Brain Research, 522 (1990) 55-62 Elsevier

55

BRES 15638

N-Methyl-o-aspartate receptors mediate responses of rat dorsal horn neurones to hindlimb ischemia Graham D. Sher and Duncan Mitchell Brain Function Research Unit, Departmentof Physiology, University of the WitwatersrandMedical School, Johannesburg (South Africa) (Accepted 26 December 1989) Key words: Ischemia; Nociception; Hyperalgesia; Dorsal horn; N-Methyl-D-aspartate; Excitatory amino acid antagonist We have investigated the role of NMDA receptors in the rat dorsal horn in mediating neuronal responses to noxious hindlimb ischemia, induced by acute occlusion of the femoral artery, as well as in the hyperalgesia evident when noxious mechanical stimuli were applied to the ischemic limb. Two specific NMDA antagonists, D-2-amino-5-phosphonovalerate (APV) and ketamine hydrochloride were applied intrathecally directly on to the spinal cord, in enflurane-anaesthetised rats. Both APV (1/aM and 100/aM) and ketamine (1 mM and 100 mM) inhibited the increase of dorsal horn neuronal firing rate induced by ischemia, but did not alter the neuronal response to noxious pinching or innocuous brushing of the receptive field. Both agents, however, abolished the hyperalgesia to noxious pinching induced by ischemia. Our results support the hypothesis that the excitatory amino acids are involved in the transmission of nociceptive information in the spinal dorsal horn, and also favour a central mechanism for hyperalgesia at the spinal level, possibly also mediated by the NMDA receptor. INTRODUCTION The excitatory amino acids ( E A A ) L-glutamate and L-aspartate are putative neurotransmitters at sites widely distributed throughout the mammalian central nervous system (CNS)2'3"9"42. The E A A s act at 3 receptor subtypes, n a m e d for the compounds which are the most effective agonists at each, N-methyl-D-aspartate (NMDA), quisqualate and kainate. Processes in which the E A A s and their receptors are involved include developmental plasticity 9, learning and m e m o r y 28, epileptiform activity 2° and mediation of neuronal damage due to ischemia 26'33. E A A s may also operate as neurotransmitters in sensory afferent pathways. Salt 35 has found that both N M D A and n o n - N M D A E A A receptors are involved in synaptic responses of ventrobasal thalamic neurones to sensory stimulation. Earlier work implicated endogenous E A A s acting at n o n - N M D A receptors in neurotransmission between vibrissae and the rat trigeminal nucleus caudalis 36. High concentrations of L-glutamate 3s and of E A A binding sites 12'16'17 have been found in the dorsal horn of the spinal cord. In vitro studies have shown that dorsal horn neurones mediate excitatory neurotransmission at least in part through the N M D A receptor 2324. The stimuli which have been used to excite afferent pathways in the studies have been either innocuous somatosensory stimuli or electrical depolarisation of afferent fibres.

E A A s may play a role in mediating nociceptive afferent information too. Micro-injection of glutamate 6 or N M D A 22 into the peri-aqueductal grey matter in rats produced potent analgesia which was reversed by the specific N M D A antagonist 2-amino-7-phosphonoheptanoate. Willcockson et al. 43 have shown that nociceptive neurones in the primate spinal cord increased firing after iontophoretic application of glutamate. Intrathecal administration of E A A agonists elicited a dose-dependent behavioural response with characteristics of the response to noxious stimulation 1, while intrathecal administration of a specific N M D A receptor antagonist inhibited behavioural responses to aversive electrical stimuli 5. A a n o n s e n and Wilcox 2 found that E A A agonists given intrathecally to conscious mice induced aversive behaviour as well as hyperalgesia to noxious stimuli. We have now investigated the role of spinal N M D A receptors in mediating another form of nociceptive information, that arising during ischemia. We also recorded the responses of neurones in the rat dorsal horn to noxious and innocuous stimuli applied to the hindlimb, in the presence and absence of hindlimb ischemia. We then examined how these responses were affected by two N M D A receptor antagonists applied to the spinal cord. The two antagonists were the competitive antagonist D-2-amino-5-phosphonovalerate and the non-competitive antagonist ketamine hydrochloride.

