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ORIGINAL ARTICLE
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Remote dose-dependent effects of dry needling at distant myofascial
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trigger spots of rabbit skeletal muscles on reduction of substance P
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levels of proximal muscle and spinal cords
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Yueh-Ling Hsieh, PT, PhD,* Chen-Chia Yang, MD, MS,† Szu-Yu Liu, PT, MS, *
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Li-Wei Chou, MD, PhD, ‡§ Chang-Zern Hong, MD¶
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Department of *Physical Therapy, Graduate Institute of Rehabilitation Science
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and ‡School of Chinese Medicine, College of Chinese Medicine, China
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Medical University, Taichung; †Department of Physical Medicine and
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Rehabilitaion, Cheng Ching General Hospital, Taichung; §Department of
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Physical Medicine and Rehabilitation, China Medical University Hospital,
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Taichung;¶Department of Physical Therapy, Hungkuang University, Taichung,
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Taiwan
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Corresponding to: Li-Wei Chou, M.D., Ph.D., Chou: Department of Physical
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Medicine and Rehabilitation, China Medical University Hospital, No. 2,
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Yuh-Der Road, Taichung, Taiwan. Tel.: +886-4-22052121 ext. 2381. E-mail
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address: chouliwe@gmail.com; and Chang-Zern Hong, M.D., Department of
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Physical Therapy, Hungkuang University, 34 Chung-Chie Road, Shalu,
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Taichung, Taiwan. Tel.: +886-4-24634393 ext. 3301. Fax: +886-4-22365051,
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E-mail address: johnczhong@yahoo.com
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Running head: Substance P in remote dry needling for trigger points
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Funding Sources: This study was supported by a grant from National Science Council
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[NSC 101-2314-B-241-001; 101-2314-B-039 -003 -MY2], China Medical University
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[CMU-101-S-35], and Cheng Ching General Hospital, Taiwan.
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Conflicts of interest: No commercial party having a direct financial interest in the
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results of the research supporting this article has or will confer a benefit upon the
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authors or upon any organization with which the authors are associated.
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What’s already known about this topic?
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Dry needling at a region some distance away from the painful myofasical trigger
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points are already often used to treat myofasical pain in clinical practice.
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The mechanisms underlying the nociceptive biochemical transmission modulated
by remote effects of dry needling are limited and unclear.
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What does this study add?
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The expressions of pain-related peptide, substance P in the proximal muscle,
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lumbosacral, thoracic and cervical dorsal horns are analyzed after dry needling
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with different dosages by needling distant myofascial trigger spots of rabbit’s
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skeletal muscle.
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Abstract
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BACKGROUND: Dry needling at distant myofascial trigger points is an effective
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pain management strategy in patients with myofascial pain. However, the biochemical
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effects of remote dry needling associated with pain relief are not well understood.
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This study evaluates the remote effects of dry needling with different dosages by
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needling distant myofascial trigger spots (MTrSs) of skeletal muscle on the
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expressions of substance P (SP) in the proximal muscle, lumbosacral, thoracic and
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cervical dorsal horns of rabbits, and explores its underlying mechanism in relieving
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myofascial pain.
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METHODS: Male New Zealand rabbits (N = 48, 2.5-3.0 kg) received either dry or
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sham-operated needling at MTrSs of an unilateral gastrocnemius muscle (distant
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muscle) for 3 minutes in one session (1D) or five daily sessions (5D). Bilateral biceps
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femoris muscles (proximal muscles) and spinal dorsal horns of L2-L5, T2-T5 and
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C2-C5 were sampled immediately and 5 days after dry needling for determining the
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levels of SP using immunoassays.
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RESULTS: Immediately after dry needling for 1D and 5D, the expressions of SP
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were significantly decreased in ipsilateral biceps femoris and bilateral superficial
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laminaes of spinal dorsal horns when compared with the levels of their sham-operated
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counterparts (p < .05). Five days after dry needling, these reduced immunoactivities
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of SP were found only in animals receiving 5D dry needling (p < .05).
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CONCLUSIONS: This biochemical effects of remote dry needling involves the
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reduction of SP levels in proximal muscle and spinal superficial laminaes, which may
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be associated with its underlying mechanism in alleviating myofascial pain.
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1. Introduction
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Myofascial trigger points (MTrPs), a source of musculoskeletal pain, has been defined
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as a hyperirritable (hypersensitive) spot in a taut band of skeletal muscle fibers and
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may play a key role in the pathophysiology of myofascial pain syndrome (Travell &
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Simons, 1983).
