Peripheral nociceptive mechanisms in an experimental rat model of fibromyalgia induced by repeated cold stress

Journal Pre-proof Peripheral nociceptive mechanisms in an experimental rat model of fibromyalgia induced by repeated cold stress Koji Wakatsuki, Yoshiko T.-Uchimura, Takanori Matsubara, Teruaki Nasu, Kazue Mizumura, Toru Taguchi

PII:

S0168-0102(19)30483-3

DOI:

https://doi.org/10.1016/j.neures.2019.12.015

Reference:

NSR 4348

To appear in:

Neuroscience Research

Received Date:

25 August 2019

Revised Date:

6 December 2019

Accepted Date:

19 December 2019

Please cite this article as: Wakatsuki K, T.-Uchimura Y, Matsubara T, Nasu T, Mizumura K, Taguchi T, Peripheral nociceptive mechanisms in an experimental rat model of fibromyalgia induced by repeated cold stress, Neuroscience Research (2019), doi: https://doi.org/10.1016/j.neures.2019.12.015

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Original Research Report

Peripheral nociceptive mechanisms in an experimental rat model of fibromyalgia induced by repeated cold stress

Koji Wakatsuki1,2), Yoshiko T.-Uchimura3), Takanori Matsubara1), Teruaki Nasu3), Kazue Mizumura3,4), Toru

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Taguchi1,5,6)

1) Department of Neuroscience II, Research Institute of Environmental Medicine, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8601, Japan.

65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, JAPAN.

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2) Department of Functional Anatomy and Neuroscience, Nagoya University Graduate School of Medicine,

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3) Department of Physical Therapy, College of Life and Health Sciences, Chubu University, 1200 Matsumoto-

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cho, Kasugai 487-8501, Japan.

4) Department of Physiology, Nihon University School of Dentistry, 1-8-13 Kandasurugadai, Chiyoda-ku, Tokyo 101-8310, Japan.

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5) Department of Physical Therapy, Faculty of Rehabilitation, Niigata University of Health and Welfare, 1398 Shimami-cho, Kita-ku, Niigata 950-3198, Japan.

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6) Institute for Human Movement and Medical Sciences, Niigata University of Health and Welfare, 1398

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Shimami-cho, Kita-ku, Niigata 950-3198, Japan.

Corresponding Author: Toru Taguchi Department of Physical Therapy, Niigata University of Health and Welfare, 1398 Shimami-cho, Kita-ku, Niigata 950-3198, Japan. Phone & Fax: +81-25-257-4767 E-mail: [email protected] 1

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27 pages, 4 tables, and 5 figures

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Highlights: ● Peripheral neural mechanisms of fibromyalgia (FM) were examined using a rat FM model. ● Muscular nociceptors were sensitized to mechanical stimulation in the FM model. ● Thermal responsiveness and general characteristics of nociceptors were unchanged. ● Muscular mRNAs of neurotrophic factors and inflammatory mediators were unchanged.

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● The facilitated muscular nociceptors could be a target for the treatment of FM.

Abstract

Fibromyalgia (FM) is a debilitating disease characterized by generalized and persistent musculoskeletal

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pain. Although central mechanisms are strongly implicated in the pathogenesis of FM, the involvement of peripheral mechanisms is poorly understood. To understand the peripheral nociceptive mechanisms, we

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examined muscular nociceptors in an FM model, which was made by exposing rats to repeated cold stress (RCS). A single muscle C-fiber nociceptors were identified through the teased fiber technique using ex vivo

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muscle-nerve preparations. Response properties of C-fibers to noxious stimuli were systematically analyzed. Messenger RNA expression of neurotrophic factors and inflammatory mediators were also studied in the

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muscle. In the RCS group, the mechanical response threshold of C-fibers, measured using a ramp mechanical stimulus, was significantly decreased, and the response magnitude was significantly increased in the RCS

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group when compared with the SHAM group, where the environmental temperature was not altered. The general characteristics of C-fibers and the responsiveness to noxious cold and heat stimuli were similar

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between the two groups. Messenger RNAs of neurotrophic factors and inflammatory mediators were not changed in the muscle during and after RCS. These results suggest that augmentation of the mechanical response of muscle C-fiber nociceptors contributes to hyperalgesia in the RCS model.

Key words: repeated cold stress, fibromyalgia, mechanical hyperalgesia, chronic widespread pain, muscular nociceptors, C-fibers, single-fiber recording, neurotrophic factors 3

Abbreviations: FM, fibromyalgia; RCS, repeated cold stress; ICS, intermittent cold stress; SART, specific alteration of rhythm in temperature; EDL, extensor digitorum longus muscle; RF, receptive field; CV, conduction velocity; NGF, nerve growth factor; GDNF, glial cell line-derived neurotrophic factor; BDNF, brain-derived neurotrophic factor; NT-3, neurotrophin-3; IL-1β, interleukin-1β; IL-6, interleukin-6; TNF-α,

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tumour necrosis factor-α; SFN, small fiber neuropathy

