ORIGINAL ARTICLE
Morphological and functional changes in regenerated primary afferent fibres following mental and inferior alveolar nerve transection Y. Tsuboi1,2*, K. Honda1,3*, Y.C. Bae4, M. Shinoda1,2, M. Kondo1,2, A. Katagiri1,2, S. Echizenya1, S. Kamakura1, J. Lee5, K. Iwata1,2 1 2 3 4 5
Department of Physiology, Nihon University School of Dentistry, Tokyo, Japan Division of Functional Morphology, Dental Research Center, Nihon University School of Dentistry, Tokyo, Japan Department of Oral and Maxillofacial Surgery, Nihon University School of Dentistry, Tokyo, Japan Department of Oral Anatomy, School of Dentistry, Kyungpook National University, Daegu, Korea Department of Prosthodontics, Nihon University School of Dentistry, Tokyo, Japan
Correspondence Koichi Iwata E-mail: [email protected] Funding sources MEXT-Supported Program for Strategic Research Foundation at Private Universities, 2013–2014, Grant-in-Aid for Challenging Exploratory Research (24659832) from the Japanese Society for Promotion of Science, Grant-in-Aid for Scientific Research (C) (24593064, 24592818 and 26460708) from the Japanese Society for Promotion of Science and a Grant-in-Aid for Young Scientists (B) (26861750) from the Japanese Society for Promotion of Science. Conflicts of interest None declared. *YT and KH have equally contributed to this paper. Accepted for publication 20 November 2014 doi:10.1002/ejp.650
Abstract Background: It is important to know the mechanisms underlying pain abnormalities associated with inferior alveolar nerve (IAN) regeneration in order to develop the appropriate treatment for orofacial neuropathic pain patients. However, peripheral mechanisms underlying orofacial pain abnormalities following IAN regeneration are not fully understood. Methods: Head withdrawal threshold (HWT), jaw opening reflex (JOR) thresholds, single-fibre recordings of the regenerated mental nerve (MN) fibres, calcitonin gene-related peptide (CGRP), isolectin B4 (IB4), peripherin, neurofilament-200 (NF-200) and transient receptor potential vanilloid 1 (TRPV1) expression in trigeminal ganglion (TG) cells, and electron microscopic (EM) observations of the regenerated MN fibres were studied in MN- and IAN-transected (M-IANX) rats. Results: HWT to mechanical or heat stimulation of the mental skin was significantly lower in M-IANX rats compared with sham rats. Mean conduction velocity of action potentials recorded from MN fibres (n = 124) was significantly slower in M-IANX rats compared with sham rats. The percentage of Fluoro-Gold (FG)-labelled CGRP-, peripherin- or TRPV1immunoreactive (IR) cells was significantly larger in M-IANX rats compared with that of sham rats, whereas that of FG-labelled IB4- and NF-200-IR cells was significantly smaller in M-IANX rats compared with sham rats. Largesized myelinated nerve fibres were rarely observed in M-IANX rats, whereas large-sized unmyelinated nerve fibres were frequently observed and were aggregated in the bundles at the distal portion of regenerated axons. Conclusions: These findings suggest that the demyelination of MN fibres following regeneration may be involved in peripheral sensitization, resulting in the orofacial neuropathic pain associated with trigeminal nerve injury.
1. Introduction It has been reported that injured nerve fibres regenerate and reinnervate the orofacial regions quite a while after the injury (Imai et al., 2003; Robinson 1258 Eur J Pain 19 (2015) 1258--1266
et al., 2004). Some possible mechanisms have been implicated in regeneration of injured nerve fibres. After degeneration (Wallerian degeneration) of the distal portion of injured nerve fibres, non-neuronal Schwann cells are reorganized along the degenerated © 2015 European Pain Federation - EFICâ
Y. Tsuboi et al.
What’s already known about this topic? • Pain abnormalities associated with peripheral nerve injury occur due to reinnervation of injured nerve fibres. What does this study add? • The demyelination of regenerated nerve fibres of the mental nerve in this case is involved in peripheral sensitization, resulting in the orofacial neuropathic pain associated with trigeminal nerve injury.
axons, and the injured nerve fibres are regenerated along the reorganized Schwann cells. It is assumed that these series of regeneration mechanisms following nerve injury are involved in pain abnormalities associated with reinnervation of regenerated nerve fibres (Saito et al., 2008; Nakagawa et al., 2010). Clinically, orofacial neuropathic pain due to tooth extraction, pulpectomy or dental implantation is difficult to treat and in some patients can last for several years. Inferior alveolar nerve (IAN) including mental nerve (MN) (third branch of the trigeminal nerve) is located in the mandibular canal in rats, and most of IAN fibres consist of sensory fibres that innervate tooth pulp, periodontal tissues, dental gum and mental skin (Carter and Keen, 1971; Kqiku et al., 2011). We hypothesized that these unique structures might be involved in the severe orofacial neuropathic pain associated with IAN injury. To evaluate the mechanisms that underlie orofacial pain abnormalities associated with IAN injury, MN and IAN transection (M-IANX) models have been developed in rats (Iwata et al., 2001; Nomura et al., 2002; Tsuboi et al., 2004; Ogawa et al., 2005; Sugiyama et al., 2013). Seven to 14 days after IANX, transected IAN was regenerated and reinnervated. The peripheral terminals of regenerated IAN were also morphologically similar to those of the originally innervated periodontal tissues (Wakisaka et al., 2000; Imai et al., 2003). Nakagawa et al. (2010) have also reported that about 70% of transected IAN fibres are regenerated 14 days after the transection in rats, and many of Aδ fibres become hyperactive with high-frequency background activity and enhanced mechanical-evoked responses after regeneration. The expression of various molecules such as neuropeptide Y, vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide (CGRP) and transient receptor potential vanilloid 1 (TRPV1) is also known to be altered in trigeminal ganglion (TG) cells follow© 2015 European Pain Federation - EFICâ
Neuropathic pain associated with nerve regeneration
ing trigeminal nerve transection. It is very important to know the mechanisms underlying pain abnormalities that are associated with IAN regeneration in order to develop the appropriate treatment for orofacial neuropathic pain patients. However, peripheral mechanisms underlying orofacial pain abnormalities associated with IAN regeneration are not fully understood. Thus, we focused on the regenerated M-IAN fibres and TG cells to clarify the peripheral mechanisms underlying orofacial pain abnormalities associated with IAN regeneration. We studied head withdrawal threshold (HWT), jaw opening reflex (JOR) elicited by MN stimulation, changes in molecular marker expression, neuronal activity and morphological changes in the regenerated MN fibres in M-IANX rats.
