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Exaggerated C-fiber activation prevents peripheral nerve injury-induced hyperinducibility of c-Fos in partially deafferented spinal dorsal horn
Neuroscience Research 27 (1997) 161 – 167
Exaggerated C-fiber activation prevents peripheral nerve injury-induced hyperinducibility of c-Fos in partially deafferented spinal dorsal horn Tomosada Sugimotoa,*, Makoto Funahashib, Chun Xiaoa, Akira Adachib, Hiroyuki Ichikawaa a
Second Department of Oral Anatomy, Okayama Uni6ersity School of Dentistry, 2 -5 -1 Shikata-cho, Okayama 700, Japan b Department of Oral Physiology, Okayama Uni6ersity School of Dentistry, 2 -5 -1 Shikata-cho, Okayama 700, Japan Received 29 August 1996; accepted 2 December 1996
Abstract Dorsal horn neurons chronically deafferented by peripheral nerve injuries acquire hypersensitivity to noxious input from outside the original receptive field. This study examines the effect of electrical nerve stimulation at the time of injury on such injury-induced hypersensitivity. The medial 3/8 of the dorsal horn laminae I/II around the junction of 4th and 5th lumbar segments (the tibial territory) was deafferented by transection of the ipsilateral tibial nerve in rats. At 2 days or 3 weeks postinjury, the hindpaw was injected with formalin to induce c-fos. At 2 days, neurons with induced c-Fos protein-like immunoreactivity (fos-neurons) were largely confined in the lateral 5/8 of laminae I/II (the peroneal and hip, thus P and H territory). At 3 weeks, fos-neurons significantly increased in the deafferented tibial territory. A similar increase was also noted in the P and H territory. Thus the dorsal horn neurons exhibited c-fos hyperinducibility, an indication of hypersensitivity. Electrical stimulation with a train of 150 shocks (10 V, 2 ms) of the proximal nerve stump immediately after transection prevented the c-fos hyperinducibility. The effect was greater with the stimulation frequency of 0.5 Hz than 0.1 Hz or 10 Hz. The stimulation had no effect on the c-fos inducibility at 2 days postinjury. © 1997 Elsevier Science Ireland Ltd. Keywords: c-Fos; Dorsal horn; Electrical stimulation; Hypersensitivity; Peripheral nerve injury; Windup
1. Introduction Peripheral nerve injuries in human cases often result in neuropathic pain. Peripherally axotomized primary neurons are known to exhibit spontaneous electrical activity, and become sensitive to mechanical and/or chemical stimuli applied to the neuroma (Wall and Gutnick, 1974; Govrin-Lippman and Devor, 1978; Korenman and Devor, 1981; Scadding, 1981). Neuropathic pain appears to depend on such pathophysiologic excitation of injured primary neurons because resection or anesthetic infiltration of painful neuromata usually brings about temporary pain relief. It is also likely, * Corresponding author. Tel.: +81 86 235 6635; fax: + 81 86 223 7053.
however, that changes in the dorsal horn neurons postsynaptic to the injured primary neurons at least partially contribute to the neuropathic pain. It has been demonstrated that superficial dorsal horn neurons chronically deafferented by peripheral nerve transection lost their original receptive field, and acquired a novel receptive field (Devor and Wall, 1978, 1981; Lisney, 1983; Markus et al., 1984; Hylden et al., 1987). Such receptive field reorganization can be demonstrated not only by an electrophysiological recording but also by a histochemical method. Dorsal horn neurons express a proto-oncogene c-fos in response to noxious stimulation of their receptive field (Hunt et al., 1984; Mene´trey et al., 1989; Bullitt, 1990; Williams et al., 1990). When a peripheral nerve was transected, the neurons in the deafferented part of the
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dorsal horn lost their receptive field (Williams et al., 1991; Sugimoto et al., 1994). Within a few weeks after the nerve injury, however, the dorsal horn neurons disconnected from their original receptive fields began to respond by c-fos expression to noxious stimulation of a skin area outside the original receptive fields (Sugimoto et al., 1993, 1994). Therefore, the chronically deafferented dorsal horn neurons exhibited hypersensitivity to somatotopically inappropriate primary nociceptive input. It is a tempting idea that such hypersensitivity is related to neuropathic pain, but evidence correlating the hypersensitivity and pain is lacking. Clinical as well as experimental studies indicate that afferent barrage immediately before or after the nerve transection increased the degree of neuropathic pain or pain-related behavior. For example, preoperative epidural lumbar blockade lowered the incidence of phantom limb pain in human amputees (Bach et al., 1988). Autotomy, a behavioral manifestation of deafferentation pain in rats, following sciatic neurotomy was suppressed by blockade of injury discharge and augmented by electrical stimulation of the nerve (Seltzer et al., 1991). Because injury discharge, under the influence of systemically administered strychnine, caused transsynaptic degeneration of dorsal horn neurons following neurotomy (Sugimoto et al., 1987), we predict that injury discharge or electrical nerve stimulation at the time of nerve injury would induce transneuronal changes that are responsible for neuropathic pain. In this study, therefore, we proceed to examine the chronic transsynaptic effect of electrical stimulation immediately after transection of the tibial nerve. At 3 weeks after injury, we deliver noxious stimulus to the hindpaw skin and evaluate the spatial pattern of c-fos induction in the partially deafferented segment of the spinal cord laminae I and II. The noxious signal is transmitted to the spinal cord through the spared peroneal nerve.