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Dry needling targeting directly the primary MTrP, if performed appropriately, is
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one of the effective therapies for inactivating MTrPs and alleviating pain (Gunn et al.,
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1980; Cummings & White, 2001; Hong, 2002; Itoh et al., 2004; Hsieh et al., 2007;
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Itoh et al., 2007; Tough et al., 2009; Fernandez-Carnero et al., 2010b; Kietrys et al.,
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2013). However, repetitive and intensive needling manipulation may cause excess
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damage and increase inflammatory nociception in skeletal muscle fibers (Hsieh et al.,
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2012). Therefore, acupuncture-like needling at a region some distance away from the
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painful MTrPs can provide an alternative approach to remote pain relief (Chou et al.,
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2009; Tsai et al., 2010; Chou et al., 2011; Chou et al., 2012; Chen et al., 2013). An
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electrophysiological study investigating the neural mechanism of remote effects of
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dry needling has found that the effects are mediated via an intact afferent neural
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pathway from the stimulated site to the spinal cord and via a normal spinal cord
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function at the segments corresponding to the innervation of the proximally affected
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muscle (Hsieh et al., 2011) and may also involve the possible effects from
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extrasegments, such as descending pain inhibitory systems (Hsieh et al., 2011). But
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the biochemical mechanisms underlying the nociception transmission modulated by
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remote effects of dry needling is still unclear.
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An animal model for MTrP study on rabbit was established by Hong and Torigoe
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(Hong & Torigoe, 1994). A certain hyperirritable spot (myofascial trigger spot, MTrS)
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in the rabbit biceps femoris muscle is similar to that in human MTrP. In this spot,
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local twitch responses can be elicited when the needle tip encountered a sensitive
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locus. As in human MTrP, spontaneous electrical activity including endplate noise
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(EPN) and endplate spike can also be frequently recorded within this sensitive spot
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(Simons et al., 1995; Simons et al., 2002). This animal model had been used for many
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studies on myofascial pain syndrome (Simons et al., 1995; Kuan et al., 2002; Kuan et
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al., 2007; Chen et al., 2008; Chen et al., 2010; Hsieh et al., 2011).
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The aim of the present study was to evaluate the remote effects of dry needling
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treatment on the pain-related peptide, substance P (SP), in a rabbit model of MTrSs.
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The analgesic mechanism of this procedure was elucidated by using
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electrophysiological EPN recordings and measurements of SP immunolabeling
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expressions in a rabbit model to assess and compare the alterations of SP levels in the
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proximally affected muscle and to explore the corresponding neuronal circuits
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affected by remote dry needling. This study is also designed to further investigate the
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dose-dependent effect of remote dry needling treatment.
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2. Materials and Methods
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2.1
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The current experiment was designed to clarify the analgesic effects of dry needling at
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a distance muscle containing MTrSs (i.e., unilateral gastrocnemius) by quantifying the
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expressions of SP in the proximally affected muscles (i.e., bilateral bicep femoris
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muscles) and in the same spinal segment as the points at which the needling
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stimulation input reaches the dorsal horns (L5-S2), as well as their suprasegments
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(C2-C5 and T2-T5). The MTrS of the gastrocnemius (i.e., distant muscles) on a
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randomly selected side was treated with predetermined dosages of dry needling. A
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total of 48 rabbits were randomly and equally assigned into one of the following two
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groups: dry needling or sham-operated dry needling treatment as experimental or
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control groups, respectively. Then the animals in each group were further divided into
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four subgroups according to the treatment dosages at distant MTrSs of gastrocnemius.
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The subgroups are (1) 1D group with animals submitted to one dosage of dry needling
General design
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(one session), (2) s1D group with animals submitted to one dosage of sham-operated
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dry needling, (3) 5D group with animals submitted to one dosage of dry needling for
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five consecutive days (five daily sessions), and (4) s5D group with animals submitted
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to one dosage of sham-operated dry needling for five consecutive days. Animals were
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sacrificed at two examined timepoints for SP immunoanalysis, half of the animals in
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each group on the day immediately after dry needling, and the remaining animals on
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the fifth day after cessation of dry needling. The experimental design is presented in
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Fig. 1.