Correspondence: G.D. Sher, Department of Physiology, University of the Witwatersrand Medical School, York Road, Parktown, Johannesburg 2193, South Africa. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

56 MATERIALS AND METHODS

Animal preparation Male Sprague-Dawley rats weighing 225-400 g were used in all experiments. Anaesthesia was induced with sodium pentobarbitone (50 mg/kg i.p.) and a tracheal cannula was inserted, through which the gaseous anaesthetic enflurane (Ethrane, Abbott Laboratories) in nitrous oxide/oxygen (67%/33%) was administered, at concentrations of 2-3% for surgery and 0.6-1% for maintenance. Rats breathed spontaneously throughout the procedures. Core body temperature was maintained at 38 °C by means of a heating blanket. The right femoral artery was exposed, and carefully dissected free from surrounding nerve fibres and the femoral vein. A snare constructed by threading a silk ligature (2/0) through a polyethylene tube was passed under the artery, and sutured to adjacent muscle to maintain its position. The femoral wound was closed with the proximal end of the snare exteriorised. A laminectomy was performed at the L1-L 3 level and the dura mater stripped off the cord. Vertebrae rostral and caudal to the exposed cord were rigidly clamped to minimise movement at the recording site. Muscle flaps were raised to form a small pool around the exposed cord for drug administration.

Recording technique and characterisation of cells Single unit extracellular recordings were made from dorsal horn neurones at depths 50-700/zm (mean = 365 + 58/~m) below the surface of the cord, the depths being determined by readings taken from a stepping microdrive. The majority of the cells recorded (58%) lay in the superficial dorsal horn (< 275 /am), with the remaining units being located in deeper laminae. Analysis of neuronal responses with respect to depth showed no difference between superficial and deep units, and the results have thus been presented as mean responses of all convergent neurones recorded. Neurones were included only if their receptive fields lay in the right hindlimb, and if they responded to both noxious pinching and innocuous brushing of the receptive field (convergent or multireceptive neurbnes). Neurones apparently responding only to either noxious or innocuous stimuli alone were not studied. The receptive field of each neurone was mapped out at the start of each experiment and again following 60 min ischemia. All recordings were made using glass-coated tungsten microelectrodes, the tips of which were coated with gold and platinum. Amplified potentials were fed through a spike processor (Digitimer D130, Digitimer Ltd., U.K.) into a laboratory interface (CED1401, Cambridge Electronic Designs, Cambridge) attached to a microcomputer for storage and analysis. To ensure that the same unit was being recorded from for the full duration of an experiment, the amplitude, shape and periods of the positive and negative phases of each impulse were monitored on a fast time base oscilloscope. Once a suitable neurone had been identified, its spontaneous firing rate was measured; only those neurones which had a background firing rate of less than 2.5 Hz were used in this study. The reason for this limitation was to eliminate the possibility of any unit exhibiting cell injury potentials. For 30 min, at approximate 5 min intervals, the receptive field was stimulated by two forms of 'natural' mechanical stimuli, one noxious and one innocuous, each of 20 s duration. A noxious pinch was applied by means of a serrated forceps to the centre of the receptive field (contact area = 26 mm2), at a pressure of 67 kPa, delivering an effective force of 1.7 N (and perceived as painful when applied to the investigators' fingers). Innocuous stimulation was achieved by gently stroking the skin in a caudal-rostral direction along the entire length of the receptive field with repeated strokes, each about 2 s in duration, using an artist's brush. Only one investigator (G.D.S.) applied the stimuli in all experiments. Hindlimb ischemia then was induced by acute occlusion of the femoral artery with the snare. The ischemia was manifest in initial pallor of the paw, with subsequent cyanosis and, in some animals, later hyperaemia. At the end of each experiment the femoral wound was exposed and the artery examined to confirm successful occlusion by the snare; animals in which there was any

evidence for incomplete or unsustained occlusion were excluded. Following the onset of ischemia, neuronal firing rate was recorded for 30 min during which no other stimuli were applied to the limb. Then, for a final period of 30 min, with ischemia still present, the pinch and brush were re-applied. In a further 6 animals, the procedure above was replicated, only ischemia was not induced in these animals (sham controls).

Drug administration The drugs were applied directly on to the exposed spinal cord in 25 /~1 boluses at the onset of ischemia, mimicking intrathecal administration. Control animals were treated with physiological saline in similar fashion. The two NMDA receptor antagonists, D-2-amino-5-phosphonovalerate (APV, Sigma) and ketamine hydrochloride (Ketalar, Parke-Davis), were dissolved in physiological saline. Two doses were employed for each drug, of the order of those shown previously to antagonise amino acid-induced excitations of mammalian spinal neurones 23'32'34'35. The concentrations were: APV 1 /~M and 100gM (pH 5.4) and ketamine 1 mM and 100 mM (pH 5.5). It has been shown using tritiated APV that intrathecal administration of the drug results in good spread into the spinal cord tissue, and that the concentration of APV which depressed excitatory transmission was in the range of 15-75/~M 5. We chose to administer ketamine intrathecally rather than systemically, since we wished to examine the role of the NMDA receptor in the spinal cord specifically. Systemic injection would not have enabled us to distinguish spinal from supraspinal sites of action, known to occur after i.v. injection 37. Statistical comparisons were made using the paired t-test with the Bonferroni correction for multiple comparisons. All procedures were approved by the Animal Ethics Committee of the University of the Witwatersrand (Clearance Certificate No. 89/16/2).

RESULTS

Neuronal response to ischernia O f t h e 8 s a l i n e - t r e a t e d a n i m a l s , 7 s h o w e d an i n c r e a s e d firing r a t e

of dorsal

horn

neurones

in r e s p o n s e

to

h i n d l i m b i s c h e m i a , t h e i n c r e a s e b e i n g s u s t a i n e d for all 60 min of ischemia. T h e m e a n p e r c e n t a g e i n c r e a s e in m e a n firing r a t e , d e t e r m i n e d as: (1/2No to 60 m i n - N _ 3 0 to 0 m i n ) x 100 N_3o to 0 m i n w h e r e N = n u m b e r of spikes, was 480 + 9 0 % ( m e a n + S . E . M . ) . F o r e a c h of t h e two successive 30 m i n i s c h e m i c p e r i o d s , the n e u r o n a l activity was significantly g r e a t e r t h a n p r e - i s c h e m i c activity ( P < 0.05, Fig. 1). T h e latency to o n s e t o f t h e e n h a n c e d activity, f o l l o w i n g institution o f i s c h e m i a , was 10.3 + 2.5 m i n , a v a l u e c o r r e s p o n d i n g well with t h e latency p r e v i o u s l y r e p o r t e d for b e h a v i o u r a l r e s p o n s e s to i s c h e m i a in c o n s c i o u s rats ~3. Fig.

2A

demonstrates

the

responses

of a typical

n e u r o n e r e c o r d e d in a s a l i n e - t r e a t e d a n i m a l b e f o r e and during ischemia. P i n c h i n g the r e c e p t i v e field p r o d u c e d a g r e a t e r spike discharge t h a n did b r u s h i n g t h e s a m e area. Two consistent f e a t u r e s w e r e n o t e d in t h e r e s p o n s e to ischemia: firstly, the firing rate i n c r e a s e d gradually, and

57 8'

secondly it tended to plateau after some time. Six of the 7 neurones which responded in such a fashion had a monophasic response, while one neurone had a biphasic response, with the second peak larger than the first, and only the latter reaching a plateau. When applied to the same area of the receptive field, a noxious pinch elicited a significantly greater response (P < 0.001) during ischemia than prior to ischemia (Fig. 3). This ischemic hyperalgesia was present in 5/8 animals. Response to an innocuous stimulus, however, was not influenced by the ischemia (Fig. 3).

d 7 6¸ "~ 5¸ o

a

"r" 2

8 ®

1

0

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b

j brush

inch [pinch

. .J .,,, .L, |,,.