2. Materials and methods Thirty-five male rats of the Sprague-Dawley strain (b.w.=200 g) were used. All the surgical procedures were performed under anesthesia by i.p. injection with a mixture of pentobarbital sodium (20 mg/kg) and ethylcarbamate (650 mg/kg). About 15 mm of a segment of the sciatic nerve was exposed between the mid-thigh level and the popliteal fossa by skin incision and blunt dissection through the biceps femoris muscle. In the proximal part of this segment, the three major divisions of the nerve (the tibial, sural and peroneal nerves) were clearly separated by individual perineurium. The distal end of the exposed segment of tibial nerve was tightly ligated with a 7-0 silk suture thread and transected
distally (chronic injury). The proximal stump of the nerve was placed on a pair of stainless steel hook electrodes (distance: 500 mm). Electrical stimulation consisting of 150 pulses (square pulses of 10 V, 2 ms) was delivered at 0.1 Hz (25 min), 0.5 Hz (5 min) or 10 Hz (15 s). One group of rats received a sham procedure in which the nerve stump was placed on the electrode for 5 min without stimulation. The nerve was returned to the original position, and the skin incision was suture-closed with a 7-0 chromic catgut. Nineteen days later, the rats were re-anesthetized and an identical transection (acute injury) was performed on the other side but electrical stimulation was omitted. Forty-eight hours after the acute injury, bilateral noxious stimulation was delivered to the hindpaw of re-anesthetized rats by four subcutaneous injections with 5% formalin (30 ml each). The tip of the hypodermic needle was located at the center of proximal and distal halves of plantar and dorsal surfaces of the hindpaw. Two hours later, anesthesia was supplemented by ether inhalation and the rats were exsanguinated and fixed by perfusion with 50 ml saline followed by 500 ml of 0.1 M sodium phosphate-buffered 4% formaldehyde (prepared fresh from paraformaldehyde, pH 7.4). Another group of rats underwent bilateral tibial nerve transection at the same time, and electrical stimulation at 0.5 Hz (5 min) was delivered only on one side. Forty-eight hours later these rats were formalin-stimulated bilaterally, and perfusion-fixed as described above. In addition, some rats received unilateral tibial neurotomy and electrical stimulation at 0.5 Hz. They survived for either 2 h, 1 day, 2 days or 3 weeks before fixation but the formalin-stimulation and contralateral injury were omitted. The spinal cord within 9 1 mm of the junction of 4th and 5th lumbar segments (L4 and L5) was dissected out. Frontal sections were cut with a freezing microtome at 50 mm in thickness and processed for histochemical demonstration of c-Fos protein-like immunoreactivity (Fos-LI). After thorough rinsing with phosphate buffered saline (PBS), they were processed for histochemical demonstration of Fos-LI using a PAP method (see Sugimoto et al., 1993 for detail). Antibodies used and the dilution were as follows; rabbit anti c-Fos protein IgG (0.2 mg protein/ml; Oncogene Science, Manhasset, NY), goat anti-rabbit IgG (1:300; Cappel, West Chester, PA), and PAP complex (1:3000; Cappel). Sections were mounted on gelatin-coated glass slides, air-dried, dehydrated in alcohols and coverslipped with Entellan (Merck). Five sections were randomly selected for each rat. Using an Olympus photomicroscope equipped with 10× objective and a camera lucida drawing tube, an outline of the dorsal horn was traced on a white paper and neurons with Fos-LI (fos-neurons) were plotted. Dark field illumination was used for drawing the out-
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Fig. 1. Medial 3/4 of the dorsal horn at the L4/5 junction. Sections (50 mm-thick) were stained for c-Fos protein-like immunoreactivity but not counterstained. Because of the section thickness, many of fos-neurons are out of focus. The number of fos-neurons can be best counted on camera lucida drawings like those shown in Fig. 2. The ipsilateral tibial nerve was cut and electrically stimulated at 0.5 Hz 2h before perfusion fixation but the formalin stimulation was omitted (A). The nerve was cut and sham-stimulated 3 weeks before formalin-stimulation (B). The nerve was cut and stimulated at 0.5 Hz 3 weeks before formalin-stimulation (C). The nerve was cut 2 days before formalin-stimulation but electrical stimulation of the nerve was omitted (D). Fig. 1(d) is the contralateral side of the section shown in Fig. 1(c), and has been reverse-printed for easier comparison. Medial to the right. Scale bar; 100 mm.
line and laminar borders of the dorsal horn, while fos-neurons were identified using bright field illumination. Objective lenses, 20 × and 40 × , were used for identification of fos-neurons when necessary. The average number per section of fos-neurons in each spinal cord area (see below) was recorded for each rat. Our quantification dealt only with the number and location of neurons with histologically detectable Fos-LI, and the staining intensity of positive cells was ignored. The analysis of our results is based on a previous observation that the dorsal horn around the L4/L5 junction receives the densest primary projection from the sciatic nerve (Swett and Woolf, 1985; LaMotte et al., 1989). In laminae I and II, the tibial primary neurons that innervate the plantar surface of the hindpaw project to the medial most 3/8, whereas the lateral 1/4 is innervated by primary neurons supplying the posterior cutaneous nerve of the thigh. The area between them receives primary projection from the superficial peroneal and sural nerves that innervate the dorsal and lateral aspects of the hindpaw (Swett and Woolf, 1985). For stating the location of neurons with Fos-LI, the medialmost 3/8 of laminae I/II will be referred to as the tibial territory and the lateral 5/8 as the P and H (peroneal and hip) territory, respectively. The rest of the spinal cord gray matter will be divided into three areas; laminae III/IV, laminae V/VI, and laminae VII-X. The laminar border between IV and V was settled by medially extending the dorsal horizontal tangent of the reticulated part of the dorsal horn neck.
The border between laminae VI and VII was settled by medially extending the ventral horizontal tangent of the reticulated part of the neck.
3. Results In all the rats examined in this study, fos-neurons were most frequent in the P and H territory of laminae I/II. Laminae VIII and IX were almost always devoid of fos-neurons. The number of formalin-induced fosneurons in the tibial territory greatly varied depending on the postinjury interval (chronic or acute) and the stimulation frequency. For example, fos-neurons significantly increased following chronic injury (without electrical stimulation) compared to acute injury (see below). On the other hand, difference in number of fos-neurons in deeper laminae was rather obscure between chronic and acute injury sides regardless of the electrical stimulation. In this study, therefore, we quantify c-fos induction in laminae I/II of the dorsal horn. Immunohistochemically demonstrable Fos-LI in these neurons are considered to have been induced by formalin-stimulation because c-fos induction by electrical stimulation was transient and almost completely disappeared within 24 h after stimulation. At 2 h after tibial nerve transection and electrical stimulation at 0.5 Hz, many fos-neurons were observed in the ipsilateral tibial territory of the dorsal horn laminae I/II (n =2) (Fig. 1(a)). At 1 day (n= 2), 2 days (n= 2) or 3 weeks (n=3)
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after such preparation, however, only few fos-neurons (less than one per section) were found in any area of the examined dorsal horn if formalin stimulation was omitted. Following chronic injury with sham-stimulation (no electrical stimulation), formalin-stimulation 2 h prior to perfusion fixation produced 19.7 fos-neurons per section of the ipsilateral tibial territory (n = 5) (Fig. 1(b)). This is about 4-fold increase over the contralateral side (4.8 fos-neurons per section) on which the tibial nerve had been acutely transected 2 days before formalin-
Fig. 2. Camera lucida drawings of dorsal horn sections through the L4/5 junction. The tibial nerve on the experimental side had been transected and sham-stimulated (A) or stimulated at 0.5 Hz (B) 3 weeks before formalin-stimulation, while the same nerve on the control side had been acutely transected 2 days before formalin-stimulation but electrical stimulation at the time of injury was omitted. The rat shown in (C) received bilateral acute tibial neurotomy 2 days before formalin-stimulation, and electrical stimulation (0.5 Hz) was delivered only on the experimental side. Many fos-neurons can be seen in the medial 3/8 of laminae I/II on the experimental side of (A) indicating chronic injury induced c-fos hyperinducibility. Electrical stimulation at 0.5 Hz has suppressed the hyperinducibility (B). Electrical stimulation at 0.5 Hz has no effect on the c-fos induction in normosensitive neurons responding to formalin-stimulation at 2 days postinjury (C).