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2.2
Animal care
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The experiments were performed on adult male New Zealand rabbits (ages from 16 to
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20 weeks, body weight of 2.5-3.0 kg). The animals were housed individually in
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standard polycarbonate tub cages lined with wood chip beddings, and had free access
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to food and water. The cages were placed in an air-conditioned room (25 ± 1°С), with
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40 dBA and a 12-hour alternating light-dark cycle (6:00 am to 6:00 pm). Each animal
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was housed and cared for according to the ethical guidelines of the International
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Association for Study of Pain in animals (Zimmermann, 1983; 1986). Effort was
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made to minimize discomfort and to reduce the number of animals used. All animal
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experiments were conducted following the procedure approved by the Animal Care
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and Use Committee of a university in accordance with the Guidelines for Animal
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Experimentation. The general experimental conditions were essentially the same as
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those previously described (Chen et al., 2008 ; Hsieh et al., 2011; Hsieh et al., 2012).
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2.3
Identification and stimulation of myofascial trigger spots
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Before an anesthetic is given, the most sensitive spots (i.e., MTrS) of biceps femoris
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and gastrocnemius muscles were identified by finger pinch. The animal’s reactions
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were observed (such as withdrawal of lower limb, head turning, and screaming) to
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confirm the exact location of an MTrS. These painful regions were marked on the
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skin with an indelible marker. Then the animals were anesthetized with 2% isoflurane
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(AErrane, Baxter Healthcare of Puerto Rico, PR, USA) in oxygen flow for induction
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followed by a 0.5% maintenance dose. Body temperature, monitored by a thermistor
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probe of a thermometer (Physiotemp Instrument, Clifton, NJ, USA) in the rectum,
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was maintained at approximately 37.5°C using a body temperature control system
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comprising a thermostatically-regulated DC current heating pad and an infrared lamp.
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The muscle of each marked site was grasped between the fingers from behind the
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muscle and the muscle is palpated by gently rubbing (rolling) it between the fingers to
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find a taut band, which feels like a clearly delineated “rope” of muscle fibers and is
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roughly 10 mm in diameter. The marked sites were areas designated for dry needling
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treatment of gastrocnemius or electrophysiological and immunolabeling studies of
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biceps femoris.
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2.4
Dry needling of gastrocnemius muscle
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Dry needling technique was similar to that used in our previous study (Hsieh et al.,
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2012). For needling in MTrS of gastrocnemius, the needle (300 μm in diameter, 1.5
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inches in length, Yu-Kuang Industrial Co., Ltd., Taiwan) was first inserted through
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the skin perpendicularly at the center of the marked spot and advanced slowly and
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gently into the muscle. Then simultaneous needle rotation was performed to facilitate
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fast “in-and-out” needle movement in order to elicit as many local twitch responses as
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possible. For sham-operated needling, the needle was inserted into the subcutaneous
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layer of the marked MTrS region at a depth approximately 1-2 mm from the skin
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surface, without penetrating into the muscle tissues. After insertion, the needle stayed
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there without further movement for the same period of duration as in dry needling.
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2.5
Recording of endplate noise
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This procedure was performed by an investigator who was blind to the group
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assignment. For EPN assessment, a digital EMG machine (Neuro-EMG-Micro;
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Neurosoft, Ivanovo, Russia) and monopolar needle electrodes (37 mm disposable
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Teflon-coated model) were used. The gain was set at 20 μV per division for
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recordings from both channels. Low-cut frequency filter was set at 100 Hz and the
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high-cut one was set at 1,000 Hz. Sweep speed was 10 ms per division.
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The search needle was inserted into the MTrS region in a direction parallel to the
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muscle fibers at an angle of approximately 60° to the surface of the muscle. After
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initial insertion just short of the depth of the MTrS, the needle was advanced very
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slowly with simultaneous slow rotation to prevent it from ‘grabbing’ and releasing the
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tissue suddenly to advance in a large jump. When the needle approached an active
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locus (EPN locus), the continuous electrical activity with amplitude higher than 10
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μV, i.e., EPN, can be recorded. Then the needle was fixed in place to ensure that this
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EPN can run continuously on the recording screen with constant amplitudes for at
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least 3 minutes.
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Five EPN recordings (25 ms each) taken before and 3 minutes after the needling
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treatment were randomly selected for all groups. The mean amplitude of the five
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random EPN recordings were analyzed and calculated for a certain measurement
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point for each animal.
