml) on Day 3 after nerve ligation reduced thermal hyperalgesia when assessed 24 h after injection. While a single injection of MK801 or bupivacaine attenuated thermal hyperalgesia for less than 48 h after injection, the combination of these two treatments extended the effective duration for at least 4 days after injection, xx P < 0.01 (ANOVA), as compared among different groups. *, P < 0.05 (WD), as compared to the saline group. (*), P < 0.05 (WD), as compared to each of 3 other groups.

saline and to lower dose groups (1.25, 2.5 nmol, W D , P < 0.05). However, unlike intralumbar injections, a single injection of 10 nmol MK-801 onto the thoracic spinal cord did not attenuate hyperalgesia ( A N O V A , P > 0.05). In addition, the procedure of i.t. catheter implantation had no influence on thermal hyperalgesia and spontaneous pain behaviors at least 5 days after surgery ( A N O V A , P > 0.05), nor did the i.t. MK-801 treatments change foot-withdrawal latencies of the hind paw contralateral to the side of nerve ligation ( A N O V A , P > 0.05). Effect o f intrathecal MK-801 on spontaneous pain behaviors W h e n observed before MK-801 treatment on D a y 3 after nerve ligation, CCI rats exhibited abnormal behaviors such as the placement of only the medial edge or the heel of the ligated hind paw on the ground, frequent elevation and occasional licking of the ligated hind paw. The mean duration (from 3 trials) over which one or more of these behaviors was present throughout a 5 min observation period was about 145 s (Fig. 4). A single injection of 10 nmol MK-801 onto the lumbar spinal cord on D a y 3 after nerve ligation reliably lowered spontaneous pain behavior rating scores when assessed 24 h after injection as c o m p a r e d to the saline group ( A N O V A , P < 0.01, Fig.4). M o r e o v e r , CCI rats receiving i.t. MK801 (10 nmol) elevated the ligated hind paws much less

259 frequently than those receiving the saline injection during the same observation period (MW, P < 0.01, Table I). Effect of local nerve anesthesia and its combination with intrathecal MK-801 on thermal hyperalgesia When tested 24 h after bupivacaine injection, CCI rats receiving the ipsilateral perinerve bupivacaine injection (0.5%, 0.6 ml) on Day 3 after nerve ligation had reliably lower foot-withdrawal latency difference scores (i.e., less hyperalgesia) as compared to those receiving the ipsilateral perinerve saline injection (ANOVA, P < 0.01, Fig. 5). In contrast, contralateral perinerve bupivacaine or contralateral intramuscular bupivacaine injections (0.5%, 0.6 ml) did not attenuate thermal hyperalgesia (ANOVA, P > 0.05), nor the absolute foot-withdrawal latencies of the hind paw contralateral to the side of nerve ligation (ANOVA, P > 0.05) when tested 24 h after injections. As indicated above, 2.5 nmol MK-801 injected i.t. on Day 3 after nerve ligation reduced thermal hyperalgesia for less than 48 h after injection. While either local nerve anesthesia or i.t. 2.5 nmol MK-801 alone reduced thermal hyperalgesia for less than 48 h after injection, the combination of both treatments extended the duration of the attenuation of thermal hyperalgesia for at least 4 days after injection (ANOVA, P < 0.01, Fig. 5).

DISCUSSION The present experiments indicate that thermal hyperalgesia and spontaneous pain behaviors following constrictive nerve injury are maintained both by abnormal peripheral input 12'23'39 and altered central activity13A5'45. MK-801 treatment initiated prior to nerve injury reduces thermal hyperalgesia produced by sciatic nerve ligation. This result indicates that NMDA receptor activation may be critical for the induction and/or maintenance of neuropathic pain behaviors following constrictive nerve injury. On the other hand, the demonstration that the postinjury application of either MK-801 or a local anesthetic bupivacaine applied to the injured nerve attenuates thermal hyperalgesia and spontaneous pain behaviors suggests that postinjury neuropathic pain behaviors may be maintained by both abnormal peripheral input and central mechanisms. Since hyperalgesia and spontaneous pain are two salient clinical features of human painful peripheral neuropathy following peripheral nerve injury3°'3s, these results also suggest new approaches for the clinical management of postinjury neuropathic pain s3)ndromes. Contributions of central N M D A receptor activation to neuropathic pain behaviors The present demonstration that systemic treatment