stimulation (Fig. 2(a), Table 1). The difference between sides was significant at the 1% level (t-test for paired samples). A tendency of chronic increase was also noted in the P and H territory, where 41.1 and 34.7 fos-neurons were counted on the chronic and acute sides, respectively. However, the difference between sides was not statistically significant. Although the average number per section of fos-neurons in deeper laminae was always greater on the chronic side than on the acute side, the difference was small. These results are consistent with a previous report that chronic tibial nerve transection induced more prominent c-fos hyperinducibility in the deafferented tibial territory than in the rest of the spinal cord areas (Sugimoto et al., 1994). Following chronic injury combined with electrical stimulation at 0.5 Hz, the number per section of fosneurons in the ipsilateral tibial territory was 5.2 (n =5). This number was similar to that on the contralateral side with the acute tibial neurotomy (5.5 fos-neurons, Fig. 1(c,d), Fig. 2(b), Table 1). The number of induced fos-neurons in the P and H territory was smaller on the chronic side (23.0) than on the acute side (34.9) though the difference was not statistically significant. The numbers of fos-neurons in both the tibial and P and H territories on the acute side of rats with 0.5 Hz stimulation were comparable with those of sham-stimulated rats. The difference in number of fos-neurons in similar territories on the chronic side was significant between the stimulated and sham-stimulated rats (PB0.01 for tibial and P B 0.05 for the P and H territories, ANOVA). Therefore, the electrical stimulation at 0.5 Hz significantly suppressed the c-fos hyperinducibility following chronic injury in laminae I/II (Fig. 3). The effect of electrical stimulation on c-fos induction in deeper laminae was obscure. Electrical stimulation at 0.1 (n = 6) and 10 Hz (n=5) also suppressed the chronic c-fos hyperinducibility but not so effectively as that at 0.5 Hz did (Fig. 3). In the tibial territory on the chronic side, 12.8 and 10.3 fosneurons were observed following stimulation at 0.1 and 10 Hz, respectively. Because 6.7 (0.1 Hz) and 6.4 (10 Hz) fos-neurons were observed on the acute side, the chronic side showed about 2-fold increase (Table 1). A t-test for paired samples revealed the difference between side to be significant only for the group stimulated at 10 Hz (P B 0.05). Difference between sides was small for the P and H territory of both groups. Because unilateral noxious stimulation has been shown to enhance c-fos induction in contralateral dorsal horn neurons in response to the second contralateral noxious stimulation delivered within 20 h (Leah et al., 1992), relative increase in number of fos-neurons on the acute side of electrically stimulated rats might have resulted in seeming suppression of c-fos hyperinducibility on the stimulated side. In rats with bilateral acute injury combined with unilateral electrical stimulation at
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Table 1 Mean numbers of fos-neurons per section on chronic and acute injury sides Stimulation frequency
Sample size
Sham
5
0.1 Hz
6
0.5 Hz
5
10 Hz
5
Side
Chronic Acute Chronic Acute Chronic Acute Chronic Acute
Mean (S.E.) of number of fos-neurons per section Tibial (I/II) P and H (I/II) III/IV
V/VI
19.7 4.8 12.8 6.7 5.2 5.5 10.3 6.4
15.6 11.9 11.8 12.2 10.4 15.4 17.0 18.5
(2.0)** (1.1) (5.9) (2.5) (1.4) (2.6) (2.3)* (1.8)
41.1 34.7 28.0 25.1 23.0 34.9 32.8 38.8
(2.6) (4.6) (4.5) (3.4) (4.3) (1.9) (3.9) (5.2)
9.0 7.8 11.5 10.8 5.6 6.8 17.1 17.6
(1.3) (1.2) (3.0) (3.0) (1.7) (1.0) (3.0) (2.9)
VII(4.4) (1.8) (3.6) (2.0) (1.4) (1.8) (2.7) (1.7)
5.9 4.4 2.7 3.4 4.2 9.4 4.3 5.9
(1.8) (1.0) (0.8) (0.7) (0.9) (2.4) (1.1) (1.7)
Numbers in parentheses indicate standard errors. Difference between sides was significant at the *5% or **1% level (t-test for paired samples).
0.5 Hz, however, c-fos induction by bilateral formalinstimulation 2 h prior to perfusion fixation was symmetrical (Fig. 2(c), Table 2). Furthermore, the number of fos-neurons on the acute side of rats, whose chronically injured tibial nerve on the contralateral side had been stimulated at 0.5 Hz, was similar to that on the acute side of sham-stimulated rats (Table 1). Therefore, contralateral effect of electrical stimulation, if any, appeared to have subsided within 2 days of electrical stimulation.
Fig. 3. Effect of electrical stimulation on the number of formalin-induced fos-neurons in the tibial and P and H territories at 3 weeks postinjury. Stimulation at 0.5 Hz significantly reduced the number of fos-neurons in both territories compared to sham-stimulation; *P B 0.05, and **P B 0.01 (ANOVA). Stimulation at 0.1 Hz and 10 Hz also showed a tendency of reduction, though the difference was not significant. The numbers of rats examined were 5 (sham-stimulation), 6 (0.1 Hz), 5 (0.5 Hz) and 5 (10 Hz).
4. Discussion We have reported that fos-neurons produced by formalin-stimulation of the whole hindpaw were limited in the peroneal territory and those in the tibial territory were rare during the first postinjury week following tibial neurotomy (Sugimoto et al., 1993, 1994). This topographic distribution and magnitude of c-fos induction are considered to be the baseline c-fos induction by selective stimulation of the peroneal primary neurons. In a chronic situation where the tibial nerve had been transected and prevented from regeneration for 2 weeks or longer before the formalin-stimulation, however, the number of induced fos-neurons significantly increased in the tibial territory and the medial part of the peroneal territory (Sugimoto et al., 1993, 1994). Such increase in number of fos-neurons is considered to reflect an increased sensitivity of dorsal horn neurons to spared peroneal primary input. The present study reproduced these results indicating that dorsal horn neurons chronically deafferented by peripheral nerve injury exhibited ectopic sensitivity to c-fos induction by somatotopically inappropriate noxious primary input. Because fos-neurons in the P and H territory (the peroneal and hip territories combined) were counted altogether, statistical significance could not be detected but there was a tendency of increase in number of fos-neurons in this territory. In this study, we used intense electrical stimulation (rectangular pulses at 10 V and 2 ms) to activate as many primary C-fibers in the proximal stump of the transected tibial nerve as possible. Indeed, such stimulation delivered at 0.5 Hz for 5 min induced Fos-LI in many laminae I/II neurons within 2 h. c-fos Expression induced by the electrical stimulation was transient and the Fos-LI disappeared by 24 h of stimulation. Thereafter, Fos-LI in the dorsal horn was almost negligible unless the hindpaw was stimulated by formalin injection. At 2 days after bilateral injury followed by unilateral electrical stimulation at 0.5 Hz, the number and topo-
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Table 2 Mean numbers of fos-neurons per section at 2 days after bilateral tibial nerve transection Sample size
5
Side
Stimulated Unstimulated
Mean (S.E.) of number of fos-neurons per section Tibial (I/II)
P and H (I/II)
III/IV
V/VI
VII-
5.0 (1.1) 4.7 (0.7)
26.7 (5.9) 28.7 (3.4)
8.8 (2.6) 11.0 (2.0)
12.9 (4.4) 14.0 (2.0)
5.6 (1.3) 6.9 (1.9)
The proximal stump on one side was stimilated at 0.5 Hz. Numbers in parentheses indicate S.E.
