How central is nitric oxide (NO) to the activation of

Brain Research 888 (2001) 172–175 www.elsevier.com / locate / bres

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How central is nitric oxide (NO) to the activation of c-fos in spinal neurones following noxious peripheral stimulation in the rat? a b c c, M. Nazli , E.S. Hismiogullari , T. Thippeswamy , R. Morris * a Department of Veterinary Anatomy, University of Kafkas, Kars, Turkey Department of Veterinary Pharmacology and Toxicology, University of Ankara, Ankara, Turkey c Department of Veterinary Preclinical Sciences, University of Liverpool, Brownlow Hill and Crown Street, Liverpool, L69 3 BX, UK b

Accepted 10 October 2000

Abstract Intrathecal application of high doses of NO-donor compounds in the anaesthetised rat was not found to cause any induction of c-fos in spinal neurones. Furthermore, intrathecal injection of a NO-synthase (NOS) blocking drug did not alter the numbers of c-fos positive neurones induced by noxious stimulation. Additionally very little colocalization between NOS and c-fos following noxious stimulation was found. Collectively these data give no support for a role for NO in the noxiously evoked induction of c-fos.  2001 Elsevier Science B.V. All rights reserved. Theme: Sensory systems Topic: Pain modulation: pharmacology Keywords: Nitric oxide; c-fos; Spinal cord; Pain; Nociception

Noxious peripheral stimulation causes the induction of the intermediate-early gene c-fos in the nuclei of cells in the appropriate somatotopically related region of the spinal cord, in particular in LII [6]. The neuronal isoform of nitric oxide synthase (nNOS) is also located in many neurones in the inner part of LII [3]. This led to the suggestion that NO is involved in nociception [9] and may have an important role in the induction of c-fos [7]. This is a very attractive proposition as it would be an elegant mechanism linking excitation of spinal neurones by nociceptors through the diffusion of NO to the induction opioids in intrinsic interneurones, resulting in a reduction in nociceptive signalling [2]. Several experiments have provided evidence for such a role of NO in c-fos induction following noxious peripheral stimulation [4,7,10], however, this has not been consistently demonstrated [1]. The present experiments have re-examined the role of NO in c-fos induction in spinal neurones following noxious peripheral stimulation. Adult rats (Wistar) of either sex were anaesthetised with *Corresponding author. Tel.: 144-151-794-4227; fax: 144-151-7944243. E-mail address: [email protected] (R. Morris).

urethane (1 mg / kg, i.p.) and prior to vascular perfusion they were given a lethal dose of pentobarbitone (60 mg / kg, i.p.). Animals were bred in the Biomedical Services Unit of the University of Liverpool and all procedures were performed under UK Home Office statutory regulation. For studies involving intrathecal drug application, large adult rats (over 300 g) were used and the intrathecal cannula was inserted in the sub-dural space via a cut in the atlanto-occipital membrane. The cannula was gently fed down this space to a level just rostral to lumbar enlargement taking great care to avoid any spinal cord irritation or damage which strongly activate c-fos. The following treatments were used: 100 nmol of 3morpholinylsydnoneimine chloride (SIN-1) (Tocris) (n5 6); 1 mmol SIN-1 to which was added 1 nmol of the cGMP/ cAMP phosphodiesterase inhibitor, 3-isobutyl-1methylxanthine (IBMX) (Sigma) (n51); 10 nmol of Snitroso-N-acetylpenicillamine (SNAP) (Tocris) (n52); vehicle controls (n53). All injections were given in 10 ml, washed in with 10 ml of saline and repeated three times at half hour intervals. In a further 15 animals either injections of N G -nitro-L-arginine methyl ester ( L-NAME) (10 nmol) or its inactive isomer D-NAME (10 nmol) or vehicle

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M. Nazli et al. / Brain Research 888 (2001) 172 – 175

