Exp Brain Res DOI 10.1007/s00221-015-4203-2

RESEARCH ARTICLE

Peripheral nerve injury activates convergent nociceptive input to dorsal horn neurons from neighboring intact nerve Ryuji Terayama · Yuya Yamamoto · Noriko Kishimoto · Kotaro Maruhama · Masahide Mizutani · Seiji Iida · Tomosada Sugimoto 

Received: 26 August 2014 / Accepted: 10 January 2015 © Springer-Verlag Berlin Heidelberg 2015

Abstract  Previous studies demonstrated that peripheral nerve injury induced excessive nociceptive response of spinal cord dorsal horn neurons and such change has been proposed to reflect the development of neuropathic pain state. The aim of this study was to examine the spinal dorsal horn for convergence of nociceptive input to second-order neurons deafferented by peripheral nerve injury. Double immunofluorescence labeling for c-Fos and phosphorylated extracellular signal-regulated kinase (p-ERK) was performed to detect convergent synaptic input to spinal dorsal horn neurons after the saphenous nerve injury. c-Fos expression and the phosphorylation of ERK were induced by noxious heat stimulation of the hindpaw and by electrical stimulation of the injured or uninjured saphenous nerve, respectively. Within the central terminal field of the saphenous nerve, the number of c-Fos protein-like immunoreactive (c-Fos-IR) cell profiles was significantly decreased at 3 days and returned to the control level by 14 days after the injury. p-ERK immunoreactive (p-ERK-IR) cell profiles were

distributed in the central terminal field of the saphenous nerve, and the topographic distribution pattern and number of such p-ERK-IR cell profiles remained unchanged after the nerve injury. The time course of changes in the number of double-labeled cell profiles was similar to that of c-Fos-IR cell profiles after the injury. These results indicate that convergent primary nociceptive input through neighboring intact nerves contributes to increased responsiveness of spinal dorsal horn nociceptive neurons. Keywords  Nerve injury · c-Fos · ERK · Spinal dorsal horn · Immunohistochemistry

R. Terayama (*) · Y. Yamamoto · N. Kishimoto · K. Maruhama · T. Sugimoto  Department of Oral Function and Anatomy, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2‑5‑1 Shikata‑cho, Kita‑ku, Okayama 700‑8525, Japan e-mail: [email protected]‑u.ac.jp

Abbreviations ANOVA Analysis of variance CNS Central nervous system DAB Diaminobenzidine ES Electrical stimulation c-Fos-IR c-Fos protein-like immunoreactive MAPK Mitogen-activated protein kinase PAP Peroxidase anti-peroxidase p-ERK Phosphorylated extracellular signal-regulated kinase p-ERK-IR p-ERK immunoreactive PB Phosphate buffer PBS Phosphate-buffered saline SNI Saphenous nerve injury

R. Terayama · K. Maruhama · M. Mizutani · S. Iida · T. Sugimoto  Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School, Okayama 700‑8525, Japan

Introduction

Y. Yamamoto · N. Kishimoto · M. Mizutani · S. Iida  Department of Oral and Maxillofacial Reconstructive Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700‑8525, Japan

Peripheral nerve injury results in a number of morphological and physiological changes in the spinal dorsal horn. Previous studies demonstrated that the somatotopic map

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of second-order neurons in the spinal dorsal horn was disrupted and that neurons disconnected from their original receptive fields began to respond to stimulation of the skin outside their original receptive field (Devor and Wall 1978; Lisney 1983; Markus et al. 1984; Hylden et al. 1987). The transection of a peripheral nerve was shown to enhance neuronal c-Fos induction and phosphorylation of extracellular signal-regulated kinase (ERK) in the spinal and medullary dorsal horns in response to the stimulation of spared primary nociceptors (Sugimoto et al. 1993; Ji et al. 1999; Nomura et al. 2002; Noma et al. 2008). We have reported exaggerated c-Fos expression in the medullary dorsal horn evoked by electrical stimulation (ES) at the C-fiber intensity of a spared uninjured nerve rather than the injured nerve itself (Fujisawa et al. 2012). These reports suggest a possibility that second-order sensory neurons acquired an ability to respond to nociceptive signals originating from somatotopically inappropriate receptive field. Such anomalous excitability may explain the clinically observed hyperalgesia spreading outside the areas denervated by the nerve injury. ERK is a member of the mitogen-activated protein kinase (MAPK) family. The phosphorylation of ERK was previously shown to be involved in the neuronal plasticity involved in learning and memory, and also contributes to pain hypersensitivity (Ji and Woolf 2001; Ji et al. 2003). Similar to c-Fos expression, phosphorylated ERK (p-ERK) expression was shown to be induced in spinal and medullary dorsal horn neurons by noxious stimulation (Ji et al. 1999; Noma et al. 2008). Therefore, p-ERK expression has also been used as a marker for neuronal activation following noxious stimulation. However, p-ERK expression in dorsal horn neurons following noxious stimulation was shown to be markedly quicker and more transient than that of c-Fos (Ji et al. 1999; Shimizu et al. 2006). Therefore, the noxious stimulation-induced p-ERK is expected to appear and disappear before c-Fos would become detectable after the same stimulation. The aim of this study was to explore a possibility that a peripheral nerve injury activates anomalous convergence of nociceptive signals to spinal dorsal horn neurons disconnected from their normal receptive field. Somatotopic representation of the sciatic and saphenous nerves in the spinal dorsal horn was elucidated by c-Fos immunohistochemistry after ES of these nerves or noxious heat stimulation of the hindpaw. Then, double immunofluorescence labeling for c-Fos and p-ERK was performed to detect convergent synaptic inputs after the saphenous nerve injury. c-Fos expression and phosphorylation of ERK were induced by noxious heat stimulation of the hindpaw 2 h before and by ES of the saphenous nerve 5 min before histochemical examination, respectively.

