Toru Taguchi

Bradykinin and Nerve Growth Factor Play Pivotal Roles in Muscular Mechanical Hyperalgesia after Exercise (Delayed-Onset Muscle Soreness)

Unaccustomed strenuous exercise that includes lengthening contraction (LC) often causes delayed-onset muscle soreness (DOMS), a kind of muscular mechanical hyperalgesia. The substances that induce this phenomenon are largely unknown. Peculiarly, DOMS is not perceived during and shortly after exercise, but rather is first perceived after ∼1 d. Using B2 bradykinin receptor antagonist HOE 140, we show here that bradykinin released during exercise plays a pivotal role in triggering the process that leads to muscular mechanical hyperalgesia. HOE 140 completely suppressed the development of muscular mechanical hyperalgesia when injected before LC, but when injected 2 d after LC failed to reverse mechanical hyperalgesia that had already developed. B1 antagonist was ineffective, regardless of the timing of its injection. Upregulation of nerve growth factor (NGF) mRNA and protein occurred in exercised muscle over a comparable time course (12 h to 2 d after LC) for muscle mechanical hyperalgesia. Antibodies to NGF injected intramuscularly 2 d after exercise reversed muscle mechanical hyperalgesia. HOE 140 inhibited the upregulation of NGF. In contrast, shortening contraction or stretching induced neither mechanical hyperalgesia nor NGF upregulation. Bradykinin together with shortening contraction, but not bradykinin alone, reproduced lasting mechanical hyperalgesia. We also showed that rat NGF sensitized thin-fiber afferents to mechanical stimulation in the periphery after 10–20 min. Thus, NGF upregulation through activation of B2 bradykinin receptors is essential (though not satisfactory) to mechanical hyperalgesia after exercise. The present observations explain why DOMS occurs with a delay, and why lengthening contraction but not shortening contraction induces DOMS.

TRP channels and ASICs mediate mechanical hyperalgesia in models of inflammatory muscle pain and delayed onset muscle soreness.

Abstract The roles of ion channels in sensory neurons were examined in experimental models of muscle pain in the rat. Rats were injected with 50 μl of 4% carrageenan or subjected to an eccentric exercise (ECC) of the gastrocnemius muscle (GM). The Randall–Selitto and von Frey tests were performed on the calves to evaluate mechanical hyperalgesia of the muscle. The changes in expression of four genes and proteins of ion channels in dorsal root ganglia were examined using quantitative PCR and immunohistochemistry, respectively. Effects of antagonists to transient receptor potential (TRP) channels and acid sensing ion channels (ASICs) on the mechanical hyperalgesia induced by carrageenan injection or ECC were evaluated. The mechanical hyperalgesia was observed 6–24 h after carrageenan injection and 1–3 days after ECC in the Randall–Selitto test. Infiltrations of the inflammatory cells in the GM were seen in carrageenan‐injected animals but not in those subjected to ECC. Expressions of genes and proteins in sensory neurons showed no changes. Intramuscular injection of antagonists to TRPV1 showed an almost complete suppressive effect on ECC‐induced muscle hyperalgesia but not a carrageenan‐induced one. Antagonists to TRP channels and ASICs showed suppressive effects for both carrageenan‐ and ECC‐induced muscle hyperalgesia. The carrageenan injection and ECC models are useful models of acute inflammatory pain and delayed onset muscle soreness (DOMS), respectively, and the time course and underlying etiology might be different. TRP channels and ASICs are closely related to the development of muscle mechanical hyperalgesia, and TRPV1 is involved in ECC‐induced DOMS.

Muscular mechanical hyperalgesia revealed by behavioural pain test and c‐Fos expression in the spinal dorsal horn after eccentric contraction in rats

Delayed onset muscle soreness (DOMS) is quite common, but the mechanism for this phenomenon is still not understood; even the existence of muscle tenderness (mechanical hyperalgesia) has not been demonstrated in experimental models. We developed an animal model of DOMS by inducing eccentric contraction (lengthening contraction, ECC) to the extensor digitorum longus muscle (EDL), and investigated the existence of mechanical hyperalgesia in the EDL by means of behavioural pain tests (Randall‐Selitto test and von Frey hair test, applied to/through the skin on the EDL muscle) and c‐Fos expression in the spinal dorsal horn. We found that the mechanical withdrawal threshold measured with the Randall‐Selitto apparatus decreased significantly between 1 and 3 days after ECC, while that measured by von Frey hairs did not. The group that underwent stretching of the muscle only (SHAM group) showed no change in mechanical pain threshold in either test. These results demonstrated that the pain threshold of deep tissues (possibly of the muscle) decreased after ECC. c‐Fos immunoