Nitric oxide mediates neuropathic pain behavior in peripherally denervated rats

ELSEVIER

Neuroscience Letters 188 (1995) 57-60

NIUROSCIINC[ IEIT[IIS

Nitric oxide mediates neuropathic pain behavior in peripherally denervated rats B e a t a N i e d b a l a I, A l b e r t o S ~ n c h e z , M a n u e l F e r i a * Department of Pharmacology, Faculty of Medicine, Universityof La Laguna, Tenerife, Spain Received 2 December 1994; revised version received 10 January 1995; accepted 13 February 1995

Abstract

The involvement of spinal cord nitric oxide (NO) in the development of autotomy, a proposed behavioral model of denervation pain, was studied in sciatic and saphenous nerves transected rats injected intrathecally, 10-15 min prior to neurectomies, with N~3-nitroL-arginine methyl ester (L.NAME, 20-500 nmol), Nt3-nitro-D-arginine methyl ester (D-NAME, 500 nmol), L- or D-arginine (5/~mol), and 8-bromoguanosine 3':5'-cyclic monophosphate sodium salt (8-Br-cGMP, 100 and 200 nmol). Self-inflicted lesions were scored daily for 8 weeks. The main effects on autotomy were: (1) a significant suppression in rats injected with L-NAME (500 nmol), but not with D-NAME; (2) a significant potentiation after L-arginine, but not D-arginine; and (3) a significant potentiation with 8-Br-cGMP, which was blocked by co-administration of L-NAME. These findings indicate that autotomy in rats can be modulated by blocking or enhancing nitroxidergic transmission at lumbosacral level, and suggest new therapeutic approaches for the prevention of certain pain syndromes, such as phantom limb pain. Keywords: Neuropathic pain; Nitric oxide; Autotomy; Injury discharge; Rat

Chronic pain conditions produced by neurectomy (i.e. phantom pain) constitute a difficult clinical problem [5]. When a peripheral nerve is cut, it emits a burst of action potentials 'injury discharge' [12] which has been suggested to play an important role as a trigger of autotomy [9]. This self-injury behavior which is induced in laboratory animals after peripheral neurectomy would simulate denervation pain in humans [1]. There is now evidence for a role of nitric oxide (NO) in spinal cord processing of nociceptive input from neuropathic origin [8]. The present study was aimed at investigating whether the inhibition or facilitation of spinal cord nitroxidergic transmission, just before arrival of injury discharge produced by peripheral neurectomy, modifies the onset or severity of autotomy behavior. The experiments were carried out on 120 male Sprague-Dawley rats, weighing 350-400 g. The animals were housed in groups of four for 1 week preceding surgical intervention and throughout the postoperative (PO) ob* Corresponding author, Tel.: +34 22 603474; Fax: +34 22 655995. 1 Present address: Department of Pharmacodynamics,Medical Academy, Bialystok, Poland.

servation period (56 days). Under sodium pentobarbitone anesthesia (50 mg/kg i.p.) the animals were cannulated intrathecally [14] so that the catheter's tip was situated at the thoraco-lumbar junction. After cannulation, the sciatic and saphenous nerves were exposed unilaterally and dissected free in the rat's right hind limb. An intrathecal (i.t.) injection of 2 pl of the drugs used or vehicle, followed by 10/tl of sterile saline was performed at a rate of 10/~1/ min. Ten minutes after L- or D-NAME (NG-nitro-L- or Darginine methyl ester) injections, or 15 min after Larginine HC1, D-arginine HC1, or 8-Br-cGMP (8-bromoguanosine 3':5'-cyclic monophosphate sodium salt) administration, the exposed sciatic and saphenous nerves were ligated with 6 - 0 silk at two locations (5 mm apart), transected, and the nerve segments between ligatures were removed. Intrathecal catheters were then taken out, and muscles and skin were closed. Rats showing any obvious sign of motor impairment during the first PO day as a consequence of cannulation were immediately euthanized with an overdose of sodium pentobarbitone and excluded from the study. Completeness of the neurectomy was inferred during the first PO week by motor deficits and by the absence of sensitivity to pinching of the toes.

0304-3940/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSD1 0304-3940(95)11394-Z

58

B. Niedbala et al. /Neuroscience Letters 188 (1995) 57-60

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The animals were randomly assigned to ten groups ( N = 12) and received one of the following treatments: phosphate buffer saline (20 mM POaHNa 2 and 150 mM NaC1; pH 7.34) as vehicle; 20, 100 or 500 nmol of LNAME, an inhibitor of NO synthesis; 500 nmol of DNAME, the inactive stereoisomer of L-NAME; 5/~mol of L- or D-arginine, the natural precursor for NO synthesis and its inactive stereoisomer, respectively; 100 or 200 nmol of 8-Br-cGMP, a stable analog of cyclic guanosine monophosphate (cGMP); and, finally, 500 nmol of LN A M E plus 200 nmol of 8-Br-cGMP. Self-induced lesions were scored daily by an observer who was blind to group assignment, using a slight modification of a previously devised scale [11]. Briefly, a