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Materials and methods Animals All surgical and experimental procedures described herein were reviewed and approved by the Animal Care and Use Committee, Okayama University, Government Animal Protection and Management Law (No. 105), Japanese Government Notification on Feeding and Safekeeping of Animals (No. 6) and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23), revised 1996. Male Sprague–Dawley rats weighing 180–200 g at the time of surgery were used. Rats were housed at 20 °C with a daily light period of 12 h and fed food and water ad libitum. Every attempt was made to minimize the number of animals used and reduce their suffering at all stages of the study. Somatotopic representation of the sciatic and saphenous nerves ES of the sciatic or saphenous nerve and noxious heat stimulation of the hindpaw Immunohistochemistry for c-Fos was employed for the determination of the topographic distribution pattern of dorsal horn neurons responding to noxious signals transmitted by the sciatic and saphenous nerves. Twelve rats underwent ES at C-fiber intensity or sham stimulation (0 mA) of either the sciatic or saphenous nerve (n = 3 for each stimulation for each nerve) under anesthesia with an i.p. injection of pentobarbital sodium (50 mg/kg). The right sciatic or saphenous nerve was exposed by skin incision (and blunt dissection through muscles for the sciatic nerve), and bipolar silver hock electrodes were placed beneath the isolated nerve. A train of rectangular pulses (5 mA, 5 ms) was delivered at 5 Hz for 10 min. These stimulation parameters were previously determined as sufficient for exciting C-fibers (Devor and Govrin-Lippmann 1983; Molander et al. 1992; Shortland and Molander 1998; Tokunaga et al. 1999; Hughes et al. 2008; Fujisawa et al. 2012). Three additional rats underwent noxious heat stimulation of the hindpaw. Under anesthesia with an i.p. injection of pentobarbital sodium, the right hindpaw was immersed in hot water (55 °C) for 10 s. Tissue preparation Two hours after ES or noxious heat stimulation, the rats were perfused transcardially with saline followed by 4 % paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The spinal cord including the first to fifth lumbar (L1– L5) segments with dorsal and ventral roots and L1–L5

Exp Brain Res

dorsal root ganglia attached was dissected out, postfixed in the same fixative for 24 h and then immersed in 20 % sucrose in 0.02 M phosphate-buffered saline (PBS, pH 7.4) for 48 h. Boundaries of each segment were confirmed by verifying the dorsal root entry zone, and the rostro-caudal length of the L1–L5 was measured. Immunohistochemistry for c‑Fos Fifty-µm-thick transverse frozen sections from the L1–L5 segments were collected serially in PBS. Alternate series of free-floating sections were assigned to one of 20 blocks; i.e., each of the L1–L5 segments was subdivided into four blocks along the rostro-caudal axis. Since the length of the L1–L5 was about 10.0 mm, produced blocks contained four or five consecutive sections each. Sections in each block were processed together as free floating for immunohistochemistry for the c-Fos protein using a peroxidase anti-peroxidase (PAP) method as described previously (Sugimoto et al. 1993). Briefly, sections were incubated for 1 h with 0.3 % H2O2 in 80 % methanol to quench endogenous peroxidase activity. After incubation with 3 % normal goat serum for 1 h, the sections were reacted with a rabbit anti-c-Fos antibody (1:8,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 72 h at 4 °C. The sections were then sequentially incubated with goat anti-rabbit IgG (1:300; Cappel West Chester, PA, USA) and PAP complex (1:3,000; Cappel). The reaction products were visualized by nickel ammonium sulfate-intensified diaminobenzidine (DAB) histochemistry. The sections were mounted on glass slides, air-dried, dehydrated in graded alcohol solutions, cleared in xylene and coverslipped. Using a camera lucida drawing tube, a dark-field image of the spinal cord section was traced and the bright-field image of the c-Fos proteinlike immunoreactive (c-Fos-IR) cell profile was plotted on white paper. Three sections were randomly selected for each block, the number of c-Fos-IR cell profiles was counted, and the average of three sections was recorded for each rat. Convergent primary inputs after nerve injury Saphenous nerve injury Double immunofluorescence labeling was used to detect convergent synaptic input to second-order neurons disconnected from the receptive field of the saphenous nerve. c-Fos translation was used for the activity marker for noxious heat stimulation of the hindpaw, while the phosphorylation of ERK for ES of the saphenous nerve. c-Fos/pERK double-labeled cell profiles after the nerve injury but not sham surgery were considered to have received convergent primary afferent inputs from intact and injured nerves.