2. Materials and methods 2.1 Animals Male Sprague Dawley rats (Japan SLC, Shizuoka, Japan) were used in this study. Rats were maintained in a climatecontrolled room on a 12 h light/dark cycle (lights on at 07:00 h) with food and water available ad libitum. Efforts were made to minimize the number of animals used and their suffering. To reduce the number of animals, single-unit recording experiments have been conducted in the same animals used for JOR threshold measurement. We used the enough number of animals for the statistical analysis in each experiment. This study was conducted in accordance with the ethical guidelines of the International Association for the Study of Pain (Zimmermann, 1983) and approved by the local animal ethics committee in accordance with the Guidelines for Animal Experiments in Nihon University.
2.2 MN and IAN transection Rats were anaesthetized with sodium pentobarbital (50 mg/ kg, i.p.) and placed on a warm mat. The left facial skin was incised 10 mm and then a small incision was made in the masseter muscle. The mandibular bone was exposed, and the bone covering the MN and IAN was removed. Subsequently, MN and IAN were cut and repositioned in the alveolar canal, and the masseter and facial skin were sutured.
2.3 JOR threshold and HWT measurement The JOR threshold was measured to make sure whether the transected M-IAN was regenerated on day 14 and was also measured to obtain the activation threshold values of regenerated MN. Rats were anaesthetized with sodium pentobarbital (50 mg/kg, i.p.) and left mental skin was incised and MN was exposed, and bipolar wire electrodes were placed on the MN for electrical stimulation (Fig. 1A). Another pair of Eur J Pain 19 (2015) 1258--1266
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Figure 1 Schematic illustration of the single-fibre recording and stimulation of the mental nerve in mental and inferior alveolar nerve-transected (M-IANX) or sham rats. Stimulus electrodes were placed at the distal stump at 9.00–11.75 mm to the transected end, the recording electrode was placed at the proximal to the stump and about 0.5 mm proximal to the stump end.
wire electrodes was inserted into the left digastric muscle to record JOR elicited by electrical stimulation of the MN (duration: 200 μs, frequency: 1 Hz). After completing the surgery, anaesthesia was maintained throughout the experiment by continuous inhalation of isoflurane (1.0%; Mylan, Canonsburg, PA, USA) mixed with 100% oxygen (2.0 L/min). JOR threshold was measured following electrical stimulation of the MN in M-IANX or sham rats. Bipolar wire electrodes were also inserted into the trapezius muscle to record electromyography (EMG) activities in isoflurane (1.0–2.0%) anaesthetized rats. Graded mechanical or heat stimuli were applied to the mental skin, and threshold intensities high enough to elicit EMG activities in trapezius muscle were recorded. The HWTs were defined as the stimulus intensity to elicit small EMG activities.
2.4 Single-fibre recording A total of 14 rats were used for single-fibre recording experiments (sham; n = 7, M-IANX; n = 7). Rats were anaesthetized with 2.0% isoflurane mixed with 100% oxygen (2.0 L/ min) and the trachea and left femoral vein were cannulated to allow artificial respiration and intravenous administration of drugs, respectively. Rats were mounted on surgical stage at a lateral position. Left facial skin was incised and the muscles over mandibular bone were pushed aside, and then the IAN and MN were exposed (Fig. 1). During single-fibre recording, rats were immobilized with pancuronium bromide (1 mg/ kg/h, i.v.), anaesthetized with continuous inhalation of isoflurane (1.0%) mixed with 100% oxygen (2.0 L/min) and ventilated artificially. Rectal temperature was maintained at 37–38 °C by a thermostatically controlled heating pad (TR 100, FST Inc., Foster City, CA, USA). An electrocardiogram was also monitored and the heart rate was maintained between 333 and 430 beats/min during the experiments. When the heart rate increased following mechanical stimulation of the face, the concentration of isoflurane was increased to 1.2%. 1260 Eur J Pain 19 (2015) 1258--1266
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The enamel-coated tungsten microelectrode (10 Mohm, FST Inc.) was advanced at 1 μm steps into the proximal site to the cut end of the MN and single-fibre activities were recorded (Fig. 1B). When single-fibre activities were isolated, the waveform of each unit was amplified using a differential amplifier (high cut-off: 3 kHz, low cut-off: 30 Hz; Plexon Inc., Dallas, TX, USA) and identified using Spike 2 software (CED, Cambridge, UK). Spontaneous activities were first recorded for 60 s before applying electrical stimulation to the MN. The spike latency in each unit following MN stimulation was measured. Responses were saved on computer disk for subsequent offline analysis of signals. After completion of recordings, animals were deeply anaesthetized with sodium pentobarbital (100 mg/kg, i.p.) and sacrificed, and the left mandibular bone was removed. The length of conduction pathway between stimulus and recording sites was measured, and conduction velocity (CV) of action potentials was calculated.
2.5 CGRP, isolectin B4 (IB4), peripherin, neurofilament-200 (NF-200) and TRPV1 immunohistochemistry Rats were anaesthetized with sodium pentobarbital (50 mg/ kg, i.p.), and 10 μL of 3% hydroxystilbamidine, Fluoro-Gold (FG, Fluorochrome, Denver, CO, USA) was injected subcutaneously into the mental skin with a 30-gauge needle 3 days before perfusion in order to label regenerated primary neurons. On day 14 after M-IANX, rats were anaesthetized with sodium pentobarbital (100 mg/kg, i.p.) and transcardially perfused with isotonic saline (500 mL) followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4, 500 mL). The ipsilateral TGs were removed and post-fixed with 4% PFA for 12 h at 4 °C. The tissues were then transferred to 20% sucrose (w/v) in phosphate buffered saline (PBS) for 12 h at 4 °C for cryoprotection. The specimens were embedded in TissueTek (Sakura Finetek, Tokyo, Japan) and stored at −20 °C until cryosectioning. TGs were cut on the horizontal plane along the long axis of the ganglion using a cryostat (Leica CM1850, Leica Microsystems, Wetzlar, Germany) at a thickness of 10 μm. The sections were thaw mounted onto MAS-coated glass slides (Matsunami, Osaka, Japan). For analysis, every 10th section (five sections per rat) was collected. The sections were incubated in 4% normal donkey serum (NDS) in 0.01 M PBS with 0.3% Triton X-100 for 2 h at room temperature (RT), and then incubated in sheep anti-CGRP (1:2000, Abcam, Cambridge, UK), rabbit anti-peripherin (1:200, EMD Millipore, Billerica, MA, USA), mouse anti-NF200 (10 μg/mL, EMD Millipore) or rabbit anti-TRPV1 antibody (1:200, Alomone labs, Jerusalem, Israel) in 0.01 M PBS with 0.3% Triton X-100 and 4% NDS for 12 h at 4 °C. After rinsing with 0.01 M PBS, sections were incubated in Alexa Fluor 488 donkey anti-sheep IgG (1:200, Invitrogen, Paisley, UK), Alexa Fluor 488 donkey anti-rabbit IgG (1:200, Invitrogen) or Alexa Fluor 488 donkey anti-mouse IgG (1:200, Invitrogen) for 2 h at RT. After rinsing with 0.01 M PBS, © 2015 European Pain Federation - EFICâ
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sections were coverslipped in mounting medium (Thermo Fisher Scientific, Waltham, MA, USA). Negative controls were prepared as above without primary antibodies. Moreover, the sections were incubated in 4% NDS in 0.01 M PBS with 0.3% Triton X-100 for 2 h at RT, and then incubated in fluorescence-conjugated Griffonia simplicifolia IB4 (FITC-IB4, 10 mg/mL, Sigma, St. Louis, MO, USA) in 0.01 M PBS with 0.3% Triton X-100 for 12 h at 4 °C. Double-labelled (FG with Alexa Fluor 488) cells were identified using a fluorescence microscope (BZ-9000, Keyence, Osaka, Japan). Mean percentage of FG-labelled CGRP-immunoreactive (IR), IB4-IR, peripherin-IR, NF-200-IR or TRPV1-IR cells was calculated with the following formula: Mean percentage = the number of FG-labelled CGRP-, IB4-, peripherin-, NF-200- or TRPV1-cells/the number of FG-labelled cells × 100.
2.6 Electron microscopic (EM) study Six male rats were used in the EM study. On day 14 after M-IANX, rats were anaesthetized with sodium pentobarbital (100 mg/kg, i.p.) and perfused transcardially with 100 mL heparinized normal saline. Portions of MN at the proximal and distal to the transection site were removed and treated with a fixative solution containing 2.5% glutaraldehyde, 1% PFA and 0.1% picric acid in phosphate buffer (PB; 0.1 M, pH 7.4) for 2 h at 4 °C, and 60 μm-thick transverse sections were cut on a Vibratome. Sections were postfixed for 1 h with 1% osmium tetroxide in PB, dehydrated in graded alcohols, flat embedded in Durcupan ACM (Fluka, Buchs, Switzerland) and cured for 48 h at 60 °C. Chips containing MN portions proximal and distal to the transection site were cut out of the wafers and glued onto blank resin blocks with cyanoacrylate. Sections from each block were collected on formvar-coated single-slot nickel grids. The grids were counterstained with both uranyl acetate and lead citrate and then examined on a Hitachi H-7500 electron microscope (Hitachi, Tokyo, Japan) at 80 kV accelerating voltage. Images were acquired using Digital Micrograph software driving a SC1000 camera (Gatan, Pleasanton, CA, USA) attached to the microscope. The images were saved as TIFF files, and brightness and contrast were adjusted in Photoshop CS3 (Adobe Systems, San Jose, CA, USA). For quantitative analysis of the fibre types contained in each proximal and distal portion of the MN, electron micrographs were taken at ×10,000 original magnifications from three areas of the sections in three MN–IANX rats. Fibre types in each section were counted and analysed using a digitizing tablet and Image J software (NIH, Bethesda, MD, USA).