with MK-801, a non-competitive NMDA receptor antagonist 2s, initiated prior to nerve injury reduces thermal hyperalgesia in CCI rats provides behavioral evidence for the involvement of NMDA-receptor activation in the development of neuropathic pain behaviors in a rat model of painful peripheral mononeuropathy. In a report using the same model ~°, preinjury treatment with MK-801 (1.0 mg/kg, i.p.) beginning immediately before nerve ligation and daily for the first 7 days after nerve ligation produces a protective effect on the development of the postinjury thermal hyperalgesia. Our data further indicate that 4 daily MK-801 injections, the first of which was initiated 15 min prior to nerve ligation, are sufficient to attenuate thermal hyperalgesia over the experimental period (up to 15 days after nerve ligation). Thus, the first few days after nerve ligation might be a critical stage for the development of neuropathic pain behaviors, and NMDA-receptor activation may play an important role at this stage. This is consistent with an observation that preinjury treatment with NMDA receptor antagonists (MK-801 or a competitive NMDA antagonist, 5-APV) prevents the development of neuropathic pain behaviors (autotomy) in a model of peripheral nerve section 36. All doses of MK-801 employed in the systemic experiments, two of which (0.03 and 0.1 mg/kg) did not attenuate hyperalgesia when given only once on Day 3 after nerve ligation, produce attenuation of hyperalgesia with multiple injections. The absence of dose-dependency following multiple injections may be due to changes of receptor sensitivity following repeated exposure to MK-801 and/or the 'floor' effects of doses of MK-801 (i.e., doses being too high) on nociceptive behaviors employed in the multiple injection experiments. In addition, it is possible that systemic MK-801 treatments exert actions at multiple sites. However, since systemic treatment with single doses of MK-801 does show dose-dependency, it is more likely that the lack of dose-dependency is due to multiple injections. The possibility that the maintenance of postinjury hyperalgesia would require NMDA-receptor activation was tested by a single postinjury injection of MK-801 on Day 3 after nerve ligation. The results support this hypothesis and reveal a dose-dependent relationship in i.t. MK801 experiments. First, a single i.t. injection of MK-801 (2.5-20 nmol) produces a prolonged reduction of hyperalgesia for at least 24 h, a duration comparable to that for its in vitro 22 and in v i v o 41 protection on ischemia-induced neuronal death. MK-801 at doses higher than 5 nmol (but not 2.5 nmol) does not further decrease the degree of thermal hyperalgesia but extends the duration of the attenuation of thermal hyperalgesia for at least 48 h after injection. This dose-dependent relationship indicates that the effect of NMDA receptor blockade by

260 MK-801 on thermal hyperalgesia may reach a plateau when i.t. doses are higher than 5 nmol, and suggests that non-NMDA receptor activation might be involved in development/maintenance of postinjury nociceptive behaviors. Finally, the site of i.t. MK-801 action appears to be central and specifically at the caudal (probably lumbar) spinal cord level, since i.t. injection of 10 nmol MK801 is effective in the reduction of hyperalgesia when injected onto the lumbar spinal cord, yet fails to produce any protective effect when injected onto the thoracic spinal cord in CCI rats. These results, together with the preinjury treatment data, suggest that central NMDA receptor activation contributes to induction and/or maintenance of hyperalgesia after peripheral nerve injury.

Contributions of abnormal peripheral input to neuropathic pain behaviors One proposed mechanism of postinjury hyperalgesia is related to peripheral changes such as the sensitization of nociceptive receptors 5'8'35 and spontaneous discharges derived from the severed nerve 17'19'4°. The demonstration of the relatively prolonged attenuation of thermal hyperalgesia by postinjury local nerve anesthesia suggests that peripheral abnormalities produced by sciatic nerve ligation may also contribute to the maintenance of thermal hyperalgesia in this model. The ineffectiveness in reduction of thermal hyperalgesia by both contralateral perinerve bupivacaine and contralateral intramuscular bupivacaine injections indicates a local action of local nerve anesthesia. This result is consistent with the clinical observation that application of local anesthetics to the injured nerve effectively reduces neuropathic pain reports 7. The abnormal peripheral input following nerve ligation in this neuropathic pain model may have a tonic influence on central nociceptive processes, since the sciatic nerve undergoes a continuously constrictive injury after the initial ligation. As also described previously4, we have observed that most ligatures (chromic gut) around the ligated sciatic nerve are not removed by enzymatic digestion during the first 10 days after nerve ligation. It is reported that ectopic discharges may arise from the ganglion of the injured nerve at the early stage (days l-3) after nerve ligation 2°. Our observation that thermal hyperalgesia was reduced after bupivacaine was applied to the injured nerve distal to the ganglion suggests that ectopic discharges arising from the ganglion may not be crucial for maintenance of postinjury thermal hyperalgesia. Thus, ongoing abnormal peripheral input may be derived from the ligated sciatic nerve due to the continuous constriction for at least the first 10 postinjury days. The consequent ongoing release of glutamate and/or aspartate from the primary afferent terminals 6'42--44 during

