Effect of colchicine on neuropeptide Y expression in rat dorsal root ganglia and spinal cord

Neuroscience Letters 259 (1999) 45–48

Effect of colchicine on neuropeptide Y expression in rat dorsal root ganglia and spinal cord Nathalie Cougnon-Aptel, Garth T. Whiteside, Rajesh Munglani* University Department of Anaesthesia, Addenbrookes Hospital, Hills Road, Cambridge, CB2 2QQ, UK Received 30 September 1998; received in revised form 5 November 1998; accepted 6 November 1998

Abstract Neuropeptide Y (NPY) expression in the spinal cord and dorsal root ganglia (DRG) was examined after application of colchicine, an axonal transport blocker, on the intact sciatic nerve or prior to axotomy or chronic constriction injury (CCI). Rats that underwent topical application of colchicine on the sciatic nerve showed decreased responsiveness to heat stimulation, ipsilaterally. CCI-induced hyperalgesia was prevented by prior application of colchicine. However, colchicine did not block axotomy-induced NPY increase when applied proximally to the injury. In fact, colchicine induced the expression of NPY in the DRG and spinal cord in an identical manner to axotomy. The present data indicates that the increase in NPY observed after nerve injury could be initiated by the suppression of retrograde transport of factors, possibly neurotrophins, rather than by the production of an active factor at the site of injury.  1999 Elsevier Science Ireland Ltd. All rights reserved

Keywords: Colchicine; Neuropeptide Y; Dorsal root ganglia; Hyperalgesia; Spinal cord; Mononeuropathy; Axotomy

Nerve injury induces massive de novo neuropeptide Y (NPY) expression in the ipsilateral medium and large neurones of dorsal root ganglia (DRG) which project to laminae 3–4 in the spinal cord [12,13,16,17]. It is not known if this change in NPY expression is due to the suppression of a factor coming from the periphery and retrogradely transported to the cell body, or to the action of an active factor generated at the site of injury. Molecules are transported to and from cell bodies by fast axonal transport. Retrograde transport of growth factors is believed to play an important role in regulation of neuropeptides in sensory neurones [10]. The increase of NPY seen after axotomy has been shown to be attenuated by application of neurotrophin-3 [14] or fibroblast growth factor [7] to the end of the injured nerve, or intrathecal administration of nerve growth factor [15]. Colchicine disrupts axonal transport by depolymerising microtubules [6]. Rats treated with colchicine are not hyperalgesic and colchicine blocks hyperalgesia in a model of neuropathic injury, the chronic constriction injury (CCI) model [1], suggesting that a factor is generated at the site * Corresponding author. Tel.: +44 1223 217890/89; fax: +44 1223 217887; e-mail: [email protected]

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of nerve injury and transported in the sciatic nerve to induce hyperalgesia [18]. In this study the effect of colchicine application to the intact and injured sciatic nerve on NPY expression and rat sensitivity to pain was investigated. Sprague–Dawley rats were anaesthetised using halothane, the left sciatic nerve was exposed and a piece of parafilm placed underneath. Colchicine (50 mM) or saline were applied to the nerve using a cotton bud. After 10 min the cotton bud was removed and the nerve washed with saline. The nerve was then either left intact, ligated distally to the colchicine application to perform an axotomy, or loosely tied with three chromic cat gut sutures to perform a CCI model of neuropathy. For further comparison a group of rats underwent sciatic nerve axotomy alone. After 7 days (saline only, colchicine only, saline + CCI, colchicine + CCI) and 40 days (colchicine + CCI, saline + CCI) the animal’s thermal nociceptive threshold was measured using a Plantar Test (Ugo Basile, Comerio, Italy).To initiate a test, the rat was placed in the box and allowed 5 min to habituate. The heat source was positioned under the plantar surface of one hindpaw and activated. This initiated a timing circuit allowing measurement of the time interval between the application of the light beam and the

 1999 Elsevier Science Ireland Ltd. All rights reserved

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N. Cougnon-Aptel et al. / Neuroscience Letters 259 (1999) 45–48

Fig. 1. Effect of application of 50 mM colchicine, alone or in combination with CCI, on the sciatic nerve on thermal nociceptive threshold. Colchicine or saline were applied on the sciatic nerve, intact or chronically constricted, as described in methods. Left (operated) and right (unoperated) hindpaw withdrawal latencies (PWL) were measured using a Plantar Test as described in methods. Results are expressed as mean ± SEM. Data were analyzed with Statview 4. ANOVA test was used. P-Values for ipsilateral side compared to contralateral side were respectively for each group: P = 0.88 (n = 4), P , 0.0001 (n = 5), P = 0.068 (n = 3), P = 0.0006 (n = 3), P = 0.079 (n = 3), P = 0.39 (n = 3).