III

brush

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/pinch

P, 30

30

~

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lechaemla (30 to 60 min.)

animals receiving the 1 ~M dose, 5 failed to show recovery of the spontaneous firing rate by the end of the recording period. In the other three, the firing rate of the neurone either reached or exceeded preischemic levels, in a response similar to that of saline-treated animals. For the higher dose of APV (100/~M), 6 neurones were still inhibited after 60 min, while two showed recovery. Neuronal responses to noxious pinching of the receptive field were the same before and during ischemia, in the presence of both doses of APV. Moreover, the responses during ischemia in both groups of APV-treated

Ai.

30

u)

L

lachaemia (0 to ,t0 rnln.)

Fig. 1. Mean firing rate of convergent dorsal horn neurones with hindlimb receptive fields, before and during hindlimb ischemia. Histogram and bars represent means _+ S.E.M. for n = 8 animals. Firing rates for both ischemic periods are significantly greater than pre-ischemic firing rates (P < 0.05).

Application of the specific NMDA antagonist APV to the spinal cord at the onset of the ischemia reduced the response to ischemia significantly (P < 0.05), in 7/8 animals treated with 1/zM APV and in 6/8 treated with 100/zM APV (Fig. 4). For both doses of the drug, there is no significant difference in the mean firing rate between the pre-ischemic and the ischemic periods. Fig. 2B shows the activity of a typical dorsal horn neurone during APV treatment. Spontaneous firing rate actually tended to decrease after application of APV, in spite of the concurrent ischemia, the mean percentage inhibition for the lower dose of APV being 19.6 + 4.3% and 49.6 + 7.4% for the higher dose. The mean latency of onset of the inhibition was 12.7 + 3.4 min for the lower dose and 11.1 + 2.7 min for the higher dose. Of the 8

++

+

Pre-lechaemla

(-30 to 0 mln.)

Effect of APV

so-

4

|

I,

pinch t ,,+y

+ +,..t...L. . . . . . I, ,.,+,J. . . . . . . ~ Ill

brush|pinch

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Fig. 2. Individual examples showing the firing patterns of convergent dorsal horn neurones before and during ischemia with and without drug treatment. Traces I represent first 15 min of pre-ischemic recordings (-30 to -15 min); II = first 15 min of ischemia (0 to 15 min); I I I = last 15 min of ischemia (45 to 60 min). Series A, saline-treated control; B, animal given 1/~M APV; C, animal given 1 mM ketamine. Vertical arrows depict time of onset of ischemia and of drug administration. Note the truncated responses to noxious pinching in Am.

58

Effect of ketamine

1200.

Spinal cord administration of k e t a m i n e hydrochloride

1000.

== "6

affected dorsal horn n e u r o n a l responses in a way very similar to APV. In 6 out of 8 animals given 1 m M and all

800.

8 given 100 m M ketamine, the increase in spontaneous

600.

firing rate during ischemia was abolished. The mean firing rate for the first 30 min of ischemia was significantly

400Z

reduced in both groups of k e t a m i n e - t r e a t e d animals

2000

Pre-lschaemta

Ischaemla

Fig. 3. Evidence for hyperaigesia to noxious mechanical stimuli during ischemia. Open bars represent total number of spikes induced by noxious pinching; the response during ischemia was significantly greater than prior to ischemia (P < 0.001). Filled bars represent response to innocuous brushing; there was no difference in the response during ischemia.