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1. Introduction Fibromyalgia (FM) is characterized by chronic widespread pain in deep tissues, and by a variety of concomitant symptoms, such as dysautonomia, psychological disorders, and hypersensitivity to external stimuli. FM is known to be triggered and exacerbated by long-term exposure to stress (Clauw, 2014; Sluka and Clauw, 2016). Many rodent models of FM have been reported (DeSantana et al., 2013), and some of them were developed by exposure to stress, such as through forced swimming (Nazeri et al., 2018), chronic restraint (Scheich et al., 2017), unpredictable sound (Khasar et al., 2009), repeated cold exposure (Nasu et al., 2010), and maternal separation (Green et al., 2011). Among these models, the repeated (intermittent) cold stress (RCS

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or ICS), originally called specific alteration of rhythm in temperature (SART) stress (Kita et al., 1975), in which environmental temperature is rapidly changed at short intervals of 30 min or 1 h between normal (22 ºC) and cold (4 ºC), is particularly useful. First, the RCS/ICS model exhibits persistent pain-related behaviors in rats

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(Nasu et al., 2010) and mice (Kita et al., 1979; Montserrat-de la Paz, 2015; Nishiyori and Ueda, 2008). In RCS rats, chronic and bilateral mechanical nociceptive hypersensitivity was induced preferentially in the deep

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tissues (e.g., muscle) compared with the skin (Nasu et al., 2010; Nasu et al., 2018). The long-lasting and

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widespread nature of nociceptive hypersensitivity in deep tissues is compatible with clinical features of FM. Second, the RCS/ICS model shows a number of comorbid symptoms related to FM, including psychological disorders such as depression and anxiety (Hata et al., 1999; Montserrat-de la Paz, 2015; Nasu et al., 2019) and

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autonomic dysfunctions in circulatory, respiratory, and digestive organs (Itomi et al., 2015; Kita et al., 1975). Third, the RCS/ICS-induced hyperalgesia and comorbidities are reduced by therapeutic drugs used for FM

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patients, including gabapentinoids (Nishiyori and Ueda, 2008; Saeki et al., 2019), serotonin and/or

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noradrenalin reuptake inhibitors (Nishiyori et al., 2011), tri- and tetra-cyclic antidepressants (Hata et al., 1995; Nishiyori et al., 2011), and neurotropin (Nasu et al., 2018; Nasu et al., 2019). Morphine or the related opioids are less effective for relieving pain in patients with FM (Schrepf et al., 2016; Sörensen et al., 1995) and the rodent FM models induced by ICS (Nishiyori et al., 2010) and monoamine depletion (Nagakura et al., 2019). The lack of morphine analgesia is likely to be due to impaired descending serotonergic pain inhibitory system (Nishiyori et al., 2010) and disturbed endogenous opioid system in several pain-processing brain regions (Schrepf et al., 2016). Taken together, the nature of pain, a variety of the concomitant symptoms, and the 5

analgesic drugs fit well with clinical features of FM, suggesting that the RCS/ICS model is useful for the study of FM. FM is assumed to be a heterogeneous condition with complicated pathologies driven by central mechanisms with or without facilitated peripheral inputs in human and animal models (Serra et al., 2014; Sluka and Clauw, 2016). In rodent FM models induced by RCS, the transcription factor interferon regulatory factor 8 (IRF8)—expressed in activated spinal microglia—is involved in tactile allodynia (Akagi et al., 2014). Intrathecal administration of antidepressants exhibited analgesia (Nishiyori et al., 2011). Impaired descending pain inhibition is proposed to be a mechanism underlying nociceptive hypersensitivity (Itomi et al., 2016;

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Kawamura et al., 1998; Nasu et al., 2018; Nishiyori et al., 2010). RCS changed the proteomes prominently in the mesencephalon and cerebellum, and this change was caused by post-translational modification of proteolysis or phosphorylation (Fujisawa et al., 2008). Collectively, central mechanisms are strongly suggested

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in the pathogenesis of FM.

Conversely, in the periphery, patients with FM exhibit signs of epidermal small fiber neuropathy (SFN)

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(Doppler et al., 2015; Evdokimov et al., 2019; Farhad, 2019; Lawson et al., 2018; Oaklander et al. 2013).

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Abnormal hyperexcitability of mechano-insensitive (silent) C-nociceptors in the skin has been reported in patients with FM and SFN (Serra et al. 2014). Sodium channel gene variants in thin-fiber afferents seem to be the common pathological mechanisms for pain phenotype in patients with FM and SFN (Martínez-Lavín.,

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2018; Sopacua et al., 2019). Morphological alterations in muscle tissue exposed to RCS are characterized (Bonaterra et al., 2016), and the expression profiles of pain-related genes are changed in the dorsal root ganglia

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(Kozaki et al., 2015). However, it is unclear whether muscular nociceptors are sensitized to cause nociceptive

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hyperexcitability, and what sensitizes nociceptors in the RCS model. Therefore, in this study, we: 1) systematically investigated alterations of muscular nociceptors; and 2) examined the expression profiles of neurotrophic factors and inflammatory mediators in the muscle, which potentially sensitize nociceptors in order to better understand peripheral neural mechanisms of the RCS-induced FM model.