2.7 Statistical analysis Data were expressed as means ± SEM. Statistical analyses were performed by Student’s t-test or Mann–Whitney U-test where appropriate. A value of p < 0.05 was considered significant. © 2015 European Pain Federation - EFICâ
Neuropathic pain associated with nerve regeneration
3. Results 3.1 HWT to mechanical and heat stimulation To obtain an accurate threshold to activate A- and C-fibres, graded mechanical or heat stimuli were applied to the mental skin in this study. In order to measure the accurate HWT onset, EMG activity was recorded from the trapezius muscle in lightly anaesthetized rats. We observed significant decrease in the HWT to mechanical or heat stimulation of the mental skin in M-IANX rats compared with sham rats on day 14 (Fig. 2A: mechanical stimulation, Fig. 2B: heat stimulation).
3.2 JOR threshold following electrical stimulation of the MN It is well known that JOR is elicited by noxious stimulation of the orofacial regions, and JOR threshold is thought to be a reliable indicator to evaluate the activation threshold of trigeminal nociceptive afferents (Iwata et al., 1998). To obtain the activation threshold values of regenerated MN, JOR threshold values were measured in each animal. EMG activities were recorded from the anterior belly of digastric muscle by electrical stimulation of regenerated MN at the distal portion to the transected site or sham-treated MN (Fig. 1A). Typical digastric EMG activities elicited by MN stimulation were shown in Fig. 3A (a: Sham, b: M-IANX). JOR threshold elicited by electrical stimulation of the MN was not different between M-IANX and
Figure 2 Head withdrawal reflex threshold (HWT) to graded mechanical (A) or heat (B) stimulation of the mental skin in lightly anaesthetized sham (open bars) or mental and inferior alveolar nerve-transected (M-IANX) (solid bars) rats. HWT was calculated as the stimulus intensities to elicited electromyography activities in trapezius muscle following graded mechanical or heat stimulation of the mental skin. **p < 0.01.
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Figure 3 Jaw opening reflex (JOR) elicited by electrical stimulation of the inferior alveolar nerve (IAN) distal portion to the transected region in mental and IAN-transected (M-IANX) and sham rats. Typical electromyography activities in sham and M-IANX rats 14 days after the surgery (A). Mean threshold intensities eliciting JOR (B). Mean relative threshold intensities evoking spikes following electrical stimulation of the IAN distal to the transected portion (C). Relative threshold values were calculated as relative values of the JOR threshold. Conduction velocity (CV) of action potentials recorded from the transected IAN fibres just proximal from the transection site. Frequency histogram of CV in sham rats (D) and M-IANX rat (E). Mean CVs of MN fibres with ≦2 m/s and those with >2 m/s in sham and M-IANX rats (F). **p < 0.01.
sham rats (Fig. 3B), indicating that transected MN has been functionally regenerated by day 14.
3.3 MN fibre activity A total of 124 single-fibre activities were recorded from the MN at the proximal portion to the transected site. We evaluated the threshold intensity evoking spikes in the MN as relative values for JOR threshold. There were no significant differences between JOR threshold and relative threshold values evoking spikes in MN between M-IANX and sham rats (Fig. 3C). CV of spikes in MN fibres distal to the M-IANX site or sham operation was calculated in M-IANX and sham rats. Many MN fibres showed slow CVs such as C- or small diameter Aδ-fibres in M-IANX rats, whereas fast CVs such as large diameter A-fibres were obtained in sham rats (Fig. 3D and E). Mean CV of MN fibres with ≦2 m/s was not different between M-IANX and sham rats, whereas that of >2 m/s MN fibres was significantly slower in M-IANX rats compared with sham rats (Fig. 3F).
3.4 CGRP, IB4, peripherin, NF-200 and TRPV1 expression in TG cells About 109 and 197 TG cells were labelled with FG injected into the mental skin subcutaneously in M-IANX and sham rats, respectively. We counted the number of FG-labelled CGRP-, IB4-, peripherin-, NF-200- or TRPV1-IR cells in the TG. Some of the 1262 Eur J Pain 19 (2015) 1258--1266
CGRP-, IB4-, peripherin-, NF-200- or TRPV1-IR cells were labelled with FG (Fig. 4A–C, E–G, I–K, M–O and Q–S). The number of FG-labelled CGRP-, IB4-, peripherin-, NF-200- or TRPV1-IR cells was also counted. Mean percentage of FG-labelled CGRP-, peripherin- and TRPV1-IR cells was significantly larger in M-IANX rats compared with that of sham rats (Fig. 4D, L and T). On the other hand, the percentage of FG-labelled IB4- and NF-200-IR cells was significantly smaller in M-IANX rats compared with sham rats (Fig. 4H and P).
