MCS EJP.pdf

European Journal of Pain xxx (2010) xxx–xxx Contents lists available at ScienceDirect European Journal of Pain journal homepage: www.EuropeanJournalPain.com Transdural motor cortex stimulation reverses neuropathic pain in rats: A profile of neuronal activation Rosana L. Pagano a,b,⇑, Danielle V. Assis a,b, Joseph A. Clara a,c, Adilson S. Alves a, Camila S. Dale b, Manoel J. Teixeira b,d, Erich T. Fonoff b,d, Luiz R. Britto a a Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil Laboratory of Neuromodulation and Experimental Pain, Hospital Sírio Libanês, São Paulo, Brazil Minority Health International Research and Training Program, Christian Brothers University, Memphis, Tennessee, United States d Division of Functional Neurosurgery, Department of Neurology, University of São Paulo Medical School, São Paulo, Brazil b c a r t i c l e i n f o Article history: Received 15 October 2009 Received in revised form 19 July 2010 Accepted 6 August 2010 Available online xxxx Keywords: Motor cortex stimulation Neuropathic pain Fos Zif268 Rats a b s t r a c t Motor cortex stimulation (MCS) has been used to treat patients with neuropathic pain resistant to other therapeutic approaches; however, the mechanisms of pain control by MCS are still not clearly understood. We have demonstrated that MCS increases the nociceptive threshold of naive conscious rats, with opioid participation. In the present study, the effect of transdural MCS on neuropathic pain in rats subjected to chronic constriction injury of the sciatic nerve was investigated. In addition, the pattern of neuronal activation, evaluated by Fos and Zif268 immunolabel, was performed in the spinal cord and brain sites associated with the modulation of persistent pain. MCS reversed the mechanical hyperalgesia and allodynia induced by peripheral neuropathy. After stimulation, Fos immunoreactivity (Fos-IR) decreased in the dorsal horn of the spinal cord and in the ventral posterior lateral and medial nuclei of the thalamus, when compared to animals with neuropathic pain. Furthermore, the MCS increased the Fos-IR in the periaqueductal gray, the anterior cingulate cortex and the central and basolateral amygdaloid nuclei. Zif268 results were similar to those obtained for Fos, although no changes were observed for Zif268 in the anterior cingulate cortex and the central amygdaloid nucleus after MCS. The present findings suggest that MCS reverts neuropathic pain phenomena in rats, mimicking the effect observed in humans, through activation of the limbic and descending pain inhibitory systems. Further investigation of the mechanisms involved in this effect may contribute to the improvement of the clinical treatment of persistent pain. Ó 2010 European Federation of International Association for the Study of Pain Chapters. Published by Elsevier Ltd. All rights reserved. Cite this article as: Rosana L. Pagano, Danielle V. Assis, Joseph A. Clara, Adilson S. Alves, Camila S. Dale, Manoel J. Teixeira, Erich T. Fonoff, Luiz R. Britto, Transdural motor cortex stimulation reverses neuropathic pain in rats: A profile of neuronal activation. Eur J Pain xxx (2010) xxx–xxx [doi:10.1016/ j.ejpain.2010.08.003] Ò Full text available online on ScienceDirect , www.sciencedirect.com. 1. Introduction Neuropathic pain, defined as a chronic or persistent pain resulting from an injury or dysfunction of nervous system, includes clinical symptoms of hyperalgesia, allodynia and spontaneous ongoing pain (Dworkin et al., 2003). A variety of therapeutic approaches, including opioid analgesics, tricyclic antidepressants, anticonvulsants, and local anesthetics have been used to treat neuropathic ⇑ Corresponding author at: Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil. Tel.: +55 11 30917242; fax: +55 11 30917426. E-mail address: ropagano@yahoo.com (R.L. Pagano). pain, without, however, complete effectiveness (Caviedes and Herranz, 2002; Dworkin et al., 2003). Transdural motor cortex stimulation (MCS) has been increasingly used to treat chronic pain unresponsive to current pharmacological approaches. It was initially used to treat post-stroke thalamic pain (Tsubokawa et al., 1991), and subsequently, with encouraging results, in the management of patients with neuropathic pain syndromes (Nguyen et al., 2000; Canavero and Bonicalzi, 2002; Nuti et al., 2005; Rasche et al., 2006). Subdural MCS inhibits the activity of neurons in the dorsal horn of the spinal cord (Senapati et al., 2005a), and central and peripheral neuropathic pain in rats (Rusina et al., 2005; Vaculin et al., 2008). It was 1090-3801/$36.00 Ó 2010 European Federation of International Association for the Study of Pain Chapters. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ejpain.2010.08.003 Please cite this article in press as: Pagano RL et al. Transdural motor cortex stimulation reverses neuropathic pain in rats: A profile of neuronal activation. Eur J Pain (2010), doi:10.1016/j.ejpain.2010.08.003 2 R.L. Pagano et al. / European Journal of Pain xxx (2010) xxx–xxx recently shown that subdural MCS induces spinal antinociception in rats with neuropathic pain, activating neurons into the locus coeruleus; however, the contribution of coeruleospinal noradrenergic pathways may not have a critical role in that MCS effect (Viisanen and Pertovaara, 2010a). Moreover, the rostroventromedial medulla and the descending serotoninergic pathway both contribute to MCS-induced antinociception, by acting on spinal 5-HT1A receptors (Viisanen and Pertovaara, 2010b). In an attempt to mimic the less invasive clinical protocol used in humans, we recently showed that transdural MCS increases the nociceptive threshold of naive rats, by acting on opioid system (Fonoff et al., 2009a). However, the neural mechanisms of MCS remain elusive. The expression of the inducible transcription factors (ITFs) zif268 (krox-24, egr-1s or zenk), c-fos and c-jun has been extensively used to map neuronal activation involved in the antinociceptive process (Herrera and Robertson, 1996; Beckmann and Wilce, 1997; Herdegen and Leah, 1998). The ITFs are induced in neurons in response to extracellular stimuli, including depolarization, neurotransmitters, and growth factors (Sheng and Greenberg, 1990). Despite the fact that Zif268 and Fos proteins share some common activation pathways, there are differences in their inducibility. The expression of zif268 is linked to ongoing synaptic activity, whereas c-fos expression is reliant upon activity, being triggered after a period of neural quiescence or after exposure to a novel stimulus (Chaudhuri, 1997). Nevertheless, the functional differences between ITFs remain largely unknown. An increased expression of Fos and Zif268 proteins has been demonstrated in different areas of the brain and the spinal cord after peripheral nociceptive stimulation (Herdegen et al., 1991; Lanteri-Minet et al., 1993; Harris, 1998). In addition, Gogas et al. (1991) suggested a direct relationship between nociception and Fos expression, since opioid-induced antinociception occurs concomitantly with Fos inhibition in the spinal cord. The present study was undertaken to investigate the effects of transdural MCS on experimental neuropathic pain in rats and to evaluate the neuronal activation profile of the structures implicated in nociceptive processing by using Fos and Zif268 detection. 2. Methods 2.1. General procedure Animals were evaluated in the nociceptive tests and subsequently, under anesthesia, peripheral neuropathy or sham operation was performed in the right hind limb of the anaesthetized rats (day 0). After 1 week, transdural electrodes were implanted over the left motor cortex involving the functional area of right hind limb (day 7). After an additional week, the nociceptive tests were again performed in awake animals (day 14). A group of rats with peripheral neuropathy was submitted to 15 min of MCS and, at the end of this period, still under stimulation, they were again evaluated in the nociceptive tests. After 1 h of the last nociceptive determination, animals were anesthetized and then subjected to immunohistochemistry assays. 2.2. Animals Male Wistar rats (180–200 g) were used throughout this study. Animals were maintained in a room with controlled temperature (22 ± 2 °C) and light/dark cycle (12/12 h), with free access to water and food. All procedures were in accordance with the guidelines for the ethical use of conscious animals in pain research published by International Association for the Study of Pain (Zimmermann, 1983) and were approved by the Ethics Committee on the Use of Animals at Hospital Sírio-Libanês (CEUA, protocol number 2007/05). 2.3. Induction of neuropathic pain Rats were anesthetized with halothane (2.5%) and subjected to chronic constriction injury (CCI) of the sciatic nerve according to the method of Bennett and Xie (1988). In the procedure, the sciatic nerve of the right paw was exposed at the middle of the thigh by blunt dissection through the biceps femoris. Proximal to the sciatic nerve’s trifurcation (about 7 mm), the nerve was freed of adhering tissue and four ligatures (4.0 chromic gut) were tied loosely around it with about 1 mm spacing. Great care was taken to tie the ligatures, so that the diameter of the nerve was seen to be just barely constricted. The incisions were sutured in layers using silk suture wire (4–0). The sham-operated rats were subjected to exposure of the sciatic nerve without nerve compression. 2.4. Procedure for implanting electrodes One week after the induction of peripheral neuropathy, rats were deeply anesthetized with ketamine hydrochloride (0.5 mg/ kg i.m.)/xylazine (2.3 mg/kg i.m.), as well as local scalp injections of 2% lidocaine (100 ll/animal). Whenever necessary, supplementary doses of ketamine hydrochloride were administered to the animals. Under stereotaxic conditions, a pair of transdural stainless steel electrodes (cylinders of 0.8 mm in diameter) was placed, through two small drilled holes, on the left motor cortex (over the functional area of hind limb – 1.0 mm rostral and 1.5 mm caudal to the bregma, and 1.5 mm lateral to the midline). The site of electrode implantation for MCS was chosen based on the functional map obtained previously by our group (Fonoff et al., 2009b). The integrity of the dura mater was an absolute condition for the placement of electrodes. A fixation screw was also placed 4–6 mm far from the site of stimulation. Acrylic polymer was used to seal the surface of the skull and to ensure electrical isolation. The contacts of each electrode pole were inserted into a connector, which was also fixed to the whole ensemble. During stimulation sessions, a cable was plugged to the connector. All implanted rats were allowed to recover for 1 week before testing. 2.5. Electrical MCS The electrical stimulation was applied according to the parameters described earlier, which could elicit changes in pain threshold without interfering with general or motor activities (Fonoff et al., 2009a). One week after surgical implantation, electrical stimulation was delivered in a single session of 15 min (amplitude 1.0 V; frequency 60 Hz; pulse duration 210 ls – Medtronic electrical stimulatorÒ). The cathode (negative pole) was always chosen to be the posterior contact of the electrode, in order to match with a greater surface area corresponding to the hind limb, according to the functional map (Fonoff et al., 2009b). Rats submitted to the surgical procedure, but not electrically stimulated, were evaluated as controls. 