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B. Niedbala et al. / Neuroscience Letters 188 (1995) 57-60

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tal pain in conscious animals [15] were followed and all experiments were approved by the Animal Care and Use Committee of our Institution. As shown in Fig. 1A, the injection of the low doses (20 and 100 nmol) of L-NAME did not alter autotomy. However, the injection of 500 nmol L-NAME significantly (P < 0.05) suppressed autotomy by 50%, and final scores decreased from 5.75 _+ 1.0 (mean _+ SEM) in the control group to 3.0 -+ 0.7 in the L-NAME 500 group (P < 0.05). In contrast, the injection of 500 nmol of D-NAME did not affect the autotomy compared to the vehicle group. No significant differences were detected in the average autotomy onset between L- and D-NAME-treated groups and their control group (Fig. 1B). Finally, L-NAME pro-

59

duced a dose-dependent decrease in the incidence of high autotomy (from 50%in the control group to 16% in the LNAME 500 group), an effect which was absent after injection of 500 nmol D-NAME (58%, Fig. 1C). The i.t. injection of 5/~mol of L-arginine produced a significant (P < 0.05) potentiation of the autotomy behavior, especially evident from the 4th PO week (Fig. 2A). The final score of the L-arginine group was 9 _+0.8, compared to 5.75 _+ 1 of the vehicle group (P < 0.05). In contrast, the injection of the same dose of D-arginine did not alter the time course of autotomy or its final score. Autotomy onset was not different among the three groups (Fig. 2B). Finally, the percentage of animals which autotomized severely increased from 50% in the vehicle and D-arginine groups to 83% in the L-arginine group (Fig. 2C). In the groups treated with 100 and 200 nmol 8-BrcGMP autotomy significantly increased (P < 0.05) by an average of 55% and 74%, respectively, compared to the vehicle group (Fig. 3A). Final scores in 8-Br-cGMPtreated groups (7.8 _+0.8) were significantly higher than in the vehicle group (5.75 + 1.0; P < 0.05). Average autotomy onset (Fig. 3B) shifted from 12.8 +_3 days in the vehicle group to 5.8 _+0.5 and 5.0 + 0.7 days in the 100 and 200 nmol 8-Br-cGMP-treated groups (P < 0.05 and P < 0.01), respectively. As Fig. 3C shows, the incidence of severe autotomy was higher in the 8-Br-cGMPtreated groups than in the vehicle group, 75% and 50% respectively. Finally, the prior administration of 500 nmol L-NAME prevented the effect of 200 nmol 8-Br-cGMP on every one of the parameters used to assess autotomy (Fig. 3A-C). Our results show that a single i.t. injection of drugs affecting nitroxidergic transmission, made a few minutes prior to neurectomy, suppresses or potentiates a painrelated behavior expressed long after nerve injury. This suggest that NO mediates some of the spinal cord morphological and functional processes that occur as a consequence of the arrival of injury discharge [10]. Although NO probably has no role in the synaptic transmission of mild acute pain states [7], it participates in the modulation of nociceptive responses of spinal dorsal horn neurones in hyperalgesic states induced by moderate inflammatory insults, like formalin injection [6], and in models of neuropathic pain [8]. Previously reported data, showing an increase in nitric oxide synthase immunoreactivity in rat dorsal root ganglion and superficial dorsal horn neurones following sciatic nerve transection [3] and hindpaw formalin injection [4], respectively, also tend to support this view. In our present study, L-NAME (but not D-NAME) significantly decreased autotomy. This suggests that spinal cord NO synthesis is involved in sensory processing of injury-related input which has been shown to be important in triggering autotomy. In contrast, L-arginine, but not D-arginine, significantly potentiated autotomy. Inter-

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B. Niedbala et al. / Neuroscience Letters 188 (1995) 57~50

estingly, the potentiating effect of L-arginine started from the 4th PO week, a time in which C-fibers spontaneous discharges from sciatic nerve n e u r o m a s peaked [2]. Although the n e u r o c h e m i c a l m e c h a n i s m s regulating the intracellular levels of L-arginine are largely u n k n o w n , a recent report [13] indicates that, in cultured neurones, uptake of L-arginine regulates its intracellular concentration, and consequently the availability for N O synthesis. Thus, c o n c e i v a b l y the design of specific inhibitors of Larginine uptake m a y e m e r g e as n e w agents for the treatm e n t of neuropathic pain. Finally, 8 - B r - c G M P administration increased autotomy severity and shortened significantly its onset, an effect which was prevented by the previous administration of L-NAME. This suggests that the triggering effect of injury discharge on autotomy via N O is mediated by c G M P levels. In s u m m a r y , our results demonstrate that autotomy following peripheral denervation in rats can be modulated by the previous lumbosacral N O levels. Further experiments are needed to assess the role of spinal cord N O transmission in the prevention or treatment of neuropathic pain in h u m a n s .

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[10]

This research was supported by Grant 92/111 from the G o v e r n m e n t of the C a n a r y Islands. W e gratefully ack n o w l e d g e the English c o m m e n t s and editorial assistance made by Dr. I. L6pez Gonz~ilez-Coviella.

[11]

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