this time period may in turn activate central NMDA receptors 6'36 which, at least partially, contribute to maintenance of neuropathic pain behaviors. Ongoing abnormal peripheral input cannot, however, exclusively account for postinjury thermal hyperalgesia in this model for several reasons. First, as seen in clinical studies 7, the attenuation of thermal hyperalgesia by local nerve anesthesia persists for a duration longer than the action of a local anesthetic. Second, CCI rats receiving 4 daily i.p. injections of MK-801 initiated prior to nerve ligation exhibit persistent attenuation of thermal hyperalgesia for at least 15 days after nerve ligation. Finally, the combination of i.t. MK-801 and local nerve anesthesia extends the attenuation of hyperalgesia for a duration longer than that resulting from either one of these two manipulations. These observations suggest that CCI produces a persistent change in central nociceptive processes which is at least partially mediated by NMDA receptor activation. Therefore, ongoing abnormal peripheral input and altered central nociceptive processes may synergistically induce and maintain neuropathic_pain behaviors following constrictive nerve injury. Thus, the combination of both peripheral nerve anesthesia and central NMDA receptor blockade may be useful clinically to treat patients with reflex sympathetic dystrophy (RSD) resulting from peripheral nerve injury.

Consequence of ongoing peripheral input, a possible central hyperactive state The influence of ongoing abnormal peripheral input on central nociceptive processing may reflect an important aspect of pathological processes in the induction and maintenance of neuropathic pain and possibly other chronic pains. The continuous abnormal peripheral input may gradually result in alterations of central neural activity due to excitatory neurotoxic effects of glutamate and/or aspartate 22'41. This altered central activity, a possible postinjury central hyperactive state x5"45'46, may be reflected in this CCI model as either increased spontaneous neural activity (spontaneous pain) or decreased pain threshold and/or increased responses to noxious stimulation (hyperalgesia), or both. Evidence for the former is that CCI rats exhibit spontaneous pain-related behaviors 3'4 that can be reduced by i.t. MK-801 treatment. Consistent with behavioral observations, our recent 2-deoxyglucose autoradiographic studies have revealed increases in neural activity in spinal cord lumbar segments of CCI rats in the absence of overt peripheral stimulation 24'33. In addition, CCI produces enhanced C-los protein expression in spinal cord dorsal horn neurons 21. Thus, it is likely that, ongoing spontaneous discharges that may derive from the injured n e r v e 12'17"19'4° and/or NMDA receptor-mediated central hyperactivity

261 contribute to spontaneous pain behaviors. Evidence for decreased pain threshold/increased responses to noxious stimulation in CCI rats is indicated by the behavioral hyperalgesia to radiant heat in CCI rats. In addition, extracellular recordings of spinothalamic tract neurons indicate a higher background firing and prolonged afterdischarges of these neurons in response to mechanical or thermal stimulation in CCI rats 14 days after nerve ligation 28. Although these studies indicate that CCI can produce enduring increases in spinal cord neural activity and behavioral changes, these alterations in CCI rats may also have been influenced by tonic input from supraspinal regions. At present, we have an incomplete understanding of the possible role(s) of supraspinal influences on spinal cord neural activity and behavioral manifestations in rats with neuropathic pain syndromes. Future studies comparing spinalized and intact CCI rats may help to elucidate this relationship. Relationships o f central N M D A receptor activation to mechanisms o f neuropathic pain Both behavioral and electrophysiological studies have implicated N M D A receptor activation in spinal cord nociceptive processing. Intrathecal administration of N M D A produces hyperalgesic responses in the mouse 2 and rat 34. Iontophoretic application of N M D A onto spinal cord nociceptive neurons increases neuronal responses to peripheral noxious mechanical or thermal stimulation 1. Conversely, N M D A receptor antagonists (AP5 or MK-801) produce antinociceptive effects in the mouse formalin test 27, and inhibit spinal nociceptive neuronal discharges elicited by peripheral formalin ap-

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Acknowledgement. This work was supported by P.H.S. Grants NS 21458 and NS 20049.

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