2.6. Determination of nociceptive response Nociceptive tests were carried out before the CCI (initial measure) and on the 14th day following the CCI (final measure). On the day of the final measure, rats were tested before and after 15 min of MCS, still under stimulation. The results were analyzed by comparing the initial and final measures. Testing was blind in regard to group designation. 2.6.1. Evaluation of mechanical hyperalgesia The rat paw pressure test (Randall and Selitto, 1957), which employs the use of a pressure apparatus (InsightÒ, São Paulo, Brazil), was used to determine hyperalgesia. In brief, a force with increas- Please cite this article in press as: Pagano RL et al. Transdural motor cortex stimulation reverses neuropathic pain in rats: A profile of neuronal activation. Eur J Pain (2010), doi:10.1016/j.ejpain.2010.08.003 R.L. Pagano et al. / European Journal of Pain xxx (2010) xxx–xxx ing magnitude (16 g/s) was applied to the right hind paw. The force (in grams) required to induce a withdrawal response represented the nociceptive threshold. To reduce stress, the rats were habituated to the testing procedure the day preceding the experiment. 3 10 objective for the DHSC (680,800 lm2) and a 6.3 objective for the PAG (671,240 lm2), VPL/VPM (935,000 lm2), CCA (441,280 lm2), CeA (72,900 lm2) and BLA (72,900 lm2). 2.8. Statistical analysis 2.6.2. Evaluation of mechanical allodynia Testing for mechanical allodynia was carried out in accordance with Milligan et al. (2000). The rats were placed individually in plastic cages with a wire bottom, which allowed access to their paws. To reduce stress, rats were habituated to the experimental environment on each of the 2 days preceding testing. On the day of the test, the animals were placed in the cages 15 min before the beginning of each measurement. In brief, mechanical allodynia was evaluated by application of von Frey hairs (VFH, Semmes– Weinstein monofilaments, StoeltingÒ, Wood Dale, IL, USA), in increasing order of force (from 407 mg to 15.136 g), to the planter surface of the right hind paw. Baseline assessment was initiated with the 2.041 g hair. In the occurrence of withdrawal of the paw from this hair, a smaller caliber hair was used. In the absence of a response, the application of the VFH was carried out in increasing fashion, until a response was elicited. The hair that induced an animal’s paw withdrawal was reapplied after 60 s. When the same monofilament is able to induce two consecutive responses, the stiffness necessary to induce a paw withdrawal response is considered to have been reached. The smallest amount of force required to elicit a response was recorded as the paw withdrawal threshold, in grams. 2.7. Immunohistochemistry After 1 h of the last nociceptive evaluation, rats were deeply anesthetized with ketamine hydrochloride and xylazine and then subjected to transcardiac perfusion, with saline solution, followed by a fixative solution composed of 4% paraformaldehyde (PFA) dissolved in 0.1 M phosphate buffer (PB, pH 7.4). Animals were submitted to perfusion 1 h after the last nociceptive stimulus since the expression of ITF proteins in general peaks around 1 h after the stimulus and fades by 3–4 h (Herdegen and Leah, 1998). The spinal cord and brain were collected and stored in PFA for 4 h, followed by cryoprotection in 30% sucrose solution in PB, overnight at 4 °C, then sectioned at 30 lm on a freezing microtome. The sections were washed in PB and incubated for 12–16 h with rabbit anti-Fos (1:2000; Ab-5; Calbiochem, CA/USA) or anti-Zif268 (anti-Egr-1; 1:1000; C-19; Santa Cruz Biotech., CA) primary antibodies diluted in 0.3% of Triton X-100, containing 50 lL normal donkey serum. Following three washes of 10 min each with PB, sections were incubated for 2 h in the biotinylated secondary antibody (donkey anti-rabbit IgG, Jackson ImmunoResearch, PA/USA, 1:200), then in avidin–biotin complex (1:100; ABC Elite kit, Vector Labs, Burlingame, CA), and visualized using a mixture of 0.05% diaminobenzidine–0.01% hydrogen peroxide. Sections were then washed in PB, mounted on slides with a glycerol-based mounting medium, air-dried, dehydrated through graded ethanol solutions followed by xylene, and then coverslipped with Permount (Fisher Scientific/USA). The immunoreactivity was analyzed using a light microscope and the Image analysis system (NIH/USA). A quantitative analysis was performed on the density of nuclei representative of the immunoreactivity for Fos (Fos-IR) or Zif268 (Zif268-IR) in the dorsal horn of the spinal cord (DHSC; laminae I–VI of the L4–L5 dorsal horn), rostral portion of the midbrain periaqueductal gray (PAG; dorsomedial, dorsolateral, lateral and ventrolateral columns), ventral posterior lateral (VPL) and medial (VPM) nuclei of the thalamus, anterior cingulate cortex (ACC) and central (CeA) and basolateral (BLA) nuclei of the amygdala. Measurements were taken from 10 different sections for each animal analyzed, including areas that were defined for each structure by using a Results are presented as the mean ± standard error of the mean (SEM). Statistical analyses of data were generated using GraphPad Prism, version 4.02 (GraphPad Software Inc., San Diego, CA, USA). Statistical comparison of more than two groups was performed using analysis of variance (ANOVA), followed by Bonferroni’s test. In all cases, p 6 0.05 was considered statistically significant. 3. Results 3.1. Nociceptive response The chronic constriction injury of the sciatic nerve caused a significant decrease of the pain threshold, inducing mechanical hyperalgesia (Fig. 1B), and lowered withdrawal thresholds, inducing mechanical allodynia (Fig. 1D) in the right hind paw. Mechanical hyperalgesia and low-threshold mechanical allodynia were observed on day 14 after surgery. The contralateral, intact paw (left paw) did not show alterations of pain threshold (Fig. 1A and C). Sham-operated rats did not present alterations in threshold measurements, as compared to basal values (Fig. 1). MCS was able to totally revert the hyperalgesic response observed in animals with neuropathic pain, compared to the initial measurements of the animals before the chronic constriction of the sciatic nerve (Fig. 1B). Likewise, the phenomenon of allodynia induced by peripheral neuropathy was reverted after cortical stimulation (Fig. 1D). 3.2. Neuronal activation The immunohistochemical tests were performed on the sections obtained from animals which had previously been evaluated with the nociceptive tests. The absolute conditions for carrying out the tests were the absence of nociception in naive and sham-operated animals, the presence of hyperalgesia and allodynia in animals with peripheral neuropathy and the reversal of both phenomena in animals with neuropathy after MCS. Results presented herein correspond to the mean of the density of nuclei labeled for Fos-IR and Zif268-IR for each structure evaluated and are presented as a supplementary table (Table S1, see the online version at 10.1016/j.ejpain.2010.08.003). Naive animals without any surgical intervention showed the same immunolabel pattern as sham-operated animals (Table S1, see the online version at 10.1016/j.ejpain.2010.08.003). No spinal cord and brain side differences were observed for Fos-IR and Zif268-IR in either naive or sham-operated animals (Figs. 2 and 3 and Table S1, see the online version at 10.1016/j.ejpain.2010.08.003). 3.2.1. DHSC Immunohistochemical assays were performed throughout the L4–L5 spinal cord segments, which receive the majority of sciatic nerve afferents (Rigaud et al., 2008), and the analysis of immunolabeling for proto-oncogenes was carried out in the DHSC, more specifically between the Rexed laminae 1 and 6. A significant increase of the Fos-IR occurred bilaterally in the DHSC, in the animals with CCI, but showed a more intense labeling on the right side, ipsilateral to the nerve injury (Fig. 2A). After MCS, a decrease of the density of Fos-IR cells on the right side was observed (Figs. 2A and 4A and B). With respect to Zif268, there was an increase of Zif268-IR in the DHSC on the right side, which was completely reverted after MCS (Figs. 2A and S1A and B, see the online version at 10.1016/j.ej- Please cite this article in press as: Pagano RL et al. Transdural motor cortex stimulation reverses neuropathic pain in rats: A profile of neuronal activation. Eur J Pain (2010), doi:10.1016/j.ejpain.2010.08.003 4 R.L. Pagano et al. / European Journal of Pain xxx (2010) xxx–xxx Fig. 1. Mechanical hyperalgesia evaluated by the rat paw pressure test (A and B) and low-threshold mechanical allodynia measured using the von Frey test (C and D), in sham-operated rats (Sham), animals submitted to chronic constriction injury of the sciatic nerve (CCI) and animals with nerve injury submitted to MCS (CCI + MCS). The CCI was performed in the right paw. The electrodes were implanted 7 days after the CCI over the motor cortex of the left hemisphere, and the nociceptive threshold was evaluated on the left hind paw (A and C) and on the right hind paw (B and D). The cortical electrodes were implanted contralaterally to CCI. The nociceptive tests were applied before the surgeries (IM, initial measure), 14 days after the CCI (FM, final measure) and again in the CCI group after 15 min of MCS, still under stimulation. Values represent the mean ± SEM of seven animals from each group. Statistically significant differences vs. IM () are indicated. pain.2010.08.003). No major differences were noted when considering the different laminae of the dorsal horn (Fig. S2, see the online version at 10.1016/j.ejpain.2010.08.003). pared with the sections obtained from animals with CCI but not stimulated (Figs. 2C and S1E and F, see the online version at 10.1016/j.ejpain.2010.08.003). 3.2.2. PAG There was no change in the density of Fos-IR cells in the PAG in animals with CCI, when compared to sham-operated animals (Fig. 2B). However, there was a significant increase in the density of Fos-IR cells in the PAG, bilaterally, after MCS in animals with peripheral neuropathy (Figs. 2B and 4C and D). There was a bilateral increase in the density of Zif268-IR cells in the PAG, in animals with CCI, when compared to sham-operated animals (Fig. 2B). Bilateral immunolabel intensification for Zif268 occurred in the PAG after MCS in animals with nerve injury, when compared to animals with neuropathic pain without stimulation (Figs. 2B and S1C and D, see the online version at 10.1016/j.ejpain.2010.08.003). No major differences were noted when considering the different PAG columns (Fig. S3, see the online version at 10.1016/ j.ejpain.2010.08.003). 3.2.4. ACC There was a more intense Fos immunoreactivity in the ACC of the animals with nerve injury, on both sides, when compared to sham-operated animals (Fig. 3A). A bilateral increase of Fos positive cells in the ACC was observed in animals after MCS, when compared to animals with neuropathic pain (Figs. 3A and 5A and B). No difference of Zif268-IR was observed in the ACC among sham-operated animals, those with neuropathic pain, and those with peripheral neuropathy submitted to MCS (Figs. 3A and S4A and B, see the online version at 10.1016/j.ejpain.2010.08.003). 