2. Materials and methods 6

2.1. Animals Fifty male Sprague-Dawley rats (8–11 wk, SLC Inc., Shizuoka, Japan) were used in the present experiment (N = 34 rats for electrophysiology, and N = 16 rats for reverse transcription polymerase chain reaction [RT-PCR] and enzyme-linked immunosorbent assay [ELISA]). The rats were kept two per cage in a clean room with air-conditioning (set temperature: 23 ˚C) under a 12 h light/dark cycle (light between 08.00– 20.00 h). The rats had free access to food and filtered clean water throughout the experiment. The present study was approved by the Institutional Animal Care and Use Committee of Nagoya University and Chubu University, and conducted according to the Ethical Guidelines of the International Association for the Study

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of Pain (Zimmermann, 1983).

2.2. Repeated cold stress

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To develop an animal model of FM, we exposed rats to repeated cold stress (RCS) using a house-made automated chamber according to the same procedure as in our previous study (Nasu et al., 2010). Briefly, rats

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were housed two to three per metal-mesh cage (30 x 29 x 20 cm) and kept in a cold compartment at 4 ºC from

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19.00 h on Day 1 to 10.00 h the next morning. They were then alternately moved to compartments set to either room (22 ºC) or cold (4 ºC) temperature at 30-min intervals from 10.00 h to 17.30 h. The caged rats were automatically transferred over 30 s from one compartment to the other with minimal disturbance to the sleep

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cycle. These procedures were repeated for 5 days. At 10.00 h on Day 6, the rats were moved from the RCS chamber to a normal cage at room temperature to await further testing.

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In electrophysiological experiments, rats in the SHAM (control) group were kept in the same style cage

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and RCS chamber and moved between two compartments like the RCS group, but without any temperature shifts. In RT-PCR and ELISA, naïve rats without any treatment served as a control.

2.3. Single-fiber electrophysiology 2.3.1. Ex vivo muscle-nerve preparations To examine whether peripheral nociceptive mechanisms are involved in RCS-induced deep tissue pain, we analyzed general characteristics and response properties of muscular nociceptors with an ex vivo 7

preparation consisting of common peroneal nerve with the extensor digitorum longus (EDL) muscle connected (Taguchi et al., 2005). Briefly, 8–14 days after the cessation of the RCS, when muscular mechanical hyperalgesia was still evident (Nasu et al., 2010), the EDL muscles with the common peroneal nerve attached were quickly excised from the left and right hindlimbs after euthanasia by inhalation of CO2 gas. The musclenerve preparation was placed in the test chamber with two compartments, and the muscle was superfused with modified Krebs-Henseleit solution, which contained (in mM) 110.9 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3, and 20.0 glucose. The perfusate was continuously bubbled and equilibrated with a gas mixture of 95% O2 and 5% CO2 and maintained at 34.0 ± 0.5 °C (pH 7.4) during the experiment.

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Single C-fiber was searched for and identified by manually probing the receptive field (RF) with a blunt glass rod (Taguchi et al., 2005). The mechanical response threshold of these C-fibers was semi-quantitatively measured using von Frey hairs (0.8–105.7 mN, 0.5 mm in diameter) by the method of limits. Each filament

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was applied twice, and the bending force of the filament that induced at least one response was considered to be the threshold. Size and location of the fibers’ RFs were drawn on a standardized chart; the size was

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calculated with ImageJ software (National Institutes of Health, USA). The following criteria were used to

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select C-fibers according to our previous study (Taguchi et al., 2005): 1) fibers that exhibit firings in response to perpendicular indentation of the RF with a glass rod, 2) fibers that showed no muscle length-dependent firings responding to a few millimeter stretch (to exclude muscle spindle afferents), and 3) fibers with a

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conduction velocity (CV) of < 2.0 m/s.

The CV was calculated based on the distance between the RF electrically stimulated and the recording

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site and conduction latency of an action potential induced (stimulus intensity was maintained just above the

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electrical activation threshold of < 50 V to obtain constant discharges with a pulse duration of 500 μs) (Taguchi et al., 2005). The discharges induced by muscle contraction were excluded using double pulse stimulation (pulse interval of 20 ms). Background activities (on-going discharges) of the fibers were calculated during the control period of 60 s, immediately before ramp mechanical stimulation using the mechanical stimulator described below. To examine the response properties of C-fibers, noxious stimuli were given to the RF as follows: 1) semiquantitative mechanical stimulation with von Frey hairs, 2) quantitative mechanical stimulation with a servo8

controlled mechanical stimulator, 3) cooling from approximately 34 ˚C to 4 ˚C, and 4) heating from approximately 34 ˚C to 52 ˚C. The intervals between stimuli varied: when a stimulus induced no excitation, the interval before the next stimulus was set at about 5 min; when a stimulus induced an excitation, the 5-min interval started at the end of the response. All of the data were stored in a computer using an A/D converter (ML870, PowerLab 8/30, ADInstruments, Sydney, Australia) with a sampling frequency of 20 kHz. Action potentials were analyzed on a computer using the DAPSYS data acquisition system (http://www.dapsys.net) (Zimmermann et al., 2009).