(saline) (n55 each treatment) were given. Injections were 10 ml volume, washed in with 10 ml of vehicle and repeated four times at 30 min intervals. Five minutes after the first injection one paw was painted with 40% mustard oil (Allylisothiocyanate, Merck) in liquid paraffin. To examine the influence of cannulation and drug injection controls were conducted in which both procedures were omitted. In a further 23 rats one of the following stimuli were applied to one hindlimb; topical application of 40% mustard oil (12 animals), subcutaneous injection of formalin (5% or 10%, 50 ml into the plantar surface, three animals each) or thermal stimulation (immersion of the paw in water at 48 or 528C for 20 s, three animals at each temperature). Two hours after the first noxious stimulus the animals were fixed by vascular perfusion with 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS) (pH 7.2). In all cannulated animals injection of 10 ml of pontamine sky blue dye was used to confirm the correct position of the cannula tip. In any animal in which damage to the spinal cord was visible at either a gross or a microscopic level the data obtained were excluded (not included in the above n-values). The lumbar tissue block was selected with reference to the L4 root entry zone and was 2.5 mm rostral and caudal to this root (i.e. 5 mm length). Sections (40 mm thick) were cut serially in the transverse plane on a freeze knife microtome and processed free floating. The sections were collected into 10 tubes of PBS such that each tube contained sections at 400 mm intervals through the complete tissue block. Counts were carried out on all the sections from one tube thus giving a systematic random sample from the whole block. Staining for nNOS and c-fos used standard immunocytochemical methods the sections being incubated in a mixture of rabbit raised anti-Fos protein (CRB, 1:1000) and sheep raised anti-nNOS (gift from P. Emson, 1:5000) overnight at 48C. They were then incubated in biotinylated anti-rabbit (1:200, 1 h RT, Jackson), streptavidin HRP (1:1000, Amersham) and chromagen stained [11]. Followed by incubation in biotinylated anti-sheep (1:200, 1 h RT, Jackson), streptavidin HRP (1:1000) and incubation with a second chromagen (Vector VIP kit). Counts were expressed as numbers of Fos nuclei / section / area. In the lumbar spinal cords of animals treated with intrathecal SIN-1 or SNAP small numbers of c-fos positive nuclei were observed on either side of the cord. The counts obtained were: SIN-1, LI–LIII, 11.664.5, rest of cord, 6.261.6; SNAP, LI–LIII, 2.861.7, rest of cord, 7.063 (mean6S.E.M., of numbers of positive nuclei per section / 2). Increasing the concentration of SIN-1 and adding IBMX made no difference and only very low numbers of Fos positive nuclei were seen in this animal. Overall the numbers of Fos nuclei were slightly more frequent than in saline treated animals (LI–LIII, 5.262.3, rest of cord, 2.661.2). No statistically significant change (ANOVA) was observed in the numbers of Fos positive nuclei induced by