animals were significantly less than those of saline controls (P < 0.05). In other words, the ischemic hyperalgesia seen in control animals was abolished by APV. The response to innocuous stimulation of the receptive field was unaltered by the administration of APV (Fig. 5). Application of APV (100 btM) to 6 sham-operated animals (no ischemia present) did not alter the neuronal response to either noxious pinching or innocuous stimulation of the receptive field. The response to noxious pinching was 576 + 89 spikes (mean + S.E.M.) after APV treatment, compared to 562 + 37 spikes induced in the pre-ischemic period. Following innocuous stimulation, the neuronal response was 301 + 45 spikes (mean + S.E.M.) after APV treatment compared to 337 + 29 in the pre-ischemic period.

compared to saline controls ( P < 0.05), while in the second 30 min period only the lower dose of ketamine resulted in a significant reduction (P < 0.05) of firing rate (Fig. 6). Over the entire 60-min period, the m e a n percentage inhibition for the lower dose of ketamine was 43.1 + 6.8% and for the higher dose was 70.6 + 21.3%. The latency of onset of inhibition was 3.6 + 0.4 min for the lower dose and 2.6 + 0.5 min for the higher dose. Fig. 2C shows the activity of a typical n e u r o n e in an animal treated with k e t a m i n e at the onset of ischemia. Noxious pinching of the receptive field before and during ischemia produced significantly different responses (P < 0.05) only for the lower dose of ketamine; however, both doses of the drug significantly diminished (P < 0.05) the hyperalgesia seen in control animals (Fig.

1200

Pinch

_I_

1000

==

800

"6

600

==

i

400 Z

200 0

8,

Pre-lschaemio

(-30 to 0 min.)

76.

500,

Brush

400. ~3-

8

Ischaemla

(30 to 60 mln.)

--]_I_

..>'C

Pre-i~haemia

Ischaemia (30 to 60 rain.)

300.

2-

0

Pre-ischaemia

Ischoemia

Ischaernla

(-30 to 0 min.)

(0 to 30 min.)

(30 to 60 min.)

Fig. 4. Spontaneous firing rate of dorsal horn neurones in rats treated with saline (open bars), 1/~M APV (hatched bars) and 100 /~M APV (filled bars) before and during ischemia. In the preischemic period, the bar represents means + S.E.M. of 24 (untreated) animals; during ischemia, each bar represents mean + S.E.M. of 8 animals given a particular treatment. In both ischemic periods, neuronal activity of APV-treated animals was significantly less (P < 0.05) than that of control animals. In APV-treated animals, there was no significant difference in the firing rate between pre-ischemic and ischemic periods.

z

100, o

(-30 to 0 mln.)

I

Fig. 5. Response of dorsal horn neurones to noxious pinching (top panel) and innocuous brushing (bottom panel) of the receptive field, in animals treated with saline (open bars), 1/~M APV (hatched bars) and 100 /~M APV (solid bars). APV abolished the ischemic hyperalgesia to noxious mechanical stimuli but did not inhibit the ordinary response to noxious pinching, nor to innocuous brushing. Sample sizes are the same as in Fig. 4.

59

Effect of ischemia on receptive field size

B,

~e 4 .

~3 ~2.

~, o

Pre-i=chaemio

N

Ischaemla

Imchaemla

(-30 to 0 rnin.)

(0 to .30 rain.)

(30 to 60 mln.)

•oa r~'l

Fig. 6. Spontaneous firing rates of dorsal horn neurones in rats treated with saline (open bars), 1 mM ketamine (cross-hatched bars) and ]00 mM ketaminc (hatched bars) before and during ischemia. For the first ischcmic period the neuronal activity of both kctaminetreated groups was significantly less than that of controls (P < 0.05), while in the second ischemic period (30 to 60 rain) the difference was significant for the lower dose only (P < 0.05). In the pre-ischemic period, n = 24; during ischemia, for each treatment n = 8.

7). There was no statistically significant effect of ketamine on the response to innocuous brushing of the hindlimb (Fig. 7).