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2.3.2. Mechanical, cold, and heat stimulation To quantitatively analyze the mechanical sensitivity of a C-fiber, we used a mechanical stimulator that enabled feedback regulation of force (PS-1 or PS-2001, manufactured by Aizawa S., Goto College of Medical

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Arts and Science, Tokyo, Japan). The stimulator was equipped with a plastic cylindrical probe of a flat circular tip (tip area 2.28 mm2). Compressive loads (up to 392 mN at constant compression speed of 9.8 mN/s) were

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applied to the most sensitive point on the identified RF.

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Similarly, cold and heat stimuli were applied to the most sensitive point on the RF using a feedbackcontrolled Peltier thermode (Intercross-2000N, Intercross, Co. Ltd., Tokyo, Japan) with a small probe (diameter of 1 mm). From a baseline temperature of 34 ˚C, the RF was gradually cooled down to 4 ˚C over 50

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2.3.3. Response criteria

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s or heated up to 52 ˚C over 30 s at a constant rate of 0.6 ˚C/s.

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The same response criteria was employed to determine whether a tested fiber was responsive to a stimulus as in our previous work (Taguchi et al., 2005): 1) the net evoked increase in the firing rate during the stimulus period (40 s in case of mechanical stimulation, 50 s in case of cold stimulation, and 30 s in case of heat stimulation) was more than 0.1 imp/s above the background discharge rate during the control period of 60 s immediately before the onset of noxious stimuli; and 2) the instantaneous discharge rate of two consecutive discharges exceeded the mean + 2 standard deviations (SD) of the background discharge rate. The response threshold to mechanical, cold, and heat stimuli was defined as the intensity or temperature that induced a 9

discharge that exceeded the mean frequency + 2 SD of the background discharges during the control period of 60 s, when two or more consecutive discharges exceeded this level.

2.3.4. Classification of receptor types As in our previous study, we classified the mechano-responsive C-fibers recorded in the present study into the following subclasses according to their responsiveness to cold and heat stimulation (Taguchi et al., 2005): 1) C-mechanical nociceptors (CM) that were responsive only to mechanical stimuli; 2) C-mechano-

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cold (CMC) units; 3) C-mechano-heat (CMH) units; and 4) C-mechano-cold-heat (CMCH) units.

2.4. RNA isolation and reverse transcription (RT)-PCR

When muscular mechanical hyperalgesia was evident on the 4th day during RCS (unpublished

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observation of our group) and 1 or 2 weeks after RCS (Nasu et al., 2010), EDL muscle tissues were removed and frozen immediately with liquid nitrogen at –80 °C. Total RNAs were extracted with RNeasy Fibrous Tissue

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Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instruction. Complimentary DNAs

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(cDNAs) were prepared using M-MLV Reverse Transcriptase (Promega, Madison, Wisconsin, USA). PCRs were performed using the set of primers listed in Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The PCR products were separated by 1.5% agarose gel

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electrophoresis and stained with ethidium bromide. The band optical density was analyzed with the ImageJ software. Expression levels of each messenger RNA (mRNA) were normalized to the level of GAPDH, and

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then the ratio to the control group was calculated.

2.5. Enzyme-linked immunosorbent assay (ELISA) Nerve growth factor (NGF) levels of tissue extracts in the EDL muscle were measured by ELISA (NGF Emax ImmunoAssay System, Promega) according to the manufacturer’s instruction.

2.6. Statistical analyses In electrophysiology, results are expressed as median with interquartile range (IQR) except for the case 10

that the mechanical response patterns of C-fibers are expressed as mean ± SEM. In PCR and ELISA assay, results are expressed as mean ± SEM. Electrophysiological data between the SHAM and the RCS group were analyzed using the Mann-Whitney U test. The incidence of fibers between the two groups was compared using Fisher’s exact probability test. Data of RT-PCR and ELISA were analyzed using Kruskal-Wallis test followed by Dunn’s multiple comparison test. A p value less than 0.05 was considered significant.

3. RESULTS

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3.1. General characteristics of muscular nociceptors A total of 53 C-fibers were recorded and analyzed (n = 22 in the SHAM and n = 31 in the RCS group). The general characteristics were summarized in Table 2. The parameters (i.e., electrical activation threshold,

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conduction velocity, background activity [both proportion of positive fibers and the discharge rate], and the

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size of the RFs) did not differ between the SHAM and the RCS group.