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2.6
Tissue preparation
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Half of the animals in each group were sacrificed on the day immediately after dry
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needling, and the remaining animals were sacrificed 5 days after dry needling for SP
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immunoassays. Animals were sacrificed under strong anesthesia by injection of
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saturated KCl (300 g/mL, intraperitoneal injection). Bilateral biceps femoris muscles,
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their corresponding segments of L5-S2, and extrasegments of T2-T5 and C2-C5
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spinal cords were harvested. The spinal cord specimens were fixed in 4%
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paraformaldehyde (diluted in 0.1 mol/L PBS) and then immersed in 30% sucrose in
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0.1 mol/L PBS for 2 days at 4°C for immunohistochemical staining. The muscle
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specimens were homogenized in T-PER™ Tissue Protein Extraction Reagent (Pierce
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Chemical Co., IL, USA) and the complete cocktail of protease inhibitors (Sigma, NY,
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USA) for Western blot immunoassay.
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2.7
Western blot analysis
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Equal amounts of protein were loaded and separated in 10% Tris-Tricine SDS-PAGE
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gels. The resolved proteins were transferred onto polyvinylidene fluoride membranes
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(Millipore, Bedford, MA, USA). The membranes were blocked in 5% non-fat milk for
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1 hour at room temperature, and incubated overnight with primary antibodies against
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SP (1:500, Cat. # orb11399, Biorbyt, Cambridge, UK) and GAPDH (1:2000, ab8245,
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Abcam Inc. MA, USA) at a dilution of 1:2500 in blocking solution. The blots were
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then incubated with the horseradish peroxidase-conjugated goat anti-rabbit and
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anti-mouse IgG secondary antibody (1:2000, Jackson ImmunoResearch Laboratories,
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Inc., West Grove, PA, USA) for 1 hour at room temperature. The signals were finally
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visualized using an enhanced chemiluminescence detection system (Fujifilm
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LAS-3000 Imager, Tokyo, Japan), and the blots were exposed to X-ray. All western
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blot analyses were performed at least three times, and consistent results were obtained.
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The immunoreactive bands were analyzed using a computer-based densitometry
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Gel-Pro Analyzer (version 6.0, Media Cybernetics, Inc. USA). Relative intensity of
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western blot band for each protein was normalized to the level of GAPDH protein and
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presented as percentage of its sham value.
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2.8
Immunohistochemistry for substance P and quantitative analysis
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The frozen spinal cord tissues were cut serially and coronally into sections of 4 μm
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thickness by a freezing microtome. Each spinal cord specimen produced
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approximately 100 sections. Each staining assay was examined in 10 alternate
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sections per rabbit, which were selected by a systematic-random series with a random
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start for analysis. The sections were first mounted on poly L-lysine (Sigma,
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P8920)-coated slides and then blocked in 10% normal goat serum (in PBS with 0.3%
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Triton X-100, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA).
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Subsequently, they were incubated overnight at 4C with rat monoclonal anti-SP
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antibody (1:200, MAB356, Millipore, CA, USA). The sections were then incubated
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with biotinylated goat anti-rat IgG secondary antibody (1:500, Jackson
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ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 1 hour at room
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temperature. After being washed, the sections were incubated with a
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streptavidin-horseradish peroxidase conjugate (Jackson ImmunoResearch
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Laboratories, Inc., West Grove, PA, USA). The sections were visualized as brown
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precipitates by adding 0.2 mg/mL 3,3′-diaminobenzidine (DAB, Pierce, Rockford, IL,
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USA) as a substrate. Negative control slides omitting either primary antibodies or
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secondary antibodies were also prepared for comparison.
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The slides were examined and photographed at three randomly selected fields of
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superficial dorsal horn (laminae I-II; superficial, the localization of nociresponsive
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neurons) at 200× magnification using a light microscope (BX43, Olympus America
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Inc. NY, USA) and a cooled digital color camera with a resolution of 1360 × 1024
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pixels (DP70, Olympus America Inc. NY, USA). Images were saved and adjusted to
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equalize contrast and brightness with Adobe Photoshop (CS3, San Jose, CA), no other
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modifications were made. The digital images were analyzed using a computer-based
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morphometry, Image-Pro Plus 4.5 software (Media Cybernetics, Silver Spring, USA).
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According to the automatically calculated parameters, the area labeled by
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DAB-stained strong-positive staining cells for SP immunoreactivity (SP-IR) was
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measured. The percentage of the positive and strong SP-IR pixels to total stained
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pixels in superficial lamina of spinal dorsal horn (%) was analyzed.
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2.9
Statistical analysis
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All data were expressed as mean ± standard deviation (SD). The differences in SP-IR
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levels in animals submitted to dry needling and sham operation were calculated.
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One-way analysis of variance (ANOVA) was employed to determine the differences
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in SP-IR levels among groups. Post hoc comparisons between groups were examined
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using Scheffe’s method. A p value of < .05 was considered statistically significant.