1200. Pinch

Fig. 8 demonstrates typical examples from each of the 5 groups of animals of the receptive field size before and during ischemia. Of the total of 40 animals, 23 (58%) exhibited receptive fields during ischemia larger than those prevailing before ischemia. Of these 23, 7/8 saline-treated animals exhibited enlarged receptive fields, while for APV (1/~M) 4/8 and A P V (100/~M) 3/8 showed enlargement. For ketamine (1 mM) 4/8 and (100 mM) 5/8 demonstrated enlargement. In some instances, the receptive field more than doubled in size, but in no animal did the receptive field spread to involve the contralateral limb or any other area removed from the supply of the occluded artery. Using the X2 statistic, with a 5 x 2 contingency table, there was no significant difference in the proportion of animals within each treatment group which demonstrated enlarged receptive fields during ischemia. In preliminary recordings from 8 convergent neurones, we monitored the response to the same noxious and innocuous stimuli used in this experiment, applied at 5

Pre-ischaemia

Ischaemia

1000.

= =

800,

"6 600, J~

E 400, z

200 Pm-lmchaemla (-30 to 0 mln.)

500,

400. = == 300. "6 .a

E=

Itmhaemla (30 to 60 rain.)

APv

(lpM)

APv

Brush

(lOOpM)

5_

200,

z

100 0

Ketamine (lrnM) Pre-iechaem|a

(-30 to 0 rain.)

Ischaernia

(30 to 80 rain.)

Fig. 7. Response of dorsal horn neurones to noxious pinching (top panel) and innocuous brushing (bottom panel) of the receptive field in animals treated with saline (open bars), 1 mM ketamine (cross-hatched bars) and 100 mM ketamine (hatched bars). Ketamine abolished the ischemic hyperalgesia to noxious mechanical stimuli. While the lower dose of ketamine also inhibited significantly the ordinary response to noxious pinching and to innocuous brushing (P < 0.05), the higher dose failed to do so.

(lOOmM) Fig. 8. Individual examples showing receptive field size before and during ischemia for animals within each group. All groups showed

an increase in receptive field size, unaffected by drug administration.

60 min intervals for 90 min, with no other conditioning stimulus (ischemia) present. In 7 of the 8 units, we found no evidence of adaptation or hyperalgesia to the afferent inputs, and in all 8 that the receptive fields were unaltered at 90 min. In 1 neurone, the response to innocuous stimulation increased by 24% at 60 min, compared to the initial response, and remained elevated at similar levels up to 90 min. DISCUSSION Our results demonstrate that occlusion of the femoral artery induces a neuronal response in the dorsal horn of the rat consistent with sustained noxious stimulation of peripheral nociceptors, even though the hindlimb remained static throughout the experiment. Early work on the nature of ischemic pain 18'25'27 suggested that pain arose in an ischemic limb only once the limb was exercised, and that noxious chemical mediators were released as a direct result of the muscle contractions. Ischemia alone was also claimed not to be an effective stimulus for activating slowly conducting (Groups III and IV) muscle afferents, whereas moderate rhythmic contractions resulted in sustained firing of dorsal root filaments 31. Previous work in our laboratory has shown that ischemia of the rat's tail (unexercised) induced by inflating a cuff at the base of the tail resulted in both an increased firing rate of ventrobasal thalamic neurones in anaesthetised animals and a coordinated escape behaviour in conscious animals, with mean latencies of 12.1 + 1.8 min and 11.9 + 2.1 min, respectively15. These latencies correlate well with our current observation that occlusion of the femoral artery produces an increased activity in dorsal horn neurones with a mean latency of 10.3 + 2.5 min. We used two agents, APV and ketamine, to investigate the role of spinal cord NMDA receptors in mediating the nociceptive response to ischemia. APV is a competitive antagonist which has been shown to antagonise the NMDA-induced excitation of ventrobasal thalamic neurones at a dose of 50 mM 34'35 in vivo, and to inhibit NMDA-induced depolarisation of spinal dorsal horn neurones at a dose of 10-250 /~M in in vitro slice preparations 23'24. The dissociative anaesthetic ketamine has been described recently as a non-competitive NMDA receptor antagonist 4'2°'37. Iontophoretic application of ketamine, in a dose of 50 mM, selectively reduced NMDA-mediated synaptic excitation of dorsal horn neurones in rats 37. Maurset et al. 3° found that low doses of ketamine given i.v. exhibited powerful analgesic effects against experimentally induced ischemic pain in humans, an effect independent of opioid mechanisms.

