Fos-like immunoreactivity increases in the lumbar spinal cord following a chronic constriction injury to the sciatic nerve of rat

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Neuroscience Letters 206 (1996) 9-12

NEUROSCIENC[ LETTERS

Fos-like immunoreactivity increases in the lumbar spinal cord following a chronic constriction injury to the sciatic nerve of rat Keith C. Kajander a,*, Anne M. Madsen", Michael J. Iadarola b, Gaetano Draisci b,1, Satoshi Wakisaka b,2 aDepartments o] Oral Science, and Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis, MN 55455, USA bNeurobiology and Anesthesiology Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892, USA

Received 14 December 1995; revised version received 25 January 1996; accepted 25 January 1996

Abstract

A chronic constriction injury (CCI), transection injury, or sham injury to the sciatic nerve was induced in 30 rats. Rats were then sacrificed at 1, 3, 5, 10, and 20 days following injury, and the number of cells immunohistochemically labeled for Fos-like immunoreactivity (Fos-LI) was determined in random sections from the lumbar 4 and 5 (L4 and L5) spinal segments. Non-parametric statistics (Wilcoxon) were used to compare the number of labeled cells ipsilateral to the injury to the number of labeled cells on the contralateral side. At 1 and 5 days following injury, in all treatment groups, significantly more labeled cells (P < 0.05) were observed ipsilaterally. In addition, at 3 and l0 days following injury, the CCI groups exhibited significantly more labeled cells ipsilaterally. The significant increases for the CCI groups ranged from 161% to 360%. Generally, increases were greater for the CCI groups. These results indicate that Fos-LI increases to a greater extent and for a longer duration following the CCI than following either a transection or sham injury. Keywords: Hyperalgesia; Nerve compression syndromes; Neuralgia; Neuroma; Neuronal plasticity; Pain; Proto-oncogene proteins c-fos

Bennett and Xie [2] introduced an animal model of peripheral neuropathy in which the rat's left sciatic nerve is loosely ligated with four chromic gut sutures; this procedure produces a chronic constriction injury (CCI). Behavioral data indicate that these rats experience neuropathic pain [2]. In another animal model o f peripheral neuropathy, the sciatic nerve is transected and a neuroma allowed to form. Both models have provided valuable information about pathological changes occurring in the nervous system following nerve injury. For example, the receptive fields of spinal neurons are altered in both size and location [9], and there are changes in neuropeptide levels in the spinal cord [4,8,11]. These alterations within

* Corresponding author. Department of Oral Science, 17-252 Moos Tower, 515 Delaware Street S.E., University of Minnesota, Minneapolis, MN 55455, USA. Tel.: +1 612 6260632; fax: +1 612 6262651; e-mail: kajander @maroon.tc.umn.edu. I Present address: lnstituto di Anesthesiologia e Rianimazione, Universit~iCattolica Sacro Cuore, 00100 Roma, Italy. 2 Present address: First Department of Oral Anatomy, Osaka University Faculty of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565, Japan.

the dorsal spinal cord suggest that other substances in the spinal cord also may exhibit changes following the CCI. A particularly good candidate substance to evaluate for alterations is the DNA-binding protein, Fos. Draisci and Iadarola [7] reported that the m R N A coding for Fos is rapidly and transiently expressed in the spinal cord after inflammation of the rat's hindpaw. It is known that dynorphin expression also increases following induction of inflammation [7,18]. In addition, Fos and dynorphin increase following transection injury to the sciatic nerve [5,6]. Since dynorphin increases after induction of the CCI [11], it is likely that Fos also increases in animals with the CCI. To evaluate if there are differences in the production of Fos in two different models o f peripheral neuropathy, we examined the immunohistochemical presence of Fos-LI in spinal cells after the CCI and after a transection injury. Some of these results have appeared in a published abstract [12]. Thirty adult, male S p r a g u e - D a w l e y rats (250-350 g, Harlan Sprague-Dawley, Inc.) purchased from either Madison, WI, or Indianapolis, IN, were used in this study. Animals were housed in groups of four in plastic cages on

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K.C. Kajander et al. / Neuroscience Letters 206 (1996) 9-12

soft bedding. All procedures were approved by the University of Minnesota Animal Care Committee. All rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and the left common sciatic nerve was exposed by blunt dissection at the level of the mid-thigh. A CCI was produced by tying four chromic gut sutures loosely around the nerve [2]. A transection injury was produced by first placing two tight silk ligatures around the sciatic nerve in the upper thigh; the nerve was then completely severed between the ligations and the ends tucked back to avoid regeneration. In animals with the sham injury, the sciatic nerve was exposed, as in the other injuries, and gently manipulated. In all animals, the wound was closed in layers, and the animals were kept warm until recovery from the anesthesia. The unexposed sciatic nerve of the right leg served as a control. After surgery, animals survived for 1, 3, 5, 10, or 20 days before sacrifice. Two rats composed each experimental group. On the appropriate day after injury, the thorax was opened and the animal was immediately perfused transcardially with ice-cold, normal saline (0.9%) followed by cold, buffered, 4% paraformaldehyde. The spinal column was removed and post-fixed in 4% paraformaldehyde for an additional 24 h. The lumbar 4 and 5 (L4 and L5) spinal segments were then identified and removed. A small section of tissue was removed from the right ventral quadrant to allow discrimination between sides ipsilateral and contralateral to the injury. Segments were cryoprotected by immersing in cold 30% sucrose until sectioned (30ktm) on a freezing microtome. Sections were collected in cold 0.1 M phosphate-buffered saline and processed for localization of Fos immunoreactive protein using a peroxidase-antiperoxidase protocol with nickel ammonium sulfate intensification. The primary antiserum was polyclonal; its characteristics have been described [ 14,18,20]. In this report, the term Fos-like immunoreactivity (FosLI) is used to describe the observed labeling. Ten spinal sections were selected from each animal for evaluation. Sections were selected randomly but with the stipulations that within a given animal five sections came from L4 and five sections came from L5, and that each section came from a different area along the rostralcaudal extent of each segment. Drawings of the spinal cord gray matter were made using the microscope's drawing tube at 4 0 x magnification. The number of labeled cells in the gray matter (both dorsal and ventral horns) on the ipsilateral side and on the contralateral side was recorded from the drawing of each section. Between treatment groups, the distributions of Fos-LI cells exhibited unequal variances (Cochran's C, P < 0.001). Thus, data were analyzed using non-parametric statistics. For all treatment groups at all days, 15 Wilcoxon tests were used to compare the number of labeled cells in the ipsilateral spinal cord to the number of labeled cells in the contralateral spinal cord. In addition, the per-

cent change (i.e., number of labeled cells on the ipsilateral s i d e - the number of labeled cells on the contralateral side/number of labeled cells on the contralateral side x 100) was determined for each section, and then the average (_+SD) percent change of all sections in a group was determined for each group at each day. Photomicrographs of transverse sections from spinal cords of animals sacrificed 3 days after a CCI (Fig. 1A), a transection injury (Fig. 1B), or a sham injury (Fig. 1C) are shown in Fig. 1. Labeled cells were present in the superficial dorsal horn, the substantia gelatinosa, the base and neck of the dorsal horn, and the central canal region. Very few labeled cells were present in the ventral horn in any section. As compared to the contralateral side, the

Fig. 1. Three photomicrographsillustrating transverse sections of spinal cords from animals with a CCI (A), transection injury (B), or sham injury (C) at 3 days after injury. Fos-Ll cells are seen as punctate black spots covering neuronal nuclei in the dorsal gray matter, which is outlined in black. In all sections, the dorsal gray matter ipsilateral to the injury is at the left of the figure. The number of Fos-LI cells is greatest in the spinal gray matter ipsilateral to the CCI (A). The gray matter contralateral to the CCI also exhibits an increase in Fos-LI cells (A).

K.C. Kajander et al. / Neuroscience Letters 206 (1996) 9-12

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Fig. 2. Histograms representing the percent difference (,u _+cr) in the number of Fos-Ll cells in the spinal gray matter after either a CCI, transection injury, or sham injury at 1, 3, 5, 10, or 20 days after injury. The ipsilateral gray matter exhibited significantly more Fos-Ll cells than the contralateral gray at l, 3, 5, and 10 days after CCI injury. The ipsilateral spinal gray matter of animals with either a transection injury or a sham injury exhibited significantly more Fos-LI cells than the contralateral gray at 1 and 5 days after injury (*P < 0.05; **P < 0.01).