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3.2. Mechanical sensitivity of muscular C-nociceptors in RCS model Figure 1A shows the distribution of RFs in C-fibers. Most of the RFs were located on the front side (the anterior side) near the musculotendinous junction than in other areas, as we reported previously (Taguchi et al.,

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2005). However, the distribution looked to be similar between the SHAM and the RCS group. The mechanical response threshold measured with von Frey hairs, which were applied to the most

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sensitive point in the RFs, was lower in the RCS group compared with the SHAM group, and the difference

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was statistically significant (p < 0.001, Mann-Whitney U test, Fig. 1B). We analyzed the mechanical sensitivity of C-fibers more precisely using a servo-controlled mechanical stimulator. As shown in Fig. 2, the mechanical response induced using a ramp mechanical stimulation (392 mN in 40 s) seemed to be greater in the RCS group (Fig. 2B) than in the SHAM group (Fig. 2A). Overall, the averaged mechanical response pattern of C-fibers in the SHAM group was in a stimulus intensity-dependent manner (Fig. 3A). The pattern in the RCS group was also in an intensity-dependent manner, but the build-up of the response were steeper and the magnitude was greater, especially during the first half of 11

the stimulation period (0~20 s). Statistical analyses revealed that the threshold of the mechanical response was significantly low in the RCS group compared with the SHAM group (p < 0.01, Mann-Whitney U test, Fig. 3B), and the response magnitude (i.e., the number of evoked spikes) was significantly greater in the RCS group than in the SHAM group (p < 0.01, Mann-Whitney U test, Fig. 3C).

3.3. Cold and heat responses of muscular C-fibers Table 3 summarizes the responsiveness of C-fibers to cold and heat stimuli. None of the parameters, such as the incidence of responding fibers, threshold temperature, and the response magnitude, differed between the

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SHAM and RCS groups.

3.4. Receptor types of muscular C-fibers

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To examine whether the RCS loading could affect the component of muscular nociceptors, we classified the receptor types according to the responsiveness to mechanical, cold, and heat stimuli. As shown in Table 4,

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the majority of C-fibers were the CM type. The incidence of the receptor types did not differ between the

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SHAM and the RCS group. Among the CM type receptors, mechanical response thresholds measured with von Frey filaments and a servo-controlled mechanical stimulator were significantly lower in the RCS group (n = 16) than in the SHAM group (n = 24), and the response magnitude (net evoked spikes) measured with the

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mechanical stimulator was significantly higher in the RCS group than in the SHAM group (data not shown). The small sample size (i.e. n = 0–5 fibers) in other receptor types made it difficult to analyze the data for each

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receptor subclasses.

3.5. RT-PCR

Among the mRNAs, the relative expression levels of neurotrophic factors and inflammatory mediators were not changed in the EDL muscle samples obtained on the 4th day during RCS and 1 or 2 weeks after RCS compared with the control (Fig. 4).

3.6. ELISA 12

We examined the protein level of NGF, one of the neurotrophic factors tested in the RT-PCR assay. The protein level in the EDL muscle was not different on the 4th day during RCS or 1 week after RCS compared with the control (Fig. 5).

4. DISCUSSION The main finding of this study was that the mechanical sensitivity of muscular C-fiber nociceptors was augmented in a putative rat model of FM induced by RCS. The mechanical response threshold was decreased,

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and the magnitude of the response was increased; however, neither the general characteristics, nor the responsiveness to noxious cold and heat was changed in the RCS model.

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4.1. FM-like model induced by stress

As exposure to stress can trigger and deteriorate FM (Clauw, 2014; Sluka and Clauw, 2016), experimental

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models of FM have been developed by loading physical and mental stress in rodents. Among the stress models,

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RCS—which alternates environmental temperatures at short intervals of 30 min between normal (22 ºC) and cold (4 ºC) compartments—has been reported to cause persistent and bilateral mechanical hyperalgesia preferentially in the muscle compared to the skin (Nasu et al., 2010). The intensity and duration of hyperalgesia

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are more prominent when the temperature in the cold compartment was set at –3 ºC. For laboratory rats, exposure to a cold environment at 4 °C or –3 ºC, coupled with rapid changes in the environmental temperatures

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at 30-min intervals as experienced in this study, is considered an aversive and exhausting stress in their life.

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The rats are forced to increase muscular tone for the heat production to keep their body core temperature within a normal physiological range. However, the persistent physical stress itself seems not to be involved in the RCS-induced hyperalgesia, since keeping the animals in a cold chamber without temperature alterations did not cause increased pain-related behaviors (Hata et al, 1986) nor did it cause anxiety-like behaviors or dysregulation of visceral functions typically associated with RCS (Hata et al, 1984; Hata et al, 1988). This means that RCS-induced hyperalgesia can be triggered by rapid alterations in the environmental temperature. The physical and psychological overload exposed during RCS can deprive rats of adequate sleep. Sleep 13

deprivation is related to mechanical allodynia and muscular hyperalgesia in a rat model of chronic fatigue syndrome (Yasui et al., 2014). Experimental sleep deprivation both exacerbated and prolonged pain-related behaviors in rats (Xue et al., 2018) and mice (Alexandre et al., 2017). Thus, inadequate sleep might be involved in the pathogenesis and prolongation of hyperalgesia induced by RCS. RCS is also associated with psychological disorders (Hata et al., 1999) and autonomic dysfunctions (Itomi et al., 2015; Kita et al., 1975). The chronic widespread nature of pain in deep tissues and a variety of the concomitant symptoms mean that the RCS model mimics clinical features of FM, and that this model is useful for the study of FM.