3.2.3. VPL/VPM CCI neuropathic animals showed a bilateral increase of the density of Fos-IR cells in thalamic nuclei (Fig. 2C). MCS decreased the Fos immunoreactivity on the left side (contralateral to the CCI), when compared to animals with peripheral neuropathy without cortical stimulation (Figs. 2C and 4E and F). There was a bilateral increase of the density of Zif268-IR cells in the VPL/VPM of the animals with nerve injury, which was reverted after MCS, when com- 3.2.5. CeA and BLA There was no change of Fos and Zif268-IR in the CeA and BLA nuclei between sham-operated and operated animals (Fig. 3B and C, respectively). MCS induced an increase of Fos-IR cells in the CeA, on both sides, in animals with peripheral neuropathy; however, this increase was only significantly different from the sham-operated group (Figs. 3B and 5C and D). There was no difference of Zif268-IR in the CeA after MCS (Figs. 3B and S4C and D, see the online version at 10.1016/j.ejpain.2010.08.003). A bilateral increase of Fos and Zif268 was observed in the BLA after MCS, which was significantly different from the sham-operated and neuropathic animals (Figs. 3C, 5C and D, S4C and D, see the online version at 10.1016/j.ejpain.2010.08.003). Please cite this article in press as: Pagano RL et al. Transdural motor cortex stimulation reverses neuropathic pain in rats: A profile of neuronal activation. Eur J Pain (2010), doi:10.1016/j.ejpain.2010.08.003 R.L. Pagano et al. / European Journal of Pain xxx (2010) xxx–xxx Fig. 2. Fos and Zif268 immunolabel in the DHSC (A), PAG (B) and VPL/VPM (C); in sham-operated rats (Sham), animals with neuropathic pain (CCI) and animals with nerve injury submitted to MCS (CCI + MCS). The data show the proto-oncogene immunoreactivity on the left (LS) and right (RS) side. The cortical electrodes were implanted in the left hemisphere, contralaterally to peripheral injury performed in the right hind paw. Values represent the mean ± SEM of seven animals from each group. Statistically significant differences vs. Sham (), vs. CCI (#) are indicated. 5 Fig. 3. Fos and Zif268 immunolabel in the ACC (A), CeA (B) and BLA (C); in shamoperated rats (Sham), animals with neuropathic pain (CCI) and animals with nerve injury submitted to MCS (CCI + MCS). The data show the proto-oncogene immunoreactivity on the left (LS) and right (RS) side. The cortical electrodes were implanted in the left hemisphere, contralaterally to peripheral injury performed in the right hind paw. Values represent the mean ± SEM of seven animals from each group. Statistically significant differences vs. Sham () and vs. CCI (#) are indicated. 4. Discussion Transdural MCS has been used in the treatment of patients suffering from severe neuropathic pain that is resistant to other therapeutic approaches (Nguyen et al., 1999, 2000; Canavero and Bonicalzi, 2002; Brow and Barbaro, 2003). Besides the report that motivated its clinical application, few experimental studies have used MCS in rats to investigate the mechanisms involved in the stimulation-induced antinociception (Rusina et al., 2005; Senapati et al., 2005a; Vaculin et al., 2008; Fonoff et al., 2009a; Viisanen and Pertovaara, 2010a,b), and thus its mediation remains elusive. We showed here that transdural MCS reverts the neuropathic pain in rats subjected to chronic constriction injury of the sciatic nerve, similar to the protocol and effect observed in humans, reinforcing the hypothesis that the motor cortex has a relevant role in the control of persistent pain. These findings corroborate those obtained by the Rokyta group, which showed that subdural MCS reverted CCI-induced hyperalgesia (Vaculin et al., 2008). In an attempt to determine the neural pathway involved in that effect, the neuronal activation profile was evaluated in the spinal cord and brain sites associated with the modulation of pain. We demonstrated that peripheral neuropathy enhances DHSC activation, corroborating other results obtained during neuropathic pain induced by CCI (Kajander et al., 1996; Yamazaki et al., 2001). MCS reverted the increase of neuronal activation in the DHSC, on the right side (ipsilateral to CCI). These findings are consistent with electrophysiological data showing inhibition of spinal wide-dynamic range neurons in response to mechanical stimulation after subdural MCS (Senapati et al., 2005a), in which, however, a simultaneous bilateral response was observed. The discrepancy between Please cite this article in press as: Pagano RL et al. Transdural motor cortex stimulation reverses neuropathic pain in rats: A profile of neuronal activation. Eur J Pain (2010), doi:10.1016/j.ejpain.2010.08.003 6 R.L. Pagano et al. / European Journal of Pain xxx (2010) xxx–xxx Fig. 4. Photomicrographs illustrating Fos immunostaining in the DHSC ((A) CCI, (B) CCI + MCS), the PAG ((C) CCI, (D) CCI + MCS), and the VPL/VPM ((E) CCI, (F) CCI + MCS). PAG and thalamic nuclei sections represent the left hemisphere, while DHSC sections represent the right dorsal horn. The cortical electrodes were implanted in the left hemisphere, contralaterally to peripheral injury performed in the right hind paw. CCI: sections obtained from a rat with neuropathic pain. CCI + MCS: sections obtained from a rat with CCI, without neuropathic pain after MCS. our results and those electrophysiological data can be attributed to different methods of evaluation (e.g., anaesthetized vs. awake rats), the technique of electrode placement, the site of stimulation within the cortical tissue, and the divergence of the electrical parameters used for cortical stimulation. Our results suggest that MCS leads to inhibition of the DHSC neurons directly and/or indirectly through activation of the descending inhibitory system. We also showed that MCS bilaterally increases PAG activation of animals that had peripheral neuropathy and showed reversion of neuropathic pain phenomena during cortical stimulation. The PAG constitutes a part of a descending pain suppressor pathway that projects via the rostral ventromedial medulla (RVM) to the DHSC (Basbaum and Fields, 1984). The PAG–RVM-spinal cord pathway comprises an essential neural circuit for opioid-based antinociception (Basbaum and Fields, 1984). MCS increases the nociceptive threshold through opioid mechanisms in rats (Fonoff et al., 2009a), and enhances secretion of endogenous opioids in the ACC and PAG in humans (Maarrawi et al., 2007), which is in agreement with our results that showed PAG activation during MCS. These sets of data suggest the involvement of the descending pain suppressor pathway in the MCS effects. The VPL/VPM thalamic nuclei are involved in the transfer of nociceptive information, playing a role in integrating the sensory-discriminative component of pain (Millan, 1999). The VPL nociceptive neurons receive major inputs from the contralateral sciatic nerve and therefore may be more active in animals that display lowered thresholds in response to mechanical and thermal stimuli after peripheral neuropathy (Guilbaud et al., 1990; Miki et al., 2000). Despite the VPM receiving trigeminothalamic, but little, if any, spinothalamic tract input (Yen et al., 1991), it proved to be highly activated in rats with CCI (Mao et al., 1993). Our results showed that peripheral nerve injury induced VPL/VPM activation. Please cite this article in press as: Pagano RL et al. Transdural motor cortex stimulation reverses neuropathic pain in rats: A profile of neuronal activation. Eur J Pain (2010), doi:10.1016/j.ejpain.2010.08.003 R.L. Pagano et al. / European Journal of Pain xxx (2010) xxx–xxx 7 Fig. 5. Photomicrographs illustrating Fos immunostaining in the ACC ((A) CCI, (B) CCI + MCS), the CeA ((Ci) CCI, (Di) CCI + MCS), and the BLA ((Cii) CCI, (Dii) CCI + MCS). Amygdaloid nuclei sections represent the left hemisphere. The cortical electrodes were implanted in the left hemisphere, contralaterally to peripheral injury performed in the right hind paw. CCI: sections obtained from a rat with neuropathic pain. CCI + MCS: sections obtained from a rat with CCI, without neuropathic pain after MCS. Previous studies have failed to detect Fos immunolabel in the ventrobasal thalamus (Pertovaara et al., 1993; Pearse et al., 2001), while others have found a small number of Fos-labeled cells (Bullitt, 1989, 1990; Gholami et al., 2006). However, some of these studies hypothesized that Fos and other ITFs may be expressed only after more complex stimulation patterns (Bullitt, 1989; Pearse et al., 2001). In this manner, the lack of Fos immunolabel in the ventrobasal thalamus in previous studies could be due, at least in part, to the fact that the stimuli used were relatively of low intensity, being insufficient to evoke detectable ITF expression. MCS reverted the ITF increase, into VPL/VPM, suggesting that motor cortex stimulation decreases the thalamic activation indirectly through inhibition of DHSC neurons. A direct action of MCS on VPL/VPM inhibition can also be suggested, considering the existence of reciprocal corticothalamic projections (Lee et al., 2008; Jones, 2009). ACC is an important limbic structure involved in the affective– emotional component of pain (Johansen et al., 2001). It has reciprocal connections with somatosensory and motor cortices (Reep et al., 1990; Conde et al., 1995) and projects to thalamic nuclei, PAG and posterior hypothalamic nucleus (Divac et al., 1978; Saphier and Feldman, 1986). ACC is consistently activated during neuropathic pain (Hsieh et al., 1995). We showed that peripheral neuropathy enhances ACC activation, evidenced by Fos-IR, confirming previous results that showed an increase of Fos expression in the ACC during persistent pain in rats (Lei et al., 2004; Takeda et al., 2009). ACC lesions selectively reduce the affective component of neuropathic pain (LaGraize et al., 2004). On the other hand, electrical stimulation of the ACC inhibits mechanical allodynia in rats with peripheral neuropathy (Park et al., 2006) and inhibits DHSC neurons in response to noxious mechanical stimuli (Senapati et al., 2005b). Similar to our results, which showed that MCS exacerbated the ACC activation in rats, PET imaging has revealed ACC activation during MCS in humans (Peyron et al., 1995; Garcia-Larrea et al., 1999). Considering these findings, we hypothesized that MCS induces ACC activation, inhibiting the DHSC neurons directly or indirectly by PAG activation and resulting in the activation of the descending pain suppressor pathway. This hypothesis corroborates that of Peyron and colleagues (2007), which showed that MCS activates the descending ACC–PAG connections. No changes were observed for Zif268-IR in the ACC. The expression of Fos and Zif268 proteins are widely used as tools to examine nociceptive neuron activity (Herdegen et al., 1991; Lanteri-Minet et al., 1993). However, a distinct regulation in the expression of these proteins is suggested following noxious stimulation (Herdegen et al., 1991, 1992; Herdegen and Leah, 1998). Herein, we compared the induction profile of Fos and Zif268 proteins. Considering the discrepancy of our results, it is plausible that the proto-oncogenes evaluated have different stimulus-specific patterns of activation and/or determine distinct populations of activated neurons. Identifying the neuronal type involved in the activation profile could contribute to a better understanding of the underlying mechanisms of MCS. The amygdala (AMY) is an important site of interaction between persistent pain and negative affective states. It integrates nociceptive-specific information from the spinal cord and brainstem with highly processed polymodal information from the thalamus and cortex through connections with the lateral (LA) and the basolateral (BLA) nuclei of AMY, which then project to the CeA nucleus (Neugebauer et al., 2004). The LA–BLA–CeA circuitry receives Please cite this article in press as: Pagano RL et al. Transdural motor cortex stimulation reverses neuropathic pain in rats: A profile of neuronal activation. Eur J Pain (2010), doi:10.1016/j.ejpain.2010.08.003 8 R.L. Pagano et al. / European Journal of Pain xxx (2010) xxx–xxx pain-related information from the thalamus, the insular cortex, and the ACC (Millan, 1999; Price, 2003). In addition to these indirect inputs, the CeA has direct connections with the PAG, thalamic nuclei, hypothalamus, reticular formation and other structures involved in pain regulation (Price, 2003; Neugebauer et al., 2004). The AMY has a role in both pain inhibition and pain facilitation (Neugebauer et al., 2004). Persistent neuropathy in rats induces a depressive-like behavior associated with proliferation (Gonçalves et al., 2008) and activation (Ikeda et al., 2007) of AMY neurons, which contributes to chronic pain through the generation and maintenance of central sensitization in the spinal cord (Neugebauer et al., 2004). Nevertheless, CeA lesions inhibit morphineinduced antinociception (Manning, 1998) and reduce or eliminate the conditioned analgesia (Helmstetter, 1992; Fox and Sorenson, 1994), leading to the concept that AMY, and CeA in particular, is part of a descending pain suppressor system (Helmstetter, 1992; Millan, 1999). Our results showed that MCS increases the CeA and BLA activation in animals with peripheral neuropathy. Considering that there are direct reciprocal projections between AMY and the PAG (Rizvi et al., 1991), we suggest that MCS induces BLA and CeA activation, which contributes to the inhibition of nociceptive response induced by neuropathic pain. The corticospinal tract is a predominantly crossed pathway. Nevertheless, the primary motor cortex (M1) is activated bilaterally during production of unilateral movements (Donchin et al., 2002), and rat studies demonstrate a bilateral M1 forelimb motor representation (Liang et al., 1993). Moreover, ipsilateral responses depend on the contralateral corticospinal system, through moderate interhemispheric facilitation and strong subcortical facilitation (Brus-Ramer et al., 2009). In the spinal cord, the predominance of contralateral projections of the motor cortex to the dorsal horn in rats (Joosten et al., 1992) is directly related to a more important inhibition of dorsal horn neurons, as presently shown. These results are also supported by data collected from electrophysiology (Senapati et al., 2005a). In addition, the less dense projections of the motor cortex to the ipsilateral dorsal horn produce also a mild neuronal inhibition in both proto-oncogene expression (present study) and electrophysiological activity (Senapati et al., 2005a). Conversely, it is noteworthy that the descending pyramidal tract becomes contralateral only below the medullary spinal cord transition. Thus, the corticofugal motor fibers within the brain and brainstem are ipsilateral to the cortical origin (Morecraft et al., 2007). Consequently, the structures involved in the antinociception induced by MCS, including the cingulate cortex, AMY, thalamus, PAG and lower brainstem nuclei receive predominantly ipsilateral projections from the motor cortex. The cingulate cortex is reciprocally connected to the primary motor cortex in both sides (Reep et al., 1990; Conde et al., 1995; Morecraft et al., 2007). These intimate connections are paralleled by the bilaterally balanced neuron activation in the cingulate cortex observed herein. The AMY is also reciprocally connected to the motor cortex; however, the strongest projections are those from the AMY to the motor cortex (Morecraft et al., 2007). The bilateral influence of MCS over the AMY may be related to some indirect connections, still anatomically uncharacterized. The direct anatomical connections between the motor cortex and the thalamus are very robust in the same side, although contralateral projections through the thalamic massa intermedia have also been evidenced (Molinari et al., 1985; Gómez-Pinilla and Villablanca, 1989; Lee et al., 2008; Jones, 2009). Consequently, a direct action of MCS on VPL/VPM inhibition is more likely to take place bilaterally. As described by Newman et al. (1989), using anterograde tract-tracing techniques, fibers originating in the motor cortex spread into diffuse terminal fields in the ipsilateral mesencephalic reticular formation and PAG. Inter- estingly, the afferents from the motor cortex are found to maintain some topography once they reach the PAG. The face area projects preferentially to the ventral PAG, the forelimb area connects to its ventrolateral aspects, while the trunk–hindlimb motor area is related to the lateral part of PAG (Newman et al., 1989). Taking our data together, we hypothesize that MCS reduces neuropathic pain by interrupting the transmission of noxious information from the spinal cord level through the activation of descending pain suppressor system, in accordance with previous ideas (Ohara et al., 2005; Xie et al., 2009; Viisanen and Pertovaara, 2010b). Limbic system participation during MCS pain modulation was also observed, suggesting that motor cortex stimulation modulates both the sensory-discriminative and the affective–motivational components of pain. Although, clinical results have been encouraging, some patients do not benefit from this form of neuromodulation therapy (Nuti et al., 2005; Rasche et al., 2006). In this regard, it appears clear that there is an urgent need for further clinical and experimental studies of the mechanisms involved in the pain relieving effects of MCS (e.g., Meyerson, 2005). 