Irrigation of the spinal cord with both APV and ketamine resulted in profound inhibition of the firing rate of dorsal horn neurones during ischemia at the lower dose of each drug. Application of the higher dose did not result in any greater degree of inhibition of the nociceptive response, but did inhibit spontaneous firing rate to a greater extent. The effects of APV and ketamine support the hypothesis that the NMDA receptor in the rat spinal cord is involved in nociceptive afferent transmission and that antagonists to the receptor inhibit the responses of convergent neurones in the dorsal horn to peripheral noxious stimulation. While it could be argued that the NMDA receptor antagonists are generally CNS depressant, and do not affect nociception specifically, previous work has shown that the agonist NMDA exerts a direct nociceptive action when applied to the spinal cord, but has no excitatory effect on spinal motor activity; the antagonist APV inhibited the nociceptive response but did not affect motor function in conscious animals 2. That the NMDA receptor has a role specific to nociception is further supported by the observation that NMDA receptor activation is not required for activation of motor neutones in the spinal cord, while sensory interneurone NMDA receptors do play a role in the spinal cord processing of afferent information 41. We have shown that dorsal horn neuronal responses to both noxious pinching and innocuous brushing of the receptive field are not altered by the NMDA receptor antagonist APV in the absence of ischemia. In the presence of the conditioning stimulus, however, both APV and ketamine reduced to pre-ischemic values the neuronal response to noxious stimulation, while neither agent inhibited the response to innocuous stimulation of the receptive field. In other words, the hyperalgesia to noxious mechanical stimulation induced by ischemia was attenuated by the NMDA receptor antagonists, as was the ischemia-induced neuronal activity, while these drugs had no effect on afferent inputs in the absence of a conditioning stimulus. These findings suggest that the NMDA receptor may be involved only in some forms of synaptic excitation in the central nervous system, specifically the neuronal events mediating inflammatory pain and hyperalgesia. Dickenson and Sullivan 11 found that APV reduced wind-up and post-stimulus discharges of deep dorsal neurones, without inducing any marked change in the A- and C-fibre evoked inputs onto the same cell. Similarly, thalamic responses to short duration physiological stimuli were not altered by APV, while APV did antagonise responses to maintained physiological stimuli 35. Formalin injection into the rat hindpaw, a recognised model of inflammatory pain, significantly increased the levels of glutamate in the spinal dorsal