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mustard oil treatment in animals treated with L-NAME compared with those treated with D-NAME (Fig. 1A and B, Table 1). Neurones showing co-localisation of nNOS and Fos irrespective of the type of stimulus used were rare (Fig. 1C and D). For example out of 5788 Fos positive nuclei observed in LI–III in mustard-oil-treated animals only one was in a nNOS positive cell. Sections were also made from more rostral lumbar levels in these studies and co-localisation of nNOS and Fos was slightly more frequent in the lateral horn (39 out of 631). Collectively these data were unable to confirm any role for NO in c-fos induction in the spinal cord. The most direct experiment of applying NO-donors to the spinal cord caused at best a very weak induction of c-fos. The NOdonors were made up fresh and were able to induce synthesis of cGMP. In a recent study microdialysis of SIN-1 in the middle of the spinal cord gave a significant and clear induction of c-fos for up to 400 mm from the dialysis fibre [13] and it is difficult to explain this disparity. We were also unable to confirm the inhibitory action of NOS-blocking drugs on c-fos induction. The first study to propose this mechanism used direct spinal injection of the antagonists in mice [7]. This and a subsequent study in the rat [10] used much higher NOS-blocker concentrations and baseline levels of c-fos in vehicle controls were much higher due to the route of drug administration. The doses of blocker used in the present study were based on those found to block thermal hyperalgesia in rats. However, in another more recent study a clear dose-related suppression of c-fos activation, induced by injection of formalin into one hindpaw, was seen using doses ranging from 0.01 nmol to 10 nmol of N v -nitro-L-arginine as the NOS inhibitor [4]. In this study c-fos induction, 1 h after the noxious stimulus was investigated in animals anaesthetised throughout with sodium pentobarbital. The drugs were delivered by an intrathecal cannula inserted via a thoracic laminectomy. Unfortunately, no figures are given in this paper for numbers of Fos neurones in cannulated animals that had a vehicle injection that were not stimulated with formalin, hence, it is difficult to assess c-fos induction due to surgical procedures. A role for NO in c-fos induction following plantar injection of formalin is also supported by the reduction in c-fos produced by intrathecal application of a cGMP-kinase Ia blocker [12]. This study was very carefully conducted with chronically implanted cannula and the figures for c-fos induction are comparable to those found in similar studies using plantar injection of formalin. An increase in spinal cyclic GMP-dependent protein kinase was also shown in this study 96 h after formalin injection into one hind paw, this elevation being reduced by blockers of cGMP, NOS and NMDA receptor antagonist MK801 [12]. Recently, using Western blot analysis a more quantitative evaluation has been conducted. This demonstrates that L-NAME dramatically reduced the expression of c-fos in the spinal cord [13].


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Fig. 1. Effects of L-NAME and D-NAME on Fos induction by mustard oil stimulation (A and B) and colocalisation of Fos and nNOS following noxious stimulation (C and D). (A, B) Typical sections from animals in which the paw ipsilateral to the section area shown was painted with 40% mustard oil. (A) L-NAME and (B) D-NAME treated animals. (C, D) Sections showing the ipsilateral LI–LIII and central canal region of mustard-oil-treated animals. The arrow heads indicate nNOS positive neurones and the arrows Fos positive nuclei. Scale bars, A and B, 250 mm; C, 100 mm; D, 50 mm.

The comparison of the distributions of NOS and Fos gave the very clear result that NO is not causing the induction of c-fos in the cells in which it is produced. This confirms similar findings made following formalin injections [5] but in another recent study 14% colocalisation between Fos and NOS was found following formalin stimulation [8]. In conclusion, whilst NO may be involved in c-fos synthesis following noxious peripheral stimulation the

influence of neuronal injury due to the route of drug administration has to be considered in some studies.

Acknowledgements This work was supported by the Wellcome Trust and Action Research. M.N. and E.H. were supported by Turkish Government Scholarships.

Table 1 Effects of NOS-blocker on c-fos induced by mustard oil stimulation a Noxious stimulus

Intrathecal drug

N-tested

N-Fos nuclei / section / region of spinal cord counted Ipsilateral to stimulus

Mustard oil

a

Saline L-NAME D-NAME No catheter or drug

5 5 5 5

Contralateral to stimulus

LI–LIII

Rest of cord

LI–LIII

Rest of cord

62.2614.7 65.4618.1 54.3610.1 65.5612.2

9.363.5 4.163.0 1.661.0 12.964.1

19.869.4 36.7619.1 22.666.4 34.1614.6

4.161.6 2.762.0 1.260.7 8.263.9

Counts of Fos positive nuclei were made in the regions indicated heading the columns. Insertion of the cannula and injection of saline made no difference to the numbers of c-fos-expressing nuclei induced by mustard oil treatment. Similarly values obtained for both L-NAME- and D-NAME-treated animals were not statistically different to one another or to the saline and non-cannulated controls (ANOVA). Figures are the means6S.E.M.


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