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Early-life stress through maternal separation, wherein litters are exposed to limited bedding between postnatal days 2 to 9, causes muscular mechanical hyperalgesia when the stressed rats reach adulthood (50–75 days old) (Green et al., 2011). Thus, physical and mental stress—or a combination of both—is strongly

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including the RCS model, can be useful for the study of FM.

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associated with persistent muscular hyperalgesia, and, therefore, stress-induced chronic muscle pain models,

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4.2. Peripheral mechanisms of pain in the RCS model

In this study, we found that muscular C-fiber nociceptors were sensitized to mechanical stimulation in the FM model induced by RCS. Additionally, early-life maternal separation of rats increases CV and decreases the

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mechanical response threshold of muscular thin-fiber nociceptors, resulting in mechanical hyperalgesia when they become an adult (Green et al., 2011). In our previous study, the mechanical response of mechano-sensitive

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C-nociceptors in the muscle and skin was increased in a rat model of FM induced by injecting reserpine, a

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monoamine depleter in the nervous system (Taguchi et al., 2015). Interestingly, single-fiber electrophysiological recordings revealed that the proportion of mechano-sensitive C-nociceptors among all Cfibers in the skin was dramatically decreased. Similar paradoxical and compensatory changes in the mechanical sensitivity would be expected in C-nociceptors in the muscle. Thus, the responsiveness of mechano-sensitive nociceptive afferents can be facilitated in the RCS-induced FM model as well as the FM model with biogenic amine depletion. In the mouse RCS model, morphological alterations are well characterized (Bonaterra et al., 2016). The 14

cross-sectional area of the gastrocnemius/soleus muscles was dramatically decreased, and this decrease was associated with an increase in inflammatory and atrogenic cells expressing macrophage migration inhibitory factor (MIF), muscle ring finger 1 (MuRF1), and F-Box Protein 32 (Fbxo32). A decrease in capillary contacts per muscle fiber and an increase in damaged mitochondria are reported in the RCS model, although it is unclear whether or not the morphological changes in the muscle are directly to pain-related behaviors in the RCS model. Kozaki and colleagues have identified eight pain-related genes upregulated or downregulated in dorsal root ganglion cells after RCS by PCR-based cDNA subtraction analysis or DNA microarray analysis

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(upregulated: Fyn, St8sia1 and Tac 1; downregulated: Ctsb, Fstl1, Itpr1, Npy, and S100a10) (Kozaki et al., 2015). However, there is no evidence at present that these genes are involved in the pathogenesis of the RCS

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model.

4.3. Unchanged neurotrophic factors in the muscle of the RCS model

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Neurotrophic factors are known to sensitize peripheral nociceptors to mechanical stimulation. In our previous studies, the intramuscular injection of NGF increased the mechanical response of C-fibers in the

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muscle (Murase et al., 2010). Similarly, the mechanical response of Aδ-, but not C-fibers, was facilitated by glial cell line-derived neurotrophic factor (GDNF) (Murase et al., 2014). Artemin is involved in tongue pain

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hypersensitivity with burning mouth syndrome both in human patients and the mouse model (Shinoda et al., 2015) and in rat models of inflammatory and neuropathic pain (Ikeda-Miyagawa et al., 2015). Artemin and

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neurturin activate and sensitize C- and Aδ-afferent fibers, and contribute to inflammatory bone pain (Nencini

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et al., 2018). In this study, the RT-PCR assay revealed that mRNAs of neurotrophic factors (NGF, GDNF, artemin, neurturin, persephin, brain-derived neurotrophic factor [BDNF], and neurotrophin-3 [NT-3]) and inflammatory mediators (interleukin-1β [IL-1β], IL-6, and tumour necrosis factor-α [TNF-α]), which potentially modulate muscular nociceptor activities (Hoheisel et al., 2005; Nencini et al., 2018), were not changed at any time points tested during and after RCS. Thus, peripherally-derived neurotrophic factors as well as inflammatory mediators are not likely to be involved in the RCS-induced hyperalgesia. Further search for substances responsible for mechanical sensitization of C-fibers in the muscle is needed. 15

4.4. Central mechanisms of pain in the RCS model In the RCS model, activated microglial cells play a role in tactile allodynia in rats and mice (Akagi et al., 2014). Transcription factor IRF8, which is expressed in the activated microglia, is involved in the pathogenesis of RCS-induced FM model. Spinal microglial activation is also observed in a rat model of chronic fatigue syndrome induced by multiple continuous stress (Yasui et al., 2014), and in a rat reserpine-induced FM model (Taguchi et al., 2015). Minocycline, a microglial activation inhibitor ameliorated mechanical hyperalgesia in the models.