graphic distribution of fos-neurons induced by formalin-stimulation was similar on both sides. This would indicate that the electrical stimulation of the transected nerve did not affect the baseline c-fos induction by activation of the spared peroneal primaries. Following chronic injury combined with a similar stimulation, the pattern of c-fos induction remained unchanged from the baseline. Therefore, the electrical stimulation prevented the chronic injury-induced hyperinducibility of c-fos. The effects of electrical stimulation at 0.1 and 10 Hz were similar to but less prominent than that at 0.5 Hz. Response of primary and dorsal horn neurons to the peripheral nerve stimulation at C-fiber intensity has been shown to vary depending on the stimulation frequency. According to Seltzer et al., C-fibers faithfully followed every shock in the 150-pulse train delivered at 0.1 and 0.5 Hz; while, at 10 Hz, the conduction time gradually increased and most C-fibers failed to follow every shock in the train (Seltzer et al., 1991). On the other hand, dorsal horn neurons are known to windup their response to primary C-fiber input when stimulated at a frequency greater than 0.3 Hz (Mendell and Wall, 1965, Price, 1972). Therefore, the prevention of chronic injury-induced hyperinducibility of c-fos observed in this study appears to involve dorsal horn neurons’ windup phenomenon. We previously demonstrated that afferent barrage produced at the time of peripheral nerve injury had potentially neurotoxic effect on the postsynaptic dorsal horn neurons and, in synergy with the systemically administered convulsant, caused degeneration of small dorsal horn neurons (Sugimoto et al., 1987). Although the electrical stimulation in the absence of convulsants may not cause morphologically discernible transsynaptic degeneration, it might have caused prolonged transsynaptic changes that were responsible for lowered c-fos inducibility. If this was the case, the tibial nerve stimulation would have reduced the number of neurons responding by c-fos expression to the tibial primary input, while neurons that respond only to the peroneal primary input would not be affected. This would explain why electrical stimulation affected the hyperinducibility but not baseline inducibility of c-fos in this study. Whereas dorsal horn neurons originally responding to the peroneal but not tibial primary input are not
damaged by the electrical stimulation and show baseline c-fos expression, those originally responding to the tibial but not peroneal primary input are damaged by the electrical stimulation and fail to develop hyperinducibility. What is the clinical implication of injury-induced c-fos hyperinducibility? The fact that nociceptive neurons in the deafferented territory of dorsal horn become sensitive to somatotopically inappropriate noxious primary input may indicate that stimulation of receptive field outside the denervated skin area causes ectopic excitation of deafferented dorsal horn neurons. Transmittal of such anomalous nociceptive signal along the neuroaxis may result in pain disorders such as phantom limb pain. However, c-fos induction in intact animals by noxious peripheral stimulation is largely limited to interneurons (Mene´trey et al., 1989; Tavares et al., 1993). In addition, downstream of c-fos expression is preprodynorphine-mRNA transcription (Naranjo et al., 1991; Noguchi et al., 1991). Therefore anomalous c-fos induction, by itself, may not be interpreted as a sign of neuropathic pain. Phantom sensation might result if deafferented projection neurons exhibited increase in responsiveness to somatotopically inappropriate primary input. Even if this was the case, a concomitant increase in responsiveness of inhibitory interneurons may counteract the signal transmittal. Suppression by electrical stimulation of injury-induced c-fos hyperinducibility, on the other hand, may underlie some neuropathic pain state. Because the Sprague-Dawley rat used in this study was autotomyinsensitive and because the escape behavior was impaired by the paralysis of hamstring muscles, pain-related behavior was not tested. However, electrical stimulation of the sciatic nerve at a C-fiber intensity and windup frequency immediately before the nerve transection augmented the autotomy that was seen in the Sabra strain rats (Seltzer et al., 1991). Temporary pain relief by epidural block preceding limb amputation was effective in prevention of phantom limb pain in human cases (Bach et al., 1988). Assuming that the interneurons are more sensitive than the projection neurons to transsynaptic damage (Sugimoto et al., 1990), the experimentally produced C-afferent barrage or sustained pain of the peripheral origin in human cases would chronically disinhibit the activity of projec-
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tion neurons. Therefore, pathophysiological activity of injured primary neurons and/or of deafferented dorsal horn projection neurons in response to spared primary afferent input would ascend the neuroaxis to cause neuropathic pain sensation.