Fig. 1  Diagram showing time course of surgery, noxious heat stimulation, ES of the saphenous nerve and perfusion for double immunofluorescence labeling

Saphenous nerve injury was performed under anesthesia with an i.p. injection of pentobarbital sodium (50 mg/ kg) as follows. The right saphenous nerve was exposed by a skin incision on the medial aspect of the thigh. The saphenous nerve was ligated firmly at two separate points with 7-0 silk suture. The nerve segment between the two ligatures was transected with fine scissors, and the skin was suture-closed. Sham-operated animals, in which the nerve was exposed but intentional nerve injury was omitted, were used as controls. Noxious heat stimulation of the hindpaw and ES of the saphenous nerve Three, 7 or 14 days after the saphenous nerve injury or sham surgery (n  = 5 in each group), reanesthetized rats received noxious heat stimulation to the hindpaw 2 h before perfusion and the injured or uninjured saphenous nerve was subsequently exposed for ES 15 min prior to perfusion (Fig. 1). For the noxious heat stimulation, the hindpaw was immersed in hot water (55 °C) for 10 s. For ES, silver hock electrodes were placed beneath the isolated saphenous nerve, and a train of rectangular pulses (5 mA, 5 ms) was delivered at 5 Hz for 10 min. Tissue preparation Two hours after the noxious heat stimulation and 5 min after ES, the rats were perfused transcardially with saline followed by 4 % paraformaldehyde in 0.1 M PB (Fig. 1). The spinal cord including the L1–L5 segments with dorsal and ventral roots and L1–L5 dorsal root ganglia attached was dissected out, postfixed in the same fixative for 24 h and then immersed in 20 % sucrose in 0.02 M PBS for 48 h. Boundaries of each segment were confirmed by verifying the dorsal root entry zone, and each of the L1–L5 segments was divided into the rostral and caudal halves thus yielding ten tissue blocks. Double immunofluorescence labeling for c‑Fos and p‑ERK Frozen 10-µm-thick sections from each block were cut on a cryostat and mounted onto silane-coated slides.

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Sections were incubated for 24 h at RT with a mixture of the rabbit polyclonal anti-c-Fos antibody (1:8,000; Santa Cruz Biotechnology) and mouse monoclonal anti-phospho-p44/42 MAP kinase antibody (1:2,000; Cell Signaling, Beverly, MA, USA). Alexa-488- and Alexa-568-conjugated secondary antibodies (Molecular Probes, Eugene, OR, USA; 1:1,000) were used to

visualize primary antibody binding. The primary antibodies were omitted for negative controls. Sections were coverslipped with a mounting medium (DAKO Fluorescent Mounting Medium, DAKO, Carpinteria, CA, USA). The labeled sections were examined with a Nikon (Tokyo, Japan) fluorescence microscope, and images were captured with a CCD spot camera. Both noxious heat stimulation to the hindpaw and ES to the saphenous nerve were expected to have excited primary neurons in the saphenous nerve of sham-operated controls. Since peripheral axons in the injured saphenous nerve were prevented from reinnervating the skin receptive field, double labeling in nerve-injured rats was considered to represent the convergence of sensory signals from both saphenous and sciatic nerves (Fig. 2). The numbers of c-Fos-IR, p-ERK immunoreactive (p-ERK-IR) and c-Fos/p-ERK double-labeled cell profiles were counted in laminae I and II (I/II) of the spinal dorsal horn at each rostro-caudal level. Three sections from each part of the spinal segments were randomly selected for statistical analysis, and the average of three sections was recorded for each rat. To confirm the reliability of double immunofluorescence labeling, a single stimulation, either

Fig. 3  Induction of c-Fos-IR cell profiles in the spinal dorsal horn at the L3 (a, c) and L5 (b, d) levels following sham ES (0 mA) of the sciatic (a, b) or saphenous (c, d) nerve in naïve rats. Dark reaction products representing c-Fos protein-like immunoreactivity were seen in the well-demarcated round to oval profile. The profiles were

judged to be neuronal nuclei from their diameters (5–8 µm). Sham ES of the sciatic or saphenous nerve induced a small number of c-Fos-IR cell profiles in superficial laminae I/II of the spinal dorsal horn. D, L, M and V in a indicate dorsal, lateral, medial and ventral directions, respectively. Scale bar 100 µm

Fig. 2  Schematic illustration of expected immunofluorescence labeling for c-Fos and p-ERK in the model of convergent nociceptive input after the saphenous nerve injury. Double-labeled cell profiles in nerve-injured rats are considered to represent the neurons receiving convergent sensory signals from both saphenous and sciatic nerves

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noxious heat stimulation or ES of the saphenous nerve, was conducted in a separate group of animals (n = 3 for each stimulation group) and processed for double immunofluorescence labeling.

Results

Statistical analysis Results are presented as the mean ± SEM. Statistical analysis was performed using a one-way analysis of variance (ANOVA) followed by the post hoc Tukey–Kramer test or Student’s t test. The criterion used for significance was P