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One possible reason for the spinal microglial activation is the increased input via peripheral nociceptors as observed in this study; another is the impaired descending pain inhibition. The 5-HT3 receptor-mediated serotonergic pathway and the α2 receptor-mediated noradrenergic pathway in the spinal cord contribute to

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mechanical hyperalgesia in the RCS model (Kawamura et al., 1998). Recently, it is reported that intramuscular—but not subcutaneous—injection of neurotropin, an extract of rabbit skin inflamed by

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inoculation with the vaccinia virus, suppressed the RCS-induced mechanical hyperalgesia in the gastrocnemius

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muscle, while it did not affect normal rats (Nasu et al., 2018). Interestingly, the mechanical hyperalgesia of the bilateral gastrocnemius muscle was reduced by unilateral injections of neurotropin into the gastrocnemius, quadriceps femoris, biceps brachii, and trapezius muscles. The analgesia seems to be mediated via activation

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of spinal serotonergic and GABAergic receptors.

RCS changed the proteomes prominently in the mesencephalon and cerebellum (Fujisawa et al., 2008).

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The proteins include the unc-18 protein homolog 67K and the collapsin response mediator proteins (CRMP)-

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2 and CRMP-4, which were reported to be involved in neurotransmitter release or axon elongation. This change was caused by post-translational modification of proteolysis or phosphorylation. Further studies are needed to clarify whether the alteration is associated with mechanical hyperalgesia in the RCS model.

5. Conclusions As shown in this study, mechanical sensitivity of peripheral C-fiber nociceptors in the muscle is facilitated in the RCS-induced FM model. Involvement of neurotrophic factors and inflammatory mediators in the muscle 16

is not expected to be involved when behavioral hyperalgesia is evident during and after RCS. In the spinal cord, impaired descending pain inhibition and microglial activation is evident (Akagi et al., 2014; Itomi et al., 2016). Thus, the peripheral and spinal mechanisms may work to intensify pain in the RCS model and,

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presumably, in patients with FM.

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Acknowledgments The authors thank Dr. T. Hata for her fruitful comments related to the RCS-induced FM model used in this study.

Author contributions K.W., T.M. and T.T. contributed to the electrophysiological experiments. Y.T-U. and T.N. were involved in RT-PCR and ELISA assay. K.M. checked the manuscript critically. The corresponding author T.T. conceived

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the study and drafted. All the authors have read and approved the final version of the manuscript.

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Funding information

This work was funded by JSPS KAKENHI (Grant Numbers: JP25282160, JP16H03202 and

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JP16K15338 to T.T.), and partly by the Japan Agency for Medical Research and Development (AMED)

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Grant (JP16gm0810010h0502 to T.T.) and the Adaptable and Seamless Technology Transfer (A-STEP) Program through Target driven R&D from AMED (JP15im0110702h00004 to K.M.). The grants did not lead

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Conflict of interest

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to conflict of interest for the persons participating in the study.

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There is no conflict of interest related to this study.

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Figure 1. Receptive fields and von Frey threshold of muscular C-fibers (A) Location of the receptive fields on the front (superficial) and back (deep) sides of the extensor

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digitorum longus (EDL) muscle. Shading shows the tendinous area of the EDL muscle. (B) Mechanical response threshold semi-quantitatively measured with von Frey hairs. Ordinate: logarithmic scale. Note that

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U test).

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the threshold is significantly lower in the RCS group than in the SHAM group (***p < 0.001, Mann-Whitney

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Figure 2. Original recordings of the mechanical responses in muscular C-fibers (A) Representative recordings in the SHAM group. (B) Representative recordings in the RCS group.

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Corresponding histograms and output force of the ramp mechanical stimulus (392 mN / 40 s) are shown under original traces of action potentials. In the SHAM group, the discharge rate of the mechanical response increased

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gradually in a stimulus-intensity dependent manner, peaked at around 200 mN, and declined thereafter. In the RCS group, on the other hand, the discharge rate increased in a stimulus-intensity dependent manner as in the

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SHAM group; however, the build-up phase and the overall response seems to be greater in the RCS group.

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Figure 3. Augmented mechanical responses of muscular C-fibers in RCS rats (A) Summarized mechanical response patterns. The peri-stimulus time histograms were made by

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calculating the mean discharge rate (black) + SEM (grey) of all fibers recorded (bin width: 1 second). Abscissa: time in seconds. Ordinate: discharge rate (imp/s). Output force of the ramp mechanical stimulus (392 mN / 40

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s) is shown under the graph. Note the larger magnitude and steeper initial build-up of the mechanical response in the RCS group than in the SHAM group. (B) Threshold of the mechanical response. Note the significantly

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lower threshold in the RCS group than in the SHAM group (**p < 0.01, Mann-Whitney U test). (C) Magnitude of the response given by net evoked discharges during the stimulus period for 40 seconds. Note the significantly greater mechanical response in the RCS group than in the SHAM group (**p < 0.01, Mann-Whitney U test).

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Figure 4. mRNA expression for neurotrophic factors and inflammatory mediators in the muscle Relative expression of the mRNAs (n = 4 samples for each time point, except for the case of GDNF in

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the control group [n = 3]). Note no significant changes in mRNAs of neurotrophic factors and inflammatory mediators in the EDL muscle samples obtained on the 4th day during RCS and 1 or 2 weeks after RCS,

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compared with the control.