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All data were analyzed using SPSS version 12.0 for Windows (SPSS Inc., IL, USA).
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3. Results
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3.1
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distant MTrSs
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Figure 2 shows the alterations of mean EPN amplitude recorded from biceps femoris
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ipsilaterally and contralaterally to the dry needling side at gastrocnemius muscle. As
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can be seen, there were significant differences among the four groups at any examined
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timepoint (ANOVA, all p < .05). Compared with the pre-treatment levels, the EPN
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amplitudes of ipsilateral biceps femoris were significantly decreased immediately
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after dry needling treatment (p < .05; Fig. 2A). However, the EPN amplitudes after
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sham-operated needling showed no marked changes (p > .05). There were also
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significant differences in EPN amplitudes recorded immediately after treatment
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between dry needling and sham-operation groups at any dosage (1D vs. s1D, 5D vs.
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s5D, all p < .05; Fig. 2A). Five days after treatment, the marked reduction in EPN
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amplitude was no longer observed in the 1D group (1D vs. s1D, p > .05). However,
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the 5D group showed significant decrease in EPN amplitude when compared with the
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s5D group (5D vs. s5D, p < .05; Fig. 2A) and the 1D group (1D vs. 5D, p < .05; Fig.
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2B). The EPN recordings from biceps femoris contralaterally to the dry needling side
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presented similar trends of amplitude changes (Fig. 2C).
Changes in amplitudes of EPN in biceps femoris induced by dry needling at
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3.2
Changes in SP-IR of biceps femoris induced by dry needling at distant MTrSs
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Figure 3 presents the SP-IR in biceps femoris immediately and 5 days after treatments
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for all groups. As can be seen, there were significant differences among the four
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groups at any examined timepoint (ANOVA, all p < .05). Immediately after treatment,
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1D and 5D groups showed significant decrease in SP-IR levels in biceps femoris
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ipsilaterally to the needling side when compared with s1D and s5D groups,
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respectively (Scheffe’s method, 1D vs. s1D, 5D vs. s5D, all p < .05; Figs 3A and 3B).
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Five days after treatment, the 5D group still showed marked decrease in SP-IR levels
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when compared with the s5D group (Scheffe’s method, p < .05) but there was no
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difference in SP-IR levels between 1D and s1D groups (p > .05; Fig. 3B). Moreover,
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there were no significant differences in SP-IR in biceps femoris contralaterally to the
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needling side between 1D and s1D, as well as 5D and s5D groups at any examined
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timepoint (Scheffe’s method, 1D vs. s1D, 5D vs. s5D, all p > .05; Fig. 3C).
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3.3
Changes in SP-IR cells of spinal dorsal horns induced by dry needling at
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distant MTrSs
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3.3.1 SP expressions in L5-S2 segments
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The SP-IR patterns in L5-S2 segments for the four groups immediately and 5 days
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after treatments are presented in Figs 4A and 4B, respectively. Qualitative analysis of
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the SP-IR in superficial laminaes of the L5-S2 dorsal horn showed different patterns
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of reactivity between dry needling and sham-operation groups (Fig. 4C). There were
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significant differences among the four groups at any examined timepoint (ANOVA,
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all p < .05). In sham-operated controls, SP-IR was expressed bilaterally in the nuclei
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of cells of the superficial laminaes in L5-S2 dorsal horn spinal cords, which showed
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no statistically significant differences between s1D and s5D groups at each examined
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timepoint (Scheffe’s method, p > .05; Fig. 4C). The nuclei of SP-IR neurons were
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visualized as brown precipitates and there was also some cytoplasmic staining. Most
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of the SP-IR cells were distributed in the bilateral superficial laminaes (I-II) of the
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dorsal horn. Immediately after treatment, 1D and 5D groups showed significant
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reduction in SP-IR levels in bilateral superficial laminaes when compared with s1D
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and s5D groups, respectively (Scheffe’s method, 1D vs. s1D, 5D vs. s5D; all p < .05;
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Fig. 4A). Overall, the spinal cord sections had faint SP labelings in bilateral
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superficial laminaes of dorsal horns in both groups submitted to dry needling, as
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confirmed by the quantitative analysis, which also showed statistically significant
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differences between 1D and 5D groups (Scheffe’s method, 1D vs. 5D, p < .05; Fig.
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4C). Five days after treatment, there was no significant difference in SP-IR level
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between the 1D group and the s1D group (Scheffe’s method, 1D vs. s1D, p > .05; Fig.
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4B). However, the 5D group still showed significant reduction in the SP-IR level in
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bilateral superficial laminaes when compared with the s5D group (Scheffe’s method,
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5D vs. s5D, p < .05; Fig. 4B) and the 1D group (Scheffe’s method, 1D vs. 5D, p < .05;
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Fig. 4C).
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3.3.2 SP expressions in C2-C5 and T2-T5 suprasegments
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A similar trend of SP-IR expression at L5-S2 segments was also observed in bilateral
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superficial laminaes at T2-T5 (Fig. 5A) and C2-C5 levels (Fig. 6A). There were
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significant differences in SP expressions among the four groups at any examined
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timepoint (ANOVA, all p < .05). Immediately after treatment, 1D and 5D groups
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showed statistically significant reduction in SP-IR levels in bilateral superficial
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laminaes at T2-T5 and C2-C5 levels when compared with s1D and s5D groups,
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respectively (Scheffe’s method, 1D vs. s1D, 5D vs. s5D, all p < .05). In contrast,
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there were no significant differences between 1D and 5D groups at any examined
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level (Scheffe’s method, 1D vs. 5D, T2-T5: p > .05; C2-C5: p > .05; Figs 5C and 6C).
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Five days after treatments, the 1D group showed no significant change in SP-IR at
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T2-T5 and C2-C5 levels when compared with the s1D group (Scheffe’s method, 1D
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vs. s1D group, p > .05). However, the spinal sections in the 5D group showed a
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reduction in SP-IR expressions when compared with those in the s5D group
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(Scheffe’s method, 5D vs. s5D, p < .05; Figs 5B and 6B). The 5D group also showed
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significant reduction in SP-IR levels in bilateral superficial laminaes when compared
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with the 1D group at each examined level (Scheffe’s method, 1D vs. 5D, T2-T5: p
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< .05; C2-C5: p < .05; Figs 5C and 6C).
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3.3.3 Differences in SP-IR patterns among spinal cord levels
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Comparison among SP-IR patterns of C2-C5, T2-T5 and L5-S2 levels shows no
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statistically significant differences in either 1D or s1D group at any examined
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timepoint (ANOVA, p > .05). However, the differences in SP-IR between 5D and s5D
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groups were significant among various spinal cord levels at all examined timepoint of
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either immediately or 5 days after treatments (ANOVA, p < .05). The reduction of
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SP-IR cells in the lumbosacral dorsal horn was significantly higher in comparison
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with that in cervical or thoracic cells (Scheffe’s method, all p < .05, Fig. 7).
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4. Discussion
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This is the first study showing that short-term and long-term dry needling at distant
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MTrSs can induce suppression of SP levels in the proximal muscle and spinal cord
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dorsal horns. The results also suggest that maintenance effects of remote dry needling
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may contribute to the extra-segmental desensitization effect. There is strong
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indication that both neural and biochemical effects are involved in the mechanisms of
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remote pain control.
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Dry needling targeting the MTrPs for pain relief has its basis in theories similar,
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but not exclusive, to traditional acupuncture. The electrophysiological outcomes
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affected by acupuncture and dry needling are similar (Hsieh et al., 2011). In our
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previous studies, decreases in EPN amplitude in proximal muscles containing MTrSs
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were found after dry needling at the MTrSs of a distant muscle when compared with
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pre-treatment baseline EPN amplitude in animals with intact neural circuits (Hsieh et
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al., 2011). Similar findings were found in humans receiving acupuncture at remote
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acupoints (Chou et al., 2009; Chou et al., 2011). Fernandez-Camero et al. also found
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an increase in spontaneous electrical activity at an MTrP region during persistent
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noxious stimulation at another distant MTrP, followed by suppression of
375
electrophysiological irritability after cessation of needling (Fernandez-Carnero et al.,
376
2010a). The evidences reveal that dry needling at distant MTrS could decrease the
22
377
irritability of the proximal MTrS by suppression of EPN in prevalence and amplitude.
378
In the present study, the results obtained are in line with those of earlier studies,
379
showing that EPN amplitudes were significantly reduced after a single or five dosages
380
of dry needling when compared with those after sham operation.