3.5 EM observation of regenerated MN fibres The MN proximal to the transection site showed normal morphological features as observed in the intact sensory nerve. It contained many large myelinated fibres of Aβ size (>20 μm2 in cross-sectional area that is equivalent to >5 μm in diameter, 19.1 ± 2.4%), small myelinated fibres of Aδ size (
Morphological and functional changes in regenerated primary afferent fibres following mental and inferior alveolar nerve transection Y. Tsuboi1,2*, K. Honda1,3*, Y.C. Bae4, M. Shinoda1,2, M. Kondo1,2, A. Katagiri1,2, S. Echizenya1, S. Kamakura1, J. Lee5, K. Iwata1,2 1 2 3 4 5
Department of Physiology, Nihon University School of Dentistry, Tokyo, Japan Division of Functional Morphology, Dental Research Center, Nihon University School of Dentistry, Tokyo, Japan Department of Oral and Maxillofacial Surgery, Nihon University School of Dentistry, Tokyo, Japan Department of Oral Anatomy, School of Dentistry, Kyungpook National University, Daegu, Korea Department of Prosthodontics, Nihon University School of Dentistry, Tokyo, Japan
Correspondence Koichi Iwata E-mail: [email protected] Funding sources MEXT-Supported Program for Strategic Research Foundation at Private Universities, 2013–2014, Grant-in-Aid for Challenging Exploratory Research (24659832) from the Japanese Society for Promotion of Science, Grant-in-Aid for Scientific Research (C) (24593064, 24592818 and 26460708) from the Japanese Society for Promotion of Science and a Grant-in-Aid for Young Scientists (B) (26861750) from the Japanese Society for Promotion of Science. Conflicts of interest None declared. *YT and KH have equally contributed to this paper. Accepted for publication 20 November 2014 doi:10.1002/ejp.650
Abstract Background: It is important to know the mechanisms underlying pain abnormalities associated with inferior alveolar nerve (IAN) regeneration in order to develop the appropriate treatment for orofacial neuropathic pain patients. However, peripheral mechanisms underlying orofacial pain abnormalities following IAN regeneration are not fully understood. Methods: Head withdrawal threshold (HWT), jaw opening reflex (JOR) thresholds, single-fibre recordings of the regenerated mental nerve (MN) fibres, calcitonin gene-related peptide (CGRP), isolectin B4 (IB4), peripherin, neurofilament-200 (NF-200) and transient receptor potential vanilloid 1 (TRPV1) expression in trigeminal ganglion (TG) cells, and electron microscopic (EM) observations of the regenerated MN fibres were studied in MN- and IAN-transected (M-IANX) rats. Results: HWT to mechanical or heat stimulation of the mental skin was significantly lower in M-IANX rats compared with sham rats. Mean conduction velocity of action potentials recorded from MN fibres (n = 124) was significantly slower in M-IANX rats compared with sham rats. The percentage of Fluoro-Gold (FG)-labelled CGRP-, peripherin- or TRPV1immunoreactive (IR) cells was significantly larger in M-IANX rats compared with that of sham rats, whereas that of FG-labelled IB4- and NF-200-IR cells was significantly smaller in M-IANX rats compared with sham rats. Largesized myelinated nerve fibres were rarely observed in M-IANX rats, whereas large-sized unmyelinated nerve fibres were frequently observed and were aggregated in the bundles at the distal portion of regenerated axons. Conclusions: These findings suggest that the demyelination of MN fibres following regeneration may be involved in peripheral sensitization, resulting in the orofacial neuropathic pain associated with trigeminal nerve injury.
1. Introduction It has been reported that injured nerve fibres regenerate and reinnervate the orofacial regions quite a while after the injury (Imai et al., 2003; Robinson 1258 Eur J Pain 19 (2015) 1258--1266
et al., 2004). Some possible mechanisms have been implicated in regeneration of injured nerve fibres. After degeneration (Wallerian degeneration) of the distal portion of injured nerve fibres, non-neuronal Schwann cells are reorganized along the degenerated © 2015 European Pain Federation - EFICâ
Y. Tsuboi et al.
What’s already known about this topic? • Pain abnormalities associated with peripheral nerve injury occur due to reinnervation of injured nerve fibres. What does this study add? • The demyelination of regenerated nerve fibres of the mental nerve in this case is involved in peripheral sensitization, resulting in the orofacial neuropathic pain associated with trigeminal nerve injury.
axons, and the injured nerve fibres are regenerated along the reorganized Schwann cells. It is assumed that these series of regeneration mechanisms following nerve injury are involved in pain abnormalities associated with reinnervation of regenerated nerve fibres (Saito et al., 2008; Nakagawa et al., 2010). Clinically, orofacial neuropathic pain due to tooth extraction, pulpectomy or dental implantation is difficult to treat and in some patients can last for several years. Inferior alveolar nerve (IAN) including mental nerve (MN) (third branch of the trigeminal nerve) is located in the mandibular canal in rats, and most of IAN fibres consist of sensory fibres that innervate tooth pulp, periodontal tissues, dental gum and mental skin (Carter and Keen, 1971; Kqiku et al., 2011). We hypothesized that these unique structures might be involved in the severe orofacial neuropathic pain associated with IAN injury. To evaluate the mechanisms that underlie orofacial pain abnormalities associated with IAN injury, MN and IAN transection (M-IANX) models have been developed in rats (Iwata et al., 2001; Nomura et al., 2002; Tsuboi et al., 2004; Ogawa et al., 2005; Sugiyama et al., 2013). Seven to 14 days after IANX, transected IAN was regenerated and reinnervated. The peripheral terminals of regenerated IAN were also morphologically similar to those of the originally innervated periodontal tissues (Wakisaka et al., 2000; Imai et al., 2003). Nakagawa et al. (2010) have also reported that about 70% of transected IAN fibres are regenerated 14 days after the transection in rats, and many of Aδ fibres become hyperactive with high-frequency background activity and enhanced mechanical-evoked responses after regeneration. The expression of various molecules such as neuropeptide Y, vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide (CGRP) and transient receptor potential vanilloid 1 (TRPV1) is also known to be altered in trigeminal ganglion (TG) cells follow© 2015 European Pain Federation - EFICâ
Neuropathic pain associated with nerve regeneration
ing trigeminal nerve transection. It is very important to know the mechanisms underlying pain abnormalities that are associated with IAN regeneration in order to develop the appropriate treatment for orofacial neuropathic pain patients. However, peripheral mechanisms underlying orofacial pain abnormalities associated with IAN regeneration are not fully understood. Thus, we focused on the regenerated M-IAN fibres and TG cells to clarify the peripheral mechanisms underlying orofacial pain abnormalities associated with IAN regeneration. We studied head withdrawal threshold (HWT), jaw opening reflex (JOR) elicited by MN stimulation, changes in molecular marker expression, neuronal activity and morphological changes in the regenerated MN fibres in M-IANX rats.