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Eur J Pain (2010), doi:10.1016/j.ejpain.2010.08.003 11 R.L. Pagano et al. / European Journal of Pain xxx (2010) xxx–xxx Table S1 Fos (A) and Zif268 (B) immunoreactivities. Naive Sham-operated CCI CCI + MCS Left Right Left Right Left Right Left Right A DHSC PAG VPL/VPM ACC CeA BLA 10 ± 1 82 ± 7 143 ± 6 88 ± 5 41 ± 5 5±2 14 ± 1 87 ± 6 171 ± 7 80 ± 5 38 ± 4 6±1 14 ± 1 83 ± 5 153 ± 8 73 ± 4 37 ± 6 6±1 13 ± 1 89 ± 5 180 ± 7 78 ± 5 37 ± 4 7±1 34 ± 2b 83 ± 5 298 ± 11b 137 ± 5b 50 ± 5 9±1 48 ± 2a’b 85 ± 5 273 ± 6b 156 ± 10b 53 ± 5 8±1 35 ± lb 143 ± 7c 178 ± 13 176 ± 9c 59 ± 4b 15 ± 2c 37 ± 1c 154 ± 7c 246 ± 8b 213 ± 10c 63 ± 5b 17 ± 2c B DHSC PAG VPL/VPM ACC CeA BLA 20 ± 1 59 ± 5 136 ± 6 225 ± 9 58 ± 5 10 ± 1 23 ± 1 56 ± 5 135 ± 5 229 ± 11 61 ± 6 6±2 26 ± 2 43 ± 2 150 ± 7 232 ± 8 62 ± 5 5±1 26 ± 1 54 ± 4 155 ± 6 236 ± 13 60 ± 5 9±1 30 ± 2 122 ± 7b 229 ± 7b 279 ± 18 73 ± 6 9±2 53 ± 3a’b 113 ± 7b 222 ± 5b 276 ± 18 81 ± 5 10 ± 2 29 ± 1 173 ± 13c 150 ± 9 246 ± 16 63 ± 6 21 ± 3c 28 ± 1 195 ± 16c 149 ± 6 253 ± 12 80 ± 4 24 ± 3c The cortical electrodes were implanted in the left hemisphere, contralaterally to peripheral injury realized in the right hind paw. CCI: chronic constriction injury of the sciatic nerve. MCS: motor cortex stimulation. Left and right are corresponding to the hemisphere side. DHSC: dorsal horn of the spinal cord; PAG: periaqueductal gray, VPL/VPM: ventral posterior lateral and medial nuclei of the thalamus, ACC: anterior cingulate cortex, CeA: central amygdaloid nucleus, BLA: basolateral amygdaloid nucleus. a p < 0.05 as compared to left side. b p < 0.05 as compared to naive and sham-operated group. c p < 0.05 as compared to naive, sham-operated and CCI groups. Please cite this article in press as: Pagano RL et al. Transdural motor cortex stimulation reverses neuropathic pain in rats: A profile of neuronal activation. Eur J Pain (2010), doi:10.1016/j.ejpain.2010.08.003 12 R.L. Pagano et al. / European Journal of Pain xxx (2010) xxx–xxx Fig. S1. Photomicrographs illustrating Zif268 immunostaining in the DHSC ((A) CCI, (B) CCI + MCS), the PAG ((C) CCI, (D) CCI + MCS), and the VPL/VPM ((E) CCI, (F) CCI + MCS). PAG and thalamic nuclei sections represent the left hemisphere, while DHSC sections represent the right dorsal horn. The cortical electrodes were implanted in the left hemisphere, contralaterally to peripheral injury performed in the right hind paw. CCI: sections obtained from a rat with neuropathic pain. CCI + MCS: sections obtained from a rat with CCI, without neuropathic pain after MCS. Please cite this article in press as: Pagano RL et al. Transdural motor cortex stimulation reverses neuropathic pain in rats: A profile of neuronal activation. Eur J Pain (2010), doi:10.1016/j.ejpain.2010.08.003 R.L. Pagano et al. / European Journal of Pain xxx (2010) xxx–xxx Fig. S2. Fos (A) and Zif268 (B) immunolabeling in the DHSC in sham-operated rats (Sham), animals with neuropathic pain (CCI) and animals with nerve injury submitted to MCS (CCI + MCS). The data show the proto-oncogene immunoreactivity on the right side. The cortical electrodes were implanted in the left hemisphere, contralaterally to peripheral injury performed in the right hind paw. Values represent the mean ± SEM of seven animals from each group. Statistically significant differences vs. Sham (), vs. CCI (#) are indicated. L1–L6: laminae I–VI of the L4–L5 dorsal horn. 13 Fig. S3. Fos (A) and Zif268 (B) immunolabeling in the PAG in sham-operated rats (Sham), animals with neuropathic pain (CCI) and animals with nerve injury submitted to MCS (CCI + MCS). The data show the proto-oncogene immunoreactivity on the left side. The cortical electrodes were implanted in the left hemisphere, contralaterally to peripheral injury performed in the right hind paw. Values represent the mean ± SEM of seven animals from each group. Statistically significant differences vs. Sham (), vs. CCI (#) are indicated. DM: dorsomedial column; DL: dorsolateral column; L: lateral column; VL: ventrolateral column. Please cite this article in press as: Pagano RL et al. Transdural motor cortex stimulation reverses neuropathic pain in rats: A profile of neuronal activation. Eur J Pain (2010), doi:10.1016/j.ejpain.2010.08.003 14 R.L. Pagano et al. / European Journal of Pain xxx (2010) xxx–xxx Fig. S4. Photomicrographs illustrating Zif268 immunostaining in the ACC ((A) CCI, (B) CCI + MCS), the CeA ((Ci) CCI, (Di) CCI + MCS) and the BLA ((Cii) CCI, (Dii) CCI + MCS). Amygdaloid nuclei sections represent the left hemisphere. The cortical electrodes were implanted in the left hemisphere, contralaterally to peripheral injury performed in the right hind paw. CCI: sections obtained from a rat with neuropathic pain. CCI + MCS: sections obtained from a rat with CCI, without neuropathic pain after MCS. Please cite this article in press as: Pagano RL et al. Transdural motor cortex stimulation reverses neuropathic pain in rats: A profile of neuronal activation. Eur J Pain (2010), doi:10.1016/j.ejpain.2010.08.003