number of labeled cells is greater on the side ipsilateral to the CCI (Fig. 1A, left side). In addition, many more labeled nuclei were present in the gray matter ipsilateral to the CCI (Fig. 1A) than in the other two treatment groups (Fig. 1B,C). There also appears to be an increase in the number of Fos-LI cells in the gray matter contralateral to the CCI (Fig. 1A, right side). The number of Fos-LI cells in the ipsilateral spinal cord was compared statistically to the number of labeled cells contralaterally within treatment groups at each postoperative time. There were significantly more labeled cells on the ipsilateral side in animals with the CCI at 1, 3, 5, and 10 days after surgery (P < 0.01, Wilcoxon). Significant differences existed also for the transection and sham injury groups at 1 and 3 days following injury. The percent change (Fig. 2) was greatest for all treatment groups at 1 day after injury (297--444%). The percent change at 3, 5, and 10 days was highest for the CCI groups (161-297%). The percent change was small for all groups (-10 to 9%) at 20 days after injury. In general, the greatest increases in Fos-LI occurred in the gray matter ipsilateral to the CCI. The pattern of labeling was similar to that seen by others using different noxious stimuli and antisera from other sources [3,15]. An ipsilateral increase in Fos-LI occurred at 1 and 5 days after the transection injury also. The increase observed after transection injury agrees with results reported by Chi et al. [5]. The increase in the number of Fos-LI cells observed qualitatively in the gray matter contralateral to the CCI agrees with other reports in which changes occur in the spinal gray matter contralateral to the CCI [13,16,19]. It is interesting that we found that the CCI produced a greater increase in Fos-LI than did the transection injury.

t1

The nature of these two peripheral nerve injuries differs dramatically, although it is not clear which injury is physiologically more severe. The transection injury produces immediate deafferentation of all axons in the sciatic nerve, whereas with the CCI there is a slow loss, distal to the injury, of large myelinated axons, followed later by loss of small myelinated axons and some unmyelinated axons [1]. After the CCI, spontaneous activity exists in the large myelinated fibers; this activity is coupled with ongoing receptor input from the peripheral tissues [10]. After the transection injury, although many of the deafferented myelinated axons exhibit spontaneous activity also [21], there is no peripheral (cutaneous or deep) receptor input. The combination of spontaneous activity and peripheral input may be a much more severe injury physiologically, and consequently, may up-regulate greater expression of the c-fos gene in central neurons after the CCI. Fos may be involved in molecular mechanisms used by the nervous system to translate injurious peripheral stimulation into permanent central changes after the CCI. Draisci and Iadarola [7] first suggested that Fos may be involved in the in vivo regulation of dynorphin. They demonstrated, after induction of acute peripheral inflammation in the hindpaw of the rat, that mRNA for Fos precedes the appearance of mRNA for dynorphin. In addition, co-localization of Fos-LI and mRNA for dynorphin has been observed in some spinal neurons following noxious stimulation [17,18]. Thus, the neuronal response to the noxious stimulus may be an immediate increase in Fos that produces an increase in dynorphin, which temporarily alters the responsiveness of nociceptive spinal neurons to acute stimulation. Using the CCI model, we reported a large (300%) increase in spinal dynorphin ten days after injury [11]. In the current study, the greatest increase in Fos-LI occurred earlier, at 1-10 days after injury. Considering this temporal relationship, Fos may regulate spinal dynorphin production in the CCI model also. The early increase in spinal Fos-LI may be a response to the nerve injury. Fos then induces an increase in spinal dynorphin, which may produce a chronic change in the response properties of spinal neurons. In this way, Fos may contribute to the chronic behavioral changes seen in the CCI as well as to the behavioral changes seen in models of inflammation. The authors thank Jon Coltz, Chris DeLisle, Mark Kleive, Young-Ok Lee, and Anie Roche for critically reviewing an earlier version of this manuscript. This research was supported in part by grant NS29567 from the National Institute of Neurological Disorders and Stroke. [1] Basbaum, A.I., Gautron, M., Jazat, F., Mayes, M. and Guilbaud, G., The spectrum of fiber loss in a model of neuropathic pain in the rat: an electron microscopic study, Pain, 47 (1991) 359-367. [2] Bennett, G.J, and Xie, Y.-K., A peripheral mononeuropathy in the

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[4]

[5]

[6]

[7]

[8]

[9]

[10]

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[12]

K.C. Kajander et al. I Neuroscience Letters 206 (1996) 9-12

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