2. Materials and methods 2.1 Animals Male Sprague Dawley rats (Japan SLC, Shizuoka, Japan) were used in this study. Rats were maintained in a climatecontrolled room on a 12 h light/dark cycle (lights on at 07:00 h) with food and water available ad libitum. Efforts were made to minimize the number of animals used and their suffering. To reduce the number of animals, single-unit recording experiments have been conducted in the same animals used for JOR threshold measurement. We used the enough number of animals for the statistical analysis in each experiment. This study was conducted in accordance with the ethical guidelines of the International Association for the Study of Pain (Zimmermann, 1983) and approved by the local animal ethics committee in accordance with the Guidelines for Animal Experiments in Nihon University.
2.2 MN and IAN transection Rats were anaesthetized with sodium pentobarbital (50 mg/ kg, i.p.) and placed on a warm mat. The left facial skin was incised 10 mm and then a small incision was made in the masseter muscle. The mandibular bone was exposed, and the bone covering the MN and IAN was removed. Subsequently, MN and IAN were cut and repositioned in the alveolar canal, and the masseter and facial skin were sutured.
2.3 JOR threshold and HWT measurement The JOR threshold was measured to make sure whether the transected M-IAN was regenerated on day 14 and was also measured to obtain the activation threshold values of regenerated MN. Rats were anaesthetized with sodium pentobarbital (50 mg/kg, i.p.) and left mental skin was incised and MN was exposed, and bipolar wire electrodes were placed on the MN for electrical stimulation (Fig. 1A). Another pair of Eur J Pain 19 (2015) 1258--1266
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Neuropathic pain associated with nerve regeneration
Figure 1 Schematic illustration of the single-fibre recording and stimulation of the mental nerve in mental and inferior alveolar nerve-transected (M-IANX) or sham rats. Stimulus electrodes were placed at the distal stump at 9.00–11.75 mm to the transected end, the recording electrode was placed at the proximal to the stump and about 0.5 mm proximal to the stump end.
wire electrodes was inserted into the left digastric muscle to record JOR elicited by electrical stimulation of the MN (duration: 200 μs, frequency: 1 Hz). After completing the surgery, anaesthesia was maintained throughout the experiment by continuous inhalation of isoflurane (1.0%; Mylan, Canonsburg, PA, USA) mixed with 100% oxygen (2.0 L/min). JOR threshold was measured following electrical stimulation of the MN in M-IANX or sham rats. Bipolar wire electrodes were also inserted into the trapezius muscle to record electromyography (EMG) activities in isoflurane (1.0–2.0%) anaesthetized rats. Graded mechanical or heat stimuli were applied to the mental skin, and threshold intensities high enough to elicit EMG activities in trapezius muscle were recorded. The HWTs were defined as the stimulus intensity to elicit small EMG activities.
2.4 Single-fibre recording A total of 14 rats were used for single-fibre recording experiments (sham; n = 7, M-IANX; n = 7). Rats were anaesthetized with 2.0% isoflurane mixed with 100% oxygen (2.0 L/ min) and the trachea and left femoral vein were cannulated to allow artificial respiration and intravenous administration of drugs, respectively. Rats were mounted on surgical stage at a lateral position. Left facial skin was incised and the muscles over mandibular bone were pushed aside, and then the IAN and MN were exposed (Fig. 1). During single-fibre recording, rats were immobilized with pancuronium bromide (1 mg/ kg/h, i.v.), anaesthetized with continuous inhalation of isoflurane (1.0%) mixed with 100% oxygen (2.0 L/min) and ventilated artificially. Rectal temperature was maintained at 37–38 °C by a thermostatically controlled heating pad (TR 100, FST Inc., Foster City, CA, USA). An electrocardiogram was also monitored and the heart rate was maintained between 333 and 430 beats/min during the experiments. When the heart rate increased following mechanical stimulation of the face, the concentration of isoflurane was increased to 1.2%. 1260 Eur J Pain 19 (2015) 1258--1266
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The enamel-coated tungsten microelectrode (10 Mohm, FST Inc.) was advanced at 1 μm steps into the proximal site to the cut end of the MN and single-fibre activities were recorded (Fig. 1B). When single-fibre activities were isolated, the waveform of each unit was amplified using a differential amplifier (high cut-off: 3 kHz, low cut-off: 30 Hz; Plexon Inc., Dallas, TX, USA) and identified using Spike 2 software (CED, Cambridge, UK). Spontaneous activities were first recorded for 60 s before applying electrical stimulation to the MN. The spike latency in each unit following MN stimulation was measured. Responses were saved on computer disk for subsequent offline analysis of signals. After completion of recordings, animals were deeply anaesthetized with sodium pentobarbital (100 mg/kg, i.p.) and sacrificed, and the left mandibular bone was removed. The length of conduction pathway between stimulus and recording sites was measured, and conduction velocity (CV) of action potentials was calculated.