381
SP has been widely proposed as being involved in delivering nociceptive
382
information from the peripheral receptors to the spinal dorsal horn and supraspinal
383
processing centers, thus leading to central sensitization (Hokfelt et al., 1975; Duggan
384
et al., 1988; Xu et al., 2000; Harrison & Geppetti, 2001; Zhang et al., 2007;
385
Kobayashi et al., 2010). Interventions that inhibit SP signaling pathways generally
386
show anti-nociceptive effects in animal models (Mantyh et al., 1997; De Felipe et al.,
387
1998). Electroacupuncture can reduce the noxious nerve stimulation-induced release
388
of SP from both central terminals and peripheral endings of the primary sensory
389
neurons (Zhu et al., 1991). Immunohistochemical studies showed that
390
electroacupuncture of ‘‘Zusanli’’ (ST-36) could depress the pain response and inhibit
391
spinal dorsal horn SP release (Du & He, 1992). Electroacupuncture can also suppress
392
immunoreactive SP accumulation induced by tooth pulp stimulation in the superficial
393
layers of the trigeminal nucleus caudalis (Yonehara et al., 1992). Therefore, SP may
394
be an important transmitter in mechanism of acupuncture analgesia. The present result
23
395
on dry needling-induced suppression of spinal dorsal horn SP was consistent with
396
those of previous studies described above, implying that short- and long-term of
397
remote dry needling probably produce analgesic effect. In addition, the finding of
398
reduction in SP expression in extrasegments, dorsal horns of C2-C5 and T2-T5 after
399
dry needling was similar to our previous results (Hsieh et al., 2011), which reported
400
supraspinal control of spinal inhibitory interneurons induced by dry needling at
401
distant MTrSs. The extrasegmental desensitization effect involving a more
402
generalized system of analgesia is probably activated by remote dry needling.
403
SP is also likely to be involved in the pathogenesis of musculoskeletal pain. A
404
recent study has also found higher SP levels in active MTrPs compared with control
405
latent or absent MTPs (Shah et al., 2008). Mean optical density of the
406
immunostaining for SP was statistically greater in trapezius muscle of patients with
407
myofascial pain syndrome when compared with specimens from patients with
408
fibromyalgia. Extensive studies demonstrate that SP accumulated in MTrPs of
409
skeletal muscles is associated with pain and inflammation (Shah et al., 2005; Shah et
410
al., 2008; Hsieh et al., 2011). The current findings of reduction in SP level in
411
proximal muscle (biceps femoris) ipsilateral to the needling side after dry needling at
412
a distant muscle containing MTrSs may be related to pain control.
24
413
Suppression of SP expressions was of a higher extent in animals submitted to 5D
414
dry needling than those submitted to 1D dry needling, indicating immediate
415
dose-dependent effects. On the other hand, five days after cessation of dry needling,
416
the suppression effect persisted and even became more pronounced in laminaes I to II
417
at L5-S2 levels in animals submitted to 5D dry needling, demonstrating prolonged
418
dose-dependent effects. The immediate pain relief by 1D dry needling is probably
419
elicited by the neural effect as demonstrated by changes in EPN. However, the
420
long-term or accumulated effect of pain relief by 5D dry needling may be
421
accompanied by and mediated via biochemical changes in addition to the neural
422
effects. Therefore, repetitive or extensive remote needling may provide better effects
423
on reduction in SP levels for control of myofascial pain. It is commonly accepted that
424
electroacupuncture of higher intensities and longer pulse durations can produce
425
persistent analgesia (Romita et al., 1997).
426
427
5. Conclusion
428
The hypothesis that dry needling at distant MTrSs could modulate irritability of
429
proximal MTrSs by altering the EPN amplitude and SP levels of muscle and spinal
430
cords is supported by the present results. The findings of this study might elucidate
25
431
the biochemical mechanisms induced by remote effects of dry needling. Accordingly,
432
the practice of dry needling at some distance away from the painful site may facilitate
433
decreased MTrP sensitivity in any muscle within the same levels of spinal
434
innervations via activating the mechanisms of segmental and extrasegmental
435
desensitization. Further understanding of the analgesic action of dry needling in
436
treating soft tissue pain can contribute to develop new therapeutic strategies for
437
treating myofascial pain syndrome.
438
439
Author contributions
440
The corresponding author declares that all co-authors have participated sufficiently in
441
the work to take public responsibility for appropriate portions of the content.
442
Associate Prof. Hsieh, Dr. Chou and Dr. Hong take responsibility for the integrity of
443
the work as a whole, from inception to published article.
444
445
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Figure legends
581
582
Fig. 1. Flow chart for the animal study. Abbreviations: 1D, one-dosage dry needling;
583
5D, five-dosage dry needling; BF, biceps femoris; Gastrocnemius, GAS; MTrS,
584
myofascial trigger spots, SP, substance P; s1D, sham one-dosage dry needling; s5D,
585
sham five-dosage dry needling.