61 horn 39. Furthermore, ketamine is an effective analgesic for both post-operative and experimental ischemic pain in man 30. Hyperalgesia, a state in which non-painful stimuli become painful, or in which latency of response to noxious stimuli decreases or the magnitude of the response increases, has been widely investigated 7'1°' 13-15,29,44,45. Gelgor et al. 13 found that reperfusion following ischemia induced hyperalgesia to noxious thermal stimuli in rats; this hyperalgesia was abolished by antihistamines and by aspirin TM as well as by other non-steroidal anti-inflammatory agents (personal communication). We have found that ischemia also induces hyperalgesia to noxious mechanical stimuli, while neuronal responses to innocuous stimulation of the receptive field remained unaltered during ischemia. Hyperalgesia is not specific to noxious thermal stimulation, and occurs in various forms of long-lasting inflammatory damage to tissues 7,19,40,44,45. In the absence of ischemia, we showed that repeated stimulation of the receptive field using the natural stimuli employed in this experiment did not alter the responses of convergent neurones to subsequent stimuli, nor was there any change in receptive field size. In other words, the intensity and duration of the mechanical stimuli used were not sufficient to produce trauma-induced hyperalgesia. The hyperalgesia evident in control animals, therefore, must have arisen as a result of the conditioning input, namely hindlimb ischemia. As a conditioning stimulus, ischemia may exert its effect peripherally, by changing the properties of cutaneous afferents within the receptive field, or centrally, by inducing a state of excitability at spinal or supraspinal levels. Much of the current work on hyperalgesia aims to clarify whether hyperalgesia results from central or peripheral mechanisms, or both. We have shown that ischemia-induced hyperaigesia is abolished by direct intrathecal application of NMDA receptor antagonists in small volumes, suggesting a local action in the spinal cord. Our observations therefore support a central mechanism for hyperalgesia, at the level of the spinal dorsal horn, which is in keeping with previous claims that hyperalgesia results from changes in the excitability of REFERENCES 1 Aanonsen, L.M. and Wilcox, G.L., Phencyclidine selectively blocks a spinal action of N-methyI-D-aspartatein mice, Neurosci. Lea., 67 (1986) 191-197. 2 Aanonsen, L.M. and Wilcox, G.L., Nociceptive action of excitatory amino acids in the mouse: effects of spinally administered opioids, phencyclidineand sigma agonists, J. Pharmacol. Exp. Ther., 243 (1987) 9-19. 3 Angst, C. and Williams, M., Excitatory amino acid receptors, Neurotransmissions, 3 (1987) 1-5.

dorsal horn interneurones 7'45. In further support of such a role for the NMDA receptor, Yaksh 46 has found that strychnine evoked hyperesthesia and tactile evoked allodynia result, in part, from the release of glutamate in the spinal dorsal horn; the phenomena are antagonised by intrathecal injections of both APV and ketamine, as well as of other specific N M D A receptor antagonists. Also, intrathecal administration of the agonist NMDA resulted in hyperalgesia to both the tail-flick and hot plate tests in mice 2. This NMDA-mediated hyperalgesia was inhibited partially by morphine, but more so by phencyclidine and APV. Phencyclidine has been shown to reduce selectively NMDA-induced excitation of mammalian spinal neurones 1'4. It is possible that the frequencydependent potentiation of deep dorsal horn nociceptive neurones (wind-up), shown to be NMDA dependent 11, may be one way in which hyperalgesia is mediated. Ischemia affected not only the sensitivity of nociceptive pathways but also receptive field size. After ischemia had been present for 60 min, 58% of dorsal horn neurones exhibited peripheral receptive fields which were larger than those prevailing before ischemia. The enlarged receptive field remained ipsilateral, and confined to tissue adjacent to the original field. Peripheral injury of diverse nature has been shown to result in expanded receptive fields. The mechanisms underlying receptive field enlargement are also thought to involve changes within the spinal dorsal horn, and not to be due to peripheral sensitisation of nociceptors 8'19'21. Whether the NMDA receptor antagonists which abolish hyperalgesia, also inhibit expansion of the receptive field remains to be investigated. While our results suggest that hyperalgesia evident during ischemia may be dependent on central mechanisms at the level of the spinal dorsal horn, we cannot rule out an additional contribution from peripheral mechanisms to hyperalgesia, especially during reperfusion of an area previously ischemic.

Acknowledgements. We thank the South African Medical Research Council and the University Brain Function Research Unit for financial assistance, as well as Mr. S. CartmeU and Mrs. L. Gelgor for their help and advice.

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N-methyl-D-aspartate receptors mediate responses of rat dorsal horn neurones to hindlimb ischemia.

We have investigated the role of NMDA receptors in the rat dorsal horn in mediating neuronal responses to noxious hindlimb ischemia, induced by acute ...
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