2.5 CGRP, isolectin B4 (IB4), peripherin, neurofilament-200 (NF-200) and TRPV1 immunohistochemistry Rats were anaesthetized with sodium pentobarbital (50 mg/ kg, i.p.), and 10 μL of 3% hydroxystilbamidine, Fluoro-Gold (FG, Fluorochrome, Denver, CO, USA) was injected subcutaneously into the mental skin with a 30-gauge needle 3 days before perfusion in order to label regenerated primary neurons. On day 14 after M-IANX, rats were anaesthetized with sodium pentobarbital (100 mg/kg, i.p.) and transcardially perfused with isotonic saline (500 mL) followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4, 500 mL). The ipsilateral TGs were removed and post-fixed with 4% PFA for 12 h at 4 °C. The tissues were then transferred to 20% sucrose (w/v) in phosphate buffered saline (PBS) for 12 h at 4 °C for cryoprotection. The specimens were embedded in TissueTek (Sakura Finetek, Tokyo, Japan) and stored at −20 °C until cryosectioning. TGs were cut on the horizontal plane along the long axis of the ganglion using a cryostat (Leica CM1850, Leica Microsystems, Wetzlar, Germany) at a thickness of 10 μm. The sections were thaw mounted onto MAS-coated glass slides (Matsunami, Osaka, Japan). For analysis, every 10th section (five sections per rat) was collected. The sections were incubated in 4% normal donkey serum (NDS) in 0.01 M PBS with 0.3% Triton X-100 for 2 h at room temperature (RT), and then incubated in sheep anti-CGRP (1:2000, Abcam, Cambridge, UK), rabbit anti-peripherin (1:200, EMD Millipore, Billerica, MA, USA), mouse anti-NF200 (10 μg/mL, EMD Millipore) or rabbit anti-TRPV1 antibody (1:200, Alomone labs, Jerusalem, Israel) in 0.01 M PBS with 0.3% Triton X-100 and 4% NDS for 12 h at 4 °C. After rinsing with 0.01 M PBS, sections were incubated in Alexa Fluor 488 donkey anti-sheep IgG (1:200, Invitrogen, Paisley, UK), Alexa Fluor 488 donkey anti-rabbit IgG (1:200, Invitrogen) or Alexa Fluor 488 donkey anti-mouse IgG (1:200, Invitrogen) for 2 h at RT. After rinsing with 0.01 M PBS, © 2015 European Pain Federation - EFICâ
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sections were coverslipped in mounting medium (Thermo Fisher Scientific, Waltham, MA, USA). Negative controls were prepared as above without primary antibodies. Moreover, the sections were incubated in 4% NDS in 0.01 M PBS with 0.3% Triton X-100 for 2 h at RT, and then incubated in fluorescence-conjugated Griffonia simplicifolia IB4 (FITC-IB4, 10 mg/mL, Sigma, St. Louis, MO, USA) in 0.01 M PBS with 0.3% Triton X-100 for 12 h at 4 °C. Double-labelled (FG with Alexa Fluor 488) cells were identified using a fluorescence microscope (BZ-9000, Keyence, Osaka, Japan). Mean percentage of FG-labelled CGRP-immunoreactive (IR), IB4-IR, peripherin-IR, NF-200-IR or TRPV1-IR cells was calculated with the following formula: Mean percentage = the number of FG-labelled CGRP-, IB4-, peripherin-, NF-200- or TRPV1-cells/the number of FG-labelled cells × 100.
2.6 Electron microscopic (EM) study Six male rats were used in the EM study. On day 14 after M-IANX, rats were anaesthetized with sodium pentobarbital (100 mg/kg, i.p.) and perfused transcardially with 100 mL heparinized normal saline. Portions of MN at the proximal and distal to the transection site were removed and treated with a fixative solution containing 2.5% glutaraldehyde, 1% PFA and 0.1% picric acid in phosphate buffer (PB; 0.1 M, pH 7.4) for 2 h at 4 °C, and 60 μm-thick transverse sections were cut on a Vibratome. Sections were postfixed for 1 h with 1% osmium tetroxide in PB, dehydrated in graded alcohols, flat embedded in Durcupan ACM (Fluka, Buchs, Switzerland) and cured for 48 h at 60 °C. Chips containing MN portions proximal and distal to the transection site were cut out of the wafers and glued onto blank resin blocks with cyanoacrylate. Sections from each block were collected on formvar-coated single-slot nickel grids. The grids were counterstained with both uranyl acetate and lead citrate and then examined on a Hitachi H-7500 electron microscope (Hitachi, Tokyo, Japan) at 80 kV accelerating voltage. Images were acquired using Digital Micrograph software driving a SC1000 camera (Gatan, Pleasanton, CA, USA) attached to the microscope. The images were saved as TIFF files, and brightness and contrast were adjusted in Photoshop CS3 (Adobe Systems, San Jose, CA, USA). For quantitative analysis of the fibre types contained in each proximal and distal portion of the MN, electron micrographs were taken at ×10,000 original magnifications from three areas of the sections in three MN–IANX rats. Fibre types in each section were counted and analysed using a digitizing tablet and Image J software (NIH, Bethesda, MD, USA).