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Mene´trey, D., Gannon, A., Levine, J.D. and Basbaum, A.I. (1989) Expression of c-fos protein in interneurons and projection neurons of the rat spinal cord in response to noxious somatic, articular, and visceral stimulation. J. Comp. Neurol., 285: 177– 195. Naranjo, J.R., Mellstro¨m, B., Achaval, M. and Sassone-Corsi, P. (1991) Molecular pathways of pain: fos/jun-mediated activation of a noncanonial AP-1 site in the prodynorphin gene. Neuron, 6: 607 – 617. Noguchi, K., Kowalski, K., Traub, R., Solodkin, A., Iadarola, M.J. and Ruda, M.A. (1991) Dynorphin expression and Fos-like immunoreactivity following inflammation induced hyperalgesia are colocalized in spinal cord neurons. Mol. Brain Res., 10: 227–233. Price, D.D. (1972) Characteristics of second pain and flexion reflexes indicative of prolonged central summation. Exp. Neurol., 37: 371 – 387. Scadding, J.W. (1981) Development of ongoing activity, mechanosensitivity, and adrenaline sensitivity in severed peripheral nerve axons. Exp. Neurol., 73: 345 – 364. Seltzer, Z., Beilin, B.-Z., Ginzburg, R., Paran, Y. and Shimko, T. (1991) The role of injury discharge in the induction of neuropathic pain behavior in rats. Pain, 46: 327 – 336. Sugimoto, T., Bennett, G.J. and Kajander, K.C. (1990) Transsynaptic degeneration in the superficial dorsal horn after sciatic nerve injury: effects of a chronic constriction injury, transection, and strychnine. Pain, 42: 205 – 213. Sugimoto, T., Ichikawa, H., Hijiya, H., Mitani, S. and Nakago, T. (1993) c-Fos expression by dorsal horn neurons chronically deafferented by peripheral nerve section in response to spared, somatotopically inappropriate nociceptive primary input. Brain Res., 621: 161 – 166. Sugimoto, T., Ichikawa, H., Mitani, S., Hitsu, A. and Nakago, T., Changes in c-fos induction in the dorsal horn neurons by hindpaw stimulation following tibial neurotomy. Brain Res., 642: 348–354. Sugimoto, T., Takemura, M., Sakai, A. and Ishimaru, M. (1987) Rapid transneuronal destruction following peripheral nerve transection in the medullary dorsal horn is enhanced by strychnine, picrotoxin and bicuculline. Pain, 30: 385 – 393. Swett, J.E. and Woolf, C.J. (1985) The somatotopic organization of primary afferent terminals in the superficial laminae of the dorsal horn of the rat spinal cord. J. Comp. Neurol., 231: 66–77. Tavares, I., Lima, D. and Coimbra, A. (1993) Neurons in the superficial dorsal horn of the rat spinal cord projecting to the medullary ventrolateral reticular formation express c-fos after noxious stimulation of the skin. Brain Res., 623: 278–286. Wall, P.D. and Gutnick, M. (1974) Ongoing activity in peripheral nerves: the physiology and pharmacology of impulses originating from a neuroma. Exp. Neurol., 43: 580 – 593. Williams, S., Evan, G.I. and Hunt, S.P. (1990) Changing patterns of c-fos induction in spinal neurons following thermal cutaneous stimulation in the rat. Neuroscience, 36: 73 – 81. Williams, S., Evan, G. and Hunt, S.P. (1991) C-fos induction in the spinal cord after peripheral nerve lesion. Eur. J. Neurosci., 3: 887 – 894.
Exaggerated C-fiber activation prevents peripheral nerve injury-induced hyperinducibility of c-Fos in partially deafferented spinal dorsal horn Tomosada Sugimotoa,*, Makoto Funahashib, Chun Xiaoa, Akira Adachib, Hiroyuki Ichikawaa a
Second Department of Oral Anatomy, Okayama Uni6ersity School of Dentistry, 2 -5 -1 Shikata-cho, Okayama 700, Japan b Department of Oral Physiology, Okayama Uni6ersity School of Dentistry, 2 -5 -1 Shikata-cho, Okayama 700, Japan Received 29 August 1996; accepted 2 December 1996
Abstract Dorsal horn neurons chronically deafferented by peripheral nerve injuries acquire hypersensitivity to noxious input from outside the original receptive field. This study examines the effect of electrical nerve stimulation at the time of injury on such injury-induced hypersensitivity. The medial 3/8 of the dorsal horn laminae I/II around the junction of 4th and 5th lumbar segments (the tibial territory) was deafferented by transection of the ipsilateral tibial nerve in rats. At 2 days or 3 weeks postinjury, the hindpaw was injected with formalin to induce c-fos. At 2 days, neurons with induced c-Fos protein-like immunoreactivity (fos-neurons) were largely confined in the lateral 5/8 of laminae I/II (the peroneal and hip, thus P and H territory). At 3 weeks, fos-neurons significantly increased in the deafferented tibial territory. A similar increase was also noted in the P and H territory. Thus the dorsal horn neurons exhibited c-fos hyperinducibility, an indication of hypersensitivity. Electrical stimulation with a train of 150 shocks (10 V, 2 ms) of the proximal nerve stump immediately after transection prevented the c-fos hyperinducibility. The effect was greater with the stimulation frequency of 0.5 Hz than 0.1 Hz or 10 Hz. The stimulation had no effect on the c-fos inducibility at 2 days postinjury. © 1997 Elsevier Science Ireland Ltd. Keywords: c-Fos; Dorsal horn; Electrical stimulation; Hypersensitivity; Peripheral nerve injury; Windup
1. Introduction Peripheral nerve injuries in human cases often result in neuropathic pain. Peripherally axotomized primary neurons are known to exhibit spontaneous electrical activity, and become sensitive to mechanical and/or chemical stimuli applied to the neuroma (Wall and Gutnick, 1974; Govrin-Lippman and Devor, 1978; Korenman and Devor, 1981; Scadding, 1981). Neuropathic pain appears to depend on such pathophysiologic excitation of injured primary neurons because resection or anesthetic infiltration of painful neuromata usually brings about temporary pain relief. It is also likely, * Corresponding author. Tel.: +81 86 235 6635; fax: + 81 86 223 7053.
however, that changes in the dorsal horn neurons postsynaptic to the injured primary neurons at least partially contribute to the neuropathic pain. It has been demonstrated that superficial dorsal horn neurons chronically deafferented by peripheral nerve transection lost their original receptive field, and acquired a novel receptive field (Devor and Wall, 1978, 1981; Lisney, 1983; Markus et al., 1984; Hylden et al., 1987). Such receptive field reorganization can be demonstrated not only by an electrophysiological recording but also by a histochemical method. Dorsal horn neurons express a proto-oncogene c-fos in response to noxious stimulation of their receptive field (Hunt et al., 1984; Mene´trey et al., 1989; Bullitt, 1990; Williams et al., 1990). When a peripheral nerve was transected, the neurons in the deafferented part of the
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dorsal horn lost their receptive field (Williams et al., 1991; Sugimoto et al., 1994). Within a few weeks after the nerve injury, however, the dorsal horn neurons disconnected from their original receptive fields began to respond by c-fos expression to noxious stimulation of a skin area outside the original receptive fields (Sugimoto et al., 1993, 1994). Therefore, the chronically deafferented dorsal horn neurons exhibited hypersensitivity to somatotopically inappropriate primary nociceptive input. It is a tempting idea that such hypersensitivity is related to neuropathic pain, but evidence correlating the hypersensitivity and pain is lacking. Clinical as well as experimental studies indicate that afferent barrage immediately before or after the nerve transection increased the degree of neuropathic pain or pain-related behavior. For example, preoperative epidural lumbar blockade lowered the incidence of phantom limb pain in human amputees (Bach et al., 1988). Autotomy, a behavioral manifestation of deafferentation pain in rats, following sciatic neurotomy was suppressed by blockade of injury discharge and augmented by electrical stimulation of the nerve (Seltzer et al., 1991). Because injury discharge, under the influence of systemically administered strychnine, caused transsynaptic degeneration of dorsal horn neurons following neurotomy (Sugimoto et al., 1987), we predict that injury discharge or electrical nerve stimulation at the time of nerve injury would induce transneuronal changes that are responsible for neuropathic pain. In this study, therefore, we proceed to examine the chronic transsynaptic effect of electrical stimulation immediately after transection of the tibial nerve. At 3 weeks after injury, we deliver noxious stimulus to the hindpaw skin and evaluate the spatial pattern of c-fos induction in the partially deafferented segment of the spinal cord laminae I and II. The noxious signal is transmitted to the spinal cord through the spared peroneal nerve.