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Figure 5. NGF protein level measured by ELISA

The concentration of NGF protein is measured in the EDL muscle. Note that the protein level was not

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different on the 4th day during RCS or 1 week after RCS when compared with the control.

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Legends Table 1. Sequence of primers and cycle conditions for RT-PCR. Gene product

F or R

Sequence of primers

Cycle conditions

NGF

Forward

5’-ttcggacactctggatttagact-3’

Reverse

5’-gatttggggctcggcacttg-3’

30 s at 94˚C, 30 s at 55˚C, 1 min at 72˚C, 33 cycles

Forward

5’-cggacgggactctaagatga-3’

Reverse

5’-cgtcatcaaactggtcagga-3’

Forward

5’-ccgacgagctgatacgtt-3’

Reverse

5’-ggtgctgttcacgtccat-3’

Forward

5’-cagctccctgctatctgtctg-3’

Reverse

5’-cagcccagggagaaagttctc-3’

Forward

5’-tgtcacaatggctgcaggaagactt-3’

Reverse

5’-agctcagccactggtagggtcagg-3’

Forward

5’-cgacgtccctggctgacact-3’

Reverse

5’-agtaagggcccgaacatacgattg-3’

Forward

5’-tgcagagcataagagtcacc-3’

Reverse

5’-aagtcagtgctcggacgtag-3’

Forward

5’-aagccaacaagtggtattctc-3’

Reverse

5’-ttgtttgggatccacactctc-3’

Forward

5’-atgttgttgacagccactgc-3'

Reverse

5’-acagtgcatcatcgctgttc-3’

Forward

5’-atgtggaactggcagaggag-3’

Reverse

5’-ggccatggaactgatgagag-3’

BDNF

NT-3

IL-1β

IL-6

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TNF-α

Forward

5’-gtgaaggtcggtgtcaacggattt-3’

Reverse

5’-cacagtcttctgagtggcagtgat-3’

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GAPDH

1 min at 94˚C, 1 min at 62˚C, 1 min at 72˚C, 36 cycles

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Persephin

1 min at 94˚C, 1 min at 61˚C, 1 min at 72˚C, 36 cycles

1 min at 94˚C, 1 min at 58˚C, 1 min at 72˚C, 37 cycles 1 min at 94˚C, 1 min at 58˚C, 1 min at 72˚C, 37 cycles

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Neurturin

1 min at 94˚C, 1 min at 58˚C, 1 min at 72˚C, 37 cycles

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Artemin

30 s at 94˚C, 30 s at 55˚C, 1 min at 72˚C, 33 cycles

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GDNF

30 s at 94˚C, 30 s at 53˚C, 1 min at 72˚C, 35 cycles 30 s at 94˚C, 30 s at 58˚C, 1 min at 72˚C, 36 cycles 30 s at 94˚C, 30 s at 60˚C, 1 min at 72˚C, 35 cycles 30 s at 94˚C, 30 s at 55˚C, 1 min at 72˚C, 21 cycles

Table 2. General characteristics of muscular C-fibres. SHAM (n = 22)

RCS (n = 31)

P-value

Activation threshold (V)

29.0 (24.3–36.3)

30.0 (24.0–38.0)

n.s.

Conduction velocity (m/s)

0.53 (0.36–0.69)

0.50 (0.36–0.67)

n.s.

positive fibres (%)

11/22 (50.0)

22/31 (71.0)

n.s.

frequency (imp/s)

0.01 (0–0.26)

0.07 (0–0.17)

n.s.

3.0 (1.9–4.0)

3.4 (1.8–5.0)

n.s.

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Outcome measures

Background activity

Receptive field size (mm2)

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Table 3. Cold and heat responses of muscular C-fibers. Stimulants

SHAM (n = 22)

RCS (n = 31)

P-value

1/22 (4.5%)

4/31 (12.9%)

n.s.

Threshold temp. (˚C) 24.6 [1]

21.3 (16.1–30.2) [4]

n.a.

Magnitude (spikes)

43.0 [1]

12.8 (8.0–37.2) [4]

n.a.

Incidence

6/22 (27.3%)

4/31 (12.9%)

n.s.

Threshold temp. (˚C) 41.5 (36.0–46.0) [6]

41.2 (37.2–46.1) [4]

n.s.

Magnitude (spikes)

13.5 (10.3–20.9) [4]

n.s.

Cold Incidence

Heat

12.3 (7.5–29.1) [6]

RCS

Receptor type

No. (%)

No. (%)

P-value

CM

16 ( 72.7)

24 ( 77.4)

n.s.

CMC

0 (

3(

9.7)

n.s.

CMH

5 ( 22.7) 3 (

9.7)

n.s.

CMCH

1 (

3.2)

n.s.

Total

22 (100.0) 31 (100.0)

1(

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4.5)

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0.0)

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SHAM

ro of

Table 4. Receptor types of muscular C-fibers.

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