withdrawal of the hindpaw. This value was assigned as the response latency. The animals were then anaesthetised with saturated aqueous chloral hydrate, 2 ml i.p., and perfused intracardially with 200 ml of phosphate buffered saline (PBS) followed by 250 ml of paraformaldhyde (PFA) 4% in 0.1 M PBS, (pH 7.4). Spinal cord and lumbar DRG were

removed and post-fixed in PFA for 2 h. Tissues were stored in 30% sucrose solution in PBS, containing 0.01% sodium azide. The fourth and fifth lumbar segments were identified and 40-mm thick sections were cut using a freezing microtome. Sections were incubated overnight with anti-NPY (Affiniti, 1:1000), anti-c-Jun antibodies (Amersham; 100 ng/ml), free floating, using standard histochemistry techniques. Peptides were visualized by 3,3′-diaminobenzidine or fluorescein-isothiocyanate. Quantification of NPY changes in the spinal cord was performed using a Bio Rad 1000 confocal microscope as described previously [12]. At least five sections per slide (one slide per animal) were measured. The absolute measurements of the unoperated side were then compared to the operated side and the results expressed as percentage of peptide change for each animal. The saline treated rats did not show any difference in thermal nociceptive threshold between left and right hindpaw. The CCI animals showed ipsilateral hyperalgesia. The ipsilateral paw withdrawal latency of the colchicine only and CCI + colchicine treated animal was increased compared to the control paw. Forty days after treatment, the CCI + saline group was still hyperalgesic whereas the CCI + colchicine group behaved normally (Fig. 1). C-Jun was induced ispsilaterally by colchicine, axotomy and the combination of both in many DRG neurones and motoneurones, both indicating nerve damage. NPY was up-regulated in medium and large neurones of the L4 L5 DRG after colchicine treatment or axotomy alone and colchicine treatment in combination with axotomy of

Fig. 2. NPY is induced in ipsilateral DRG after axotomy (B), axotomy + 50 mM colchicine (C) and 50 mM colchicine (D) treatments. Thirtymicrometer thick DRG sections were cut on a freezing microtome, immunostained and visualized using FITC. (A) is contralateral side. Scale bar, 50 mm.

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Fig. 3. NPY is increased in ipsilateral laminae 3–4 of the spinal cord (left) after axotomy (A), axotomy + 50 mM colchicine (B) and 50 mM colchicine (C) treatments. Forty-micrometer thick cord sections were cut on a freezing microtome, immunostained and visualized using FITC. Scale bar, 100 mm.

the sciatic nerve (Fig. 2). Saline application alone did not induce NPY. Axotomy induces NPY expression in laminae 3–4 of the lumbar section of the spinal cord ipsilaterally to the injury (Fig. 3) and is likely to arise from the large myelinated afferent fibres of the DRG projecting in laminae 3–4. NPY expression was increased from 60 to 75% in the spinal cord, when compared to the contralateral side, in the colchicine, colchicine + axotomy and axotomy only groups. The saline only group did not show any change in NPY expression (Fig. 4). No increase was observed in lamina 1–2. Rats treated with colchicine regardless of whether they underwent CCI showed increased paw withdrawal latency (PWL) equivalent to the cut-off time of the meter indicating that some nerve damaged occurred. This was confirmed by the presence of c-Jun in motoneurones. However, when the rats where tested 40 days after surgery, ipsilateral (colchicine treated) PWL was no longer different from the contral-

ateral PWL. In contrast, rats that underwent CCI showed a trend toward an hyperalgesic state. (The failure to reach significance being probably due to only three animals being used). These suggests that the integrity of nerve function is required for the development of hyperalgesia. Despite the reduction in hyperalgesia, blockade of axonal transport in the sciatic nerve by colchicine did not prevent NPY increase induced by axotomy. Furthermore, colchicine applied alone on the sciatic nerve induced NPY expression in DRG and laminae 3–4 of the spinal cord. Application of colchicine on the sciatic nerve induces NPY expression in large and medium DRG neurones and laminae 3–4 of the spinal cord in the same way as axotomy and CCI. However, the nerve degeneration that seems to occur after colchicine treatment suggests that the increase of NPY could be caused by the axotomy-like action of colchicine. Indeed, colchicine has been shown to cause C-fibre degeneration [9]. The presence of NPY in large and medium DRG neurones indicates that Ab fibres also sustained some

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[2]

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Fig. 4. Percentage changes in NPY staining in laminae 1–2 and 3–4, 7 day post-treatment. Fluorescence intensity was measured using a Bio Rad 1000 confocal microscope. The ipsilateral (operated) side is compared to the contralateral (unoperated) side of the animal. Results are expressed as mean ± SEM, n = 4 for axotomy and n = 3 for other groups.

degeneration. However, the combination of nerve degeneration and transport blocking makes it improbable that the colchicine-induced NPY increase is due to the transport of a factor generated at the site of degeneration, but rather to the suppression of retrogradely transported factor(s) which would inhibit NPY expression in the normal state. Blockade of axonal transport has previously been shown to induce vasoactive intestinal polypeptide (VIP) and galanin expression in DRG neurones [8], and VIP expression in enteric neurones [4]. This study provides further evidence that neuropeptide expression could be altered by blockade of axonal transport. Application of colchicine on the sciatic nerve induced cJun in the DRG. C-Jun has previously been shown to be controlled by target-derived NGF [5]. Blockade of axonal transport results in a loss of target-derived NGF and consequently c-Jun induction. This parallel between c-Jun and NPY expression suggest that c-Jun could be involved in the control of NPY expression. Indeed, microinjection of c-Jun antisense in sensory neurones in vitro decreased partially NPY expression [11]. In conclusion, NPY expression in the DRG and laminae 3–4 of the spinal cord is more likely to be initiated by the suppression of a retrogradely transported factor rather than by the production of an active factor at the site of injury. The mechanism of NPY induction seems to be multifactorial [3], and the role of the different factors remains unclear [2]. Financial support from Smithkline Beecham is gratefully acknowledged. [1] Bennett, G.J. and Xie, Y.-K., A peripheral neuropathy in rat that

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