2. Materials and methods Thirty-five male rats of the Sprague-Dawley strain (b.w.=200 g) were used. All the surgical procedures were performed under anesthesia by i.p. injection with a mixture of pentobarbital sodium (20 mg/kg) and ethylcarbamate (650 mg/kg). About 15 mm of a segment of the sciatic nerve was exposed between the mid-thigh level and the popliteal fossa by skin incision and blunt dissection through the biceps femoris muscle. In the proximal part of this segment, the three major divisions of the nerve (the tibial, sural and peroneal nerves) were clearly separated by individual perineurium. The distal end of the exposed segment of tibial nerve was tightly ligated with a 7-0 silk suture thread and transected
distally (chronic injury). The proximal stump of the nerve was placed on a pair of stainless steel hook electrodes (distance: 500 mm). Electrical stimulation consisting of 150 pulses (square pulses of 10 V, 2 ms) was delivered at 0.1 Hz (25 min), 0.5 Hz (5 min) or 10 Hz (15 s). One group of rats received a sham procedure in which the nerve stump was placed on the electrode for 5 min without stimulation. The nerve was returned to the original position, and the skin incision was suture-closed with a 7-0 chromic catgut. Nineteen days later, the rats were re-anesthetized and an identical transection (acute injury) was performed on the other side but electrical stimulation was omitted. Forty-eight hours after the acute injury, bilateral noxious stimulation was delivered to the hindpaw of re-anesthetized rats by four subcutaneous injections with 5% formalin (30 ml each). The tip of the hypodermic needle was located at the center of proximal and distal halves of plantar and dorsal surfaces of the hindpaw. Two hours later, anesthesia was supplemented by ether inhalation and the rats were exsanguinated and fixed by perfusion with 50 ml saline followed by 500 ml of 0.1 M sodium phosphate-buffered 4% formaldehyde (prepared fresh from paraformaldehyde, pH 7.4). Another group of rats underwent bilateral tibial nerve transection at the same time, and electrical stimulation at 0.5 Hz (5 min) was delivered only on one side. Forty-eight hours later these rats were formalin-stimulated bilaterally, and perfusion-fixed as described above. In addition, some rats received unilateral tibial neurotomy and electrical stimulation at 0.5 Hz. They survived for either 2 h, 1 day, 2 days or 3 weeks before fixation but the formalin-stimulation and contralateral injury were omitted. The spinal cord within 9 1 mm of the junction of 4th and 5th lumbar segments (L4 and L5) was dissected out. Frontal sections were cut with a freezing microtome at 50 mm in thickness and processed for histochemical demonstration of c-Fos protein-like immunoreactivity (Fos-LI). After thorough rinsing with phosphate buffered saline (PBS), they were processed for histochemical demonstration of Fos-LI using a PAP method (see Sugimoto et al., 1993 for detail). Antibodies used and the dilution were as follows; rabbit anti c-Fos protein IgG (0.2 mg protein/ml; Oncogene Science, Manhasset, NY), goat anti-rabbit IgG (1:300; Cappel, West Chester, PA), and PAP complex (1:3000; Cappel). Sections were mounted on gelatin-coated glass slides, air-dried, dehydrated in alcohols and coverslipped with Entellan (Merck). Five sections were randomly selected for each rat. Using an Olympus photomicroscope equipped with 10× objective and a camera lucida drawing tube, an outline of the dorsal horn was traced on a white paper and neurons with Fos-LI (fos-neurons) were plotted. Dark field illumination was used for drawing the out-
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Fig. 1. Medial 3/4 of the dorsal horn at the L4/5 junction. Sections (50 mm-thick) were stained for c-Fos protein-like immunoreactivity but not counterstained. Because of the section thickness, many of fos-neurons are out of focus. The number of fos-neurons can be best counted on camera lucida drawings like those shown in Fig. 2. The ipsilateral tibial nerve was cut and electrically stimulated at 0.5 Hz 2h before perfusion fixation but the formalin stimulation was omitted (A). The nerve was cut and sham-stimulated 3 weeks before formalin-stimulation (B). The nerve was cut and stimulated at 0.5 Hz 3 weeks before formalin-stimulation (C). The nerve was cut 2 days before formalin-stimulation but electrical stimulation of the nerve was omitted (D). Fig. 1(d) is the contralateral side of the section shown in Fig. 1(c), and has been reverse-printed for easier comparison. Medial to the right. Scale bar; 100 mm.
line and laminar borders of the dorsal horn, while fos-neurons were identified using bright field illumination. Objective lenses, 20 × and 40 × , were used for identification of fos-neurons when necessary. The average number per section of fos-neurons in each spinal cord area (see below) was recorded for each rat. Our quantification dealt only with the number and location of neurons with histologically detectable Fos-LI, and the staining intensity of positive cells was ignored. The analysis of our results is based on a previous observation that the dorsal horn around the L4/L5 junction receives the densest primary projection from the sciatic nerve (Swett and Woolf, 1985; LaMotte et al., 1989). In laminae I and II, the tibial primary neurons that innervate the plantar surface of the hindpaw project to the medial most 3/8, whereas the lateral 1/4 is innervated by primary neurons supplying the posterior cutaneous nerve of the thigh. The area between them receives primary projection from the superficial peroneal and sural nerves that innervate the dorsal and lateral aspects of the hindpaw (Swett and Woolf, 1985). For stating the location of neurons with Fos-LI, the medialmost 3/8 of laminae I/II will be referred to as the tibial territory and the lateral 5/8 as the P and H (peroneal and hip) territory, respectively. The rest of the spinal cord gray matter will be divided into three areas; laminae III/IV, laminae V/VI, and laminae VII-X. The laminar border between IV and V was settled by medially extending the dorsal horizontal tangent of the reticulated part of the dorsal horn neck.
The border between laminae VI and VII was settled by medially extending the ventral horizontal tangent of the reticulated part of the neck.