2.7 Statistical analysis Data were expressed as means ± SEM. Statistical analyses were performed by Student’s t-test or Mann–Whitney U-test where appropriate. A value of p < 0.05 was considered significant. © 2015 European Pain Federation - EFICâ
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3. Results 3.1 HWT to mechanical and heat stimulation To obtain an accurate threshold to activate A- and C-fibres, graded mechanical or heat stimuli were applied to the mental skin in this study. In order to measure the accurate HWT onset, EMG activity was recorded from the trapezius muscle in lightly anaesthetized rats. We observed significant decrease in the HWT to mechanical or heat stimulation of the mental skin in M-IANX rats compared with sham rats on day 14 (Fig. 2A: mechanical stimulation, Fig. 2B: heat stimulation).
3.2 JOR threshold following electrical stimulation of the MN It is well known that JOR is elicited by noxious stimulation of the orofacial regions, and JOR threshold is thought to be a reliable indicator to evaluate the activation threshold of trigeminal nociceptive afferents (Iwata et al., 1998). To obtain the activation threshold values of regenerated MN, JOR threshold values were measured in each animal. EMG activities were recorded from the anterior belly of digastric muscle by electrical stimulation of regenerated MN at the distal portion to the transected site or sham-treated MN (Fig. 1A). Typical digastric EMG activities elicited by MN stimulation were shown in Fig. 3A (a: Sham, b: M-IANX). JOR threshold elicited by electrical stimulation of the MN was not different between M-IANX and
Figure 2 Head withdrawal reflex threshold (HWT) to graded mechanical (A) or heat (B) stimulation of the mental skin in lightly anaesthetized sham (open bars) or mental and inferior alveolar nerve-transected (M-IANX) (solid bars) rats. HWT was calculated as the stimulus intensities to elicited electromyography activities in trapezius muscle following graded mechanical or heat stimulation of the mental skin. **p < 0.01.
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Figure 3 Jaw opening reflex (JOR) elicited by electrical stimulation of the inferior alveolar nerve (IAN) distal portion to the transected region in mental and IAN-transected (M-IANX) and sham rats. Typical electromyography activities in sham and M-IANX rats 14 days after the surgery (A). Mean threshold intensities eliciting JOR (B). Mean relative threshold intensities evoking spikes following electrical stimulation of the IAN distal to the transected portion (C). Relative threshold values were calculated as relative values of the JOR threshold. Conduction velocity (CV) of action potentials recorded from the transected IAN fibres just proximal from the transection site. Frequency histogram of CV in sham rats (D) and M-IANX rat (E). Mean CVs of MN fibres with ≦2 m/s and those with >2 m/s in sham and M-IANX rats (F). **p < 0.01.
sham rats (Fig. 3B), indicating that transected MN has been functionally regenerated by day 14.
3.3 MN fibre activity A total of 124 single-fibre activities were recorded from the MN at the proximal portion to the transected site. We evaluated the threshold intensity evoking spikes in the MN as relative values for JOR threshold. There were no significant differences between JOR threshold and relative threshold values evoking spikes in MN between M-IANX and sham rats (Fig. 3C). CV of spikes in MN fibres distal to the M-IANX site or sham operation was calculated in M-IANX and sham rats. Many MN fibres showed slow CVs such as C- or small diameter Aδ-fibres in M-IANX rats, whereas fast CVs such as large diameter A-fibres were obtained in sham rats (Fig. 3D and E). Mean CV of MN fibres with ≦2 m/s was not different between M-IANX and sham rats, whereas that of >2 m/s MN fibres was significantly slower in M-IANX rats compared with sham rats (Fig. 3F).
3.4 CGRP, IB4, peripherin, NF-200 and TRPV1 expression in TG cells About 109 and 197 TG cells were labelled with FG injected into the mental skin subcutaneously in M-IANX and sham rats, respectively. We counted the number of FG-labelled CGRP-, IB4-, peripherin-, NF-200- or TRPV1-IR cells in the TG. Some of the 1262 Eur J Pain 19 (2015) 1258--1266
CGRP-, IB4-, peripherin-, NF-200- or TRPV1-IR cells were labelled with FG (Fig. 4A–C, E–G, I–K, M–O and Q–S). The number of FG-labelled CGRP-, IB4-, peripherin-, NF-200- or TRPV1-IR cells was also counted. Mean percentage of FG-labelled CGRP-, peripherin- and TRPV1-IR cells was significantly larger in M-IANX rats compared with that of sham rats (Fig. 4D, L and T). On the other hand, the percentage of FG-labelled IB4- and NF-200-IR cells was significantly smaller in M-IANX rats compared with sham rats (Fig. 4H and P).
3.5 EM observation of regenerated MN fibres The MN proximal to the transection site showed normal morphological features as observed in the intact sensory nerve. It contained many large myelinated fibres of Aβ size (>20 μm2 in cross-sectional area that is equivalent to >5 μm in diameter, 19.1 ± 2.4%), small myelinated fibres of Aδ size (