586
587
Fig. 2. Changes in EPN amplitude measured at MTrS of biceps femoris before,
588
immediately and 5 days after one- (1D) and five-dosage (5D) of dry needling at
589
gastrocnemius in experimental (1D and 5D groups) and sham-operated groups (s1D
590
and s5D groups). Sample EPN recordings (A) in rabbits from 1D, s1D, 5D and s5D
591
groups. (B) Quantification of EPN amplitude in biceps femoris ipsilateral (B) and
592
contralateral (C) to dry needling side expressed as mean ± SD. * represents significant
593
difference (p < .05) compared with sham-operated groups (s1D and s5D). # represents
594
significant difference (p < .05) between 1D and 5D groups. †: p < .05, represent
595
significant differences compared with pre-treatment values.
596
597
Fig. 3. Alterations of substance P (SP) level at biceps femoris muscle after one- (1D)
35
598
and five-dosage (5D) of dry needling at gastrocnemius in experimental (1D and 5D
599
groups) and sham-operated groups (s1D and s5D groups). Representative western blot
600
photographs in ipsilateral biceps femoris immediately and 5 days after dry needling at
601
gastrocnemius were shown. Quantification of SP levels in biceps femoris ipsilateral
602
(B) and contralateral (C) to dry needling side expressed as mean ± SD. * represents
603
significant difference (p < .05) compared with sham-operated groups (s1D and s5D).
604
# represents significant difference (p < .05) between 1D and 5D groups.
605
606
Fig. 4. Alterations of substance P (SP) levels at superficial laminaes of L5-S2 after
607
one- (1D) and five-dosage (5D) of dry needling at gastrocnemius in experimental (1D
608
and 5D groups) and sham-operated groups (s1D and s5D groups). The representative
609
photomicrographs indicate immunohistochemical labeling for SP in ipsilateral dorsal
610
horns of lumbosacral spinal cords at the timepoints immediately (A) and 5 days (B)
611
after dry needling. Histograms indicate the quantitative analysis of SP
612
immunoreactivity in ipsilateral and contralateral spinal cords (C). * represents
613
significant difference (p < .05) compared with sham-operated groups (s1D and s5D).
614
# represents significant difference (p < .05) between 1D and 5D groups. (scale bar =
615
100 μm)
36
616
617
Fig. 5. Alterations of substance P (SP) levels at superficial laminaes of T2-T5 after
618
one- (1D) and five-dosage (5D) of dry needling at gastrocnemius in experimental (1D
619
and 5D groups) and sham-operated groups (s1D and s5D groups). The representative
620
photomicrographs indicate immunohistochemical labeling for SP in ipsilateral dorsal
621
horns of lumbosacral spinal cords at the timepoints immediately (A) and 5 days (B)
622
after dry needling. Histograms indicate the quantitative analysis of SP
623
immunoreactivity in ipsilateral and contralateral spinal cords (C). * represents
624
significant difference (p < .05) compared with sham-operated groups (s1D and s5D).
625
# represents significant difference (p < .05) between 1D and 5D groups. (scale bar =
626
100 μm)
627
628
Fig. 6. Alterations of substance P (SP) levels at superficial laminaes of C2-C5 after
629
one- (1D) and five-dosage (5D) of dry needling at gastrocnemius in experimental (1D
630
and 5D groups) and sham-operated groups (s1D and s5D groups). The representative
631
photomicrographs indicate immunohistochemical labeling for SP in ipsilateral dorsal
632
horns of lumbosacral spinal cords at the timepoints immediately (A) and 5 days (B)
633
after dry needling. Histograms indicate the quantitative analysis of SP
37
634
immunoreactivity in ipsilateral and contralateral spinal cords (C). * represents
635
significant difference (p < .05) compared with sham-operated groups (s1D and s5D).
636
# represents significant difference (p < .05) between 1D and 5D groups. (scale bar =
637
100 μm)
638
639
Fig. 7. Differences in substance P (SP) levels at ipsilateral and contralateral
640
superficial laminaes of L5-S2 (L), T2-T5 (T) and C2-C5 (C) between animals after
641
one- (1D) and five-dosage (5D) of dry needling at gastrocnemius in experimental (1D
642
and 5D groups) and sham-operated groups (s1D and s5D groups). * represents
643
significant difference (p < .05) compared with SP levels of thoracic and cervical
644
dorsal horns.
38