3. Results In all the rats examined in this study, fos-neurons were most frequent in the P and H territory of laminae I/II. Laminae VIII and IX were almost always devoid of fos-neurons. The number of formalin-induced fosneurons in the tibial territory greatly varied depending on the postinjury interval (chronic or acute) and the stimulation frequency. For example, fos-neurons significantly increased following chronic injury (without electrical stimulation) compared to acute injury (see below). On the other hand, difference in number of fos-neurons in deeper laminae was rather obscure between chronic and acute injury sides regardless of the electrical stimulation. In this study, therefore, we quantify c-fos induction in laminae I/II of the dorsal horn. Immunohistochemically demonstrable Fos-LI in these neurons are considered to have been induced by formalin-stimulation because c-fos induction by electrical stimulation was transient and almost completely disappeared within 24 h after stimulation. At 2 h after tibial nerve transection and electrical stimulation at 0.5 Hz, many fos-neurons were observed in the ipsilateral tibial territory of the dorsal horn laminae I/II (n =2) (Fig. 1(a)). At 1 day (n= 2), 2 days (n= 2) or 3 weeks (n=3)
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after such preparation, however, only few fos-neurons (less than one per section) were found in any area of the examined dorsal horn if formalin stimulation was omitted. Following chronic injury with sham-stimulation (no electrical stimulation), formalin-stimulation 2 h prior to perfusion fixation produced 19.7 fos-neurons per section of the ipsilateral tibial territory (n = 5) (Fig. 1(b)). This is about 4-fold increase over the contralateral side (4.8 fos-neurons per section) on which the tibial nerve had been acutely transected 2 days before formalin-
Fig. 2. Camera lucida drawings of dorsal horn sections through the L4/5 junction. The tibial nerve on the experimental side had been transected and sham-stimulated (A) or stimulated at 0.5 Hz (B) 3 weeks before formalin-stimulation, while the same nerve on the control side had been acutely transected 2 days before formalin-stimulation but electrical stimulation at the time of injury was omitted. The rat shown in (C) received bilateral acute tibial neurotomy 2 days before formalin-stimulation, and electrical stimulation (0.5 Hz) was delivered only on the experimental side. Many fos-neurons can be seen in the medial 3/8 of laminae I/II on the experimental side of (A) indicating chronic injury induced c-fos hyperinducibility. Electrical stimulation at 0.5 Hz has suppressed the hyperinducibility (B). Electrical stimulation at 0.5 Hz has no effect on the c-fos induction in normosensitive neurons responding to formalin-stimulation at 2 days postinjury (C).
stimulation (Fig. 2(a), Table 1). The difference between sides was significant at the 1% level (t-test for paired samples). A tendency of chronic increase was also noted in the P and H territory, where 41.1 and 34.7 fos-neurons were counted on the chronic and acute sides, respectively. However, the difference between sides was not statistically significant. Although the average number per section of fos-neurons in deeper laminae was always greater on the chronic side than on the acute side, the difference was small. These results are consistent with a previous report that chronic tibial nerve transection induced more prominent c-fos hyperinducibility in the deafferented tibial territory than in the rest of the spinal cord areas (Sugimoto et al., 1994). Following chronic injury combined with electrical stimulation at 0.5 Hz, the number per section of fosneurons in the ipsilateral tibial territory was 5.2 (n =5). This number was similar to that on the contralateral side with the acute tibial neurotomy (5.5 fos-neurons, Fig. 1(c,d), Fig. 2(b), Table 1). The number of induced fos-neurons in the P and H territory was smaller on the chronic side (23.0) than on the acute side (34.9) though the difference was not statistically significant. The numbers of fos-neurons in both the tibial and P and H territories on the acute side of rats with 0.5 Hz stimulation were comparable with those of sham-stimulated rats. The difference in number of fos-neurons in similar territories on the chronic side was significant between the stimulated and sham-stimulated rats (PB0.01 for tibial and P B 0.05 for the P and H territories, ANOVA). Therefore, the electrical stimulation at 0.5 Hz significantly suppressed the c-fos hyperinducibility following chronic injury in laminae I/II (Fig. 3). The effect of electrical stimulation on c-fos induction in deeper laminae was obscure. Electrical stimulation at 0.1 (n = 6) and 10 Hz (n=5) also suppressed the chronic c-fos hyperinducibility but not so effectively as that at 0.5 Hz did (Fig. 3). In the tibial territory on the chronic side, 12.8 and 10.3 fosneurons were observed following stimulation at 0.1 and 10 Hz, respectively. Because 6.7 (0.1 Hz) and 6.4 (10 Hz) fos-neurons were observed on the acute side, the chronic side showed about 2-fold increase (Table 1). A t-test for paired samples revealed the difference between side to be significant only for the group stimulated at 10 Hz (P B 0.05). Difference between sides was small for the P and H territory of both groups. Because unilateral noxious stimulation has been shown to enhance c-fos induction in contralateral dorsal horn neurons in response to the second contralateral noxious stimulation delivered within 20 h (Leah et al., 1992), relative increase in number of fos-neurons on the acute side of electrically stimulated rats might have resulted in seeming suppression of c-fos hyperinducibility on the stimulated side. In rats with bilateral acute injury combined with unilateral electrical stimulation at
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Table 1 Mean numbers of fos-neurons per section on chronic and acute injury sides Stimulation frequency
Sample size
Sham
5
0.1 Hz
6
0.5 Hz
5
10 Hz
5
Side
Chronic Acute Chronic Acute Chronic Acute Chronic Acute
Mean (S.E.) of number of fos-neurons per section Tibial (I/II) P and H (I/II) III/IV
V/VI
19.7 4.8 12.8 6.7 5.2 5.5 10.3 6.4
15.6 11.9 11.8 12.2 10.4 15.4 17.0 18.5
(2.0)** (1.1) (5.9) (2.5) (1.4) (2.6) (2.3)* (1.8)
41.1 34.7 28.0 25.1 23.0 34.9 32.8 38.8
(2.6) (4.6) (4.5) (3.4) (4.3) (1.9) (3.9) (5.2)
9.0 7.8 11.5 10.8 5.6 6.8 17.1 17.6
(1.3) (1.2) (3.0) (3.0) (1.7) (1.0) (3.0) (2.9)
VII(4.4) (1.8) (3.6) (2.0) (1.4) (1.8) (2.7) (1.7)
5.9 4.4 2.7 3.4 4.2 9.4 4.3 5.9
(1.8) (1.0) (0.8) (0.7) (0.9) (2.4) (1.1) (1.7)
Numbers in parentheses indicate standard errors. Difference between sides was significant at the *5% or **1% level (t-test for paired samples).
0.5 Hz, however, c-fos induction by bilateral formalinstimulation 2 h prior to perfusion fixation was symmetrical (Fig. 2(c), Table 2). Furthermore, the number of fos-neurons on the acute side of rats, whose chronically injured tibial nerve on the contralateral side had been stimulated at 0.5 Hz, was similar to that on the acute side of sham-stimulated rats (Table 1). Therefore, contralateral effect of electrical stimulation, if any, appeared to have subsided within 2 days of electrical stimulation.
Fig. 3. Effect of electrical stimulation on the number of formalin-induced fos-neurons in the tibial and P and H territories at 3 weeks postinjury. Stimulation at 0.5 Hz significantly reduced the number of fos-neurons in both territories compared to sham-stimulation; *P B 0.05, and **P B 0.01 (ANOVA). Stimulation at 0.1 Hz and 10 Hz also showed a tendency of reduction, though the difference was not significant. The numbers of rats examined were 5 (sham-stimulation), 6 (0.1 Hz), 5 (0.5 Hz) and 5 (10 Hz).
4. Discussion We have reported that fos-neurons produced by formalin-stimulation of the whole hindpaw were limited in the peroneal territory and those in the tibial territory were rare during the first postinjury week following tibial neurotomy (Sugimoto et al., 1993, 1994). This topographic distribution and magnitude of c-fos induction are considered to be the baseline c-fos induction by selective stimulation of the peroneal primary neurons. In a chronic situation where the tibial nerve had been transected and prevented from regeneration for 2 weeks or longer before the formalin-stimulation, however, the number of induced fos-neurons significantly increased in the tibial territory and the medial part of the peroneal territory (Sugimoto et al., 1993, 1994). Such increase in number of fos-neurons is considered to reflect an increased sensitivity of dorsal horn neurons to spared peroneal primary input. The present study reproduced these results indicating that dorsal horn neurons chronically deafferented by peripheral nerve injury exhibited ectopic sensitivity to c-fos induction by somatotopically inappropriate noxious primary input. Because fos-neurons in the P and H territory (the peroneal and hip territories combined) were counted altogether, statistical significance could not be detected but there was a tendency of increase in number of fos-neurons in this territory. In this study, we used intense electrical stimulation (rectangular pulses at 10 V and 2 ms) to activate as many primary C-fibers in the proximal stump of the transected tibial nerve as possible. Indeed, such stimulation delivered at 0.5 Hz for 5 min induced Fos-LI in many laminae I/II neurons within 2 h. c-fos Expression induced by the electrical stimulation was transient and the Fos-LI disappeared by 24 h of stimulation. Thereafter, Fos-LI in the dorsal horn was almost negligible unless the hindpaw was stimulated by formalin injection. At 2 days after bilateral injury followed by unilateral electrical stimulation at 0.5 Hz, the number and topo-
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Table 2 Mean numbers of fos-neurons per section at 2 days after bilateral tibial nerve transection Sample size
5
Side
Stimulated Unstimulated
Mean (S.E.) of number of fos-neurons per section Tibial (I/II)
P and H (I/II)
III/IV
V/VI
VII-
5.0 (1.1) 4.7 (0.7)
26.7 (5.9) 28.7 (3.4)
8.8 (2.6) 11.0 (2.0)
12.9 (4.4) 14.0 (2.0)
5.6 (1.3) 6.9 (1.9)
The proximal stump on one side was stimilated at 0.5 Hz. Numbers in parentheses indicate S.E.
graphic distribution of fos-neurons induced by formalin-stimulation was similar on both sides. This would indicate that the electrical stimulation of the transected nerve did not affect the baseline c-fos induction by activation of the spared peroneal primaries. Following chronic injury combined with a similar stimulation, the pattern of c-fos induction remained unchanged from the baseline. Therefore, the electrical stimulation prevented the chronic injury-induced hyperinducibility of c-fos. The effects of electrical stimulation at 0.1 and 10 Hz were similar to but less prominent than that at 0.5 Hz. Response of primary and dorsal horn neurons to the peripheral nerve stimulation at C-fiber intensity has been shown to vary depending on the stimulation frequency. According to Seltzer et al., C-fibers faithfully followed every shock in the 150-pulse train delivered at 0.1 and 0.5 Hz; while, at 10 Hz, the conduction time gradually increased and most C-fibers failed to follow every shock in the train (Seltzer et al., 1991). On the other hand, dorsal horn neurons are known to windup their response to primary C-fiber input when stimulated at a frequency greater than 0.3 Hz (Mendell and Wall, 1965, Price, 1972). Therefore, the prevention of chronic injury-induced hyperinducibility of c-fos observed in this study appears to involve dorsal horn neurons’ windup phenomenon. We previously demonstrated that afferent barrage produced at the time of peripheral nerve injury had potentially neurotoxic effect on the postsynaptic dorsal horn neurons and, in synergy with the systemically administered convulsant, caused degeneration of small dorsal horn neurons (Sugimoto et al., 1987). Although the electrical stimulation in the absence of convulsants may not cause morphologically discernible transsynaptic degeneration, it might have caused prolonged transsynaptic changes that were responsible for lowered c-fos inducibility. If this was the case, the tibial nerve stimulation would have reduced the number of neurons responding by c-fos expression to the tibial primary input, while neurons that respond only to the peroneal primary input would not be affected. This would explain why electrical stimulation affected the hyperinducibility but not baseline inducibility of c-fos in this study. Whereas dorsal horn neurons originally responding to the peroneal but not tibial primary input are not
damaged by the electrical stimulation and show baseline c-fos expression, those originally responding to the tibial but not peroneal primary input are damaged by the electrical stimulation and fail to develop hyperinducibility. What is the clinical implication of injury-induced c-fos hyperinducibility? The fact that nociceptive neurons in the deafferented territory of dorsal horn become sensitive to somatotopically inappropriate noxious primary input may indicate that stimulation of receptive field outside the denervated skin area causes ectopic excitation of deafferented dorsal horn neurons. Transmittal of such anomalous nociceptive signal along the neuroaxis may result in pain disorders such as phantom limb pain. However, c-fos induction in intact animals by noxious peripheral stimulation is largely limited to interneurons (Mene´trey et al., 1989; Tavares et al., 1993). In addition, downstream of c-fos expression is preprodynorphine-mRNA transcription (Naranjo et al., 1991; Noguchi et al., 1991). Therefore anomalous c-fos induction, by itself, may not be interpreted as a sign of neuropathic pain. Phantom sensation might result if deafferented projection neurons exhibited increase in responsiveness to somatotopically inappropriate primary input. Even if this was the case, a concomitant increase in responsiveness of inhibitory interneurons may counteract the signal transmittal. Suppression by electrical stimulation of injury-induced c-fos hyperinducibility, on the other hand, may underlie some neuropathic pain state. Because the Sprague-Dawley rat used in this study was autotomyinsensitive and because the escape behavior was impaired by the paralysis of hamstring muscles, pain-related behavior was not tested. However, electrical stimulation of the sciatic nerve at a C-fiber intensity and windup frequency immediately before the nerve transection augmented the autotomy that was seen in the Sabra strain rats (Seltzer et al., 1991). Temporary pain relief by epidural block preceding limb amputation was effective in prevention of phantom limb pain in human cases (Bach et al., 1988). Assuming that the interneurons are more sensitive than the projection neurons to transsynaptic damage (Sugimoto et al., 1990), the experimentally produced C-afferent barrage or sustained pain of the peripheral origin in human cases would chronically disinhibit the activity of projec-
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tion neurons. Therefore, pathophysiological activity of injured primary neurons and/or of deafferented dorsal horn projection neurons in response to spared primary afferent input would ascend the neuroaxis to cause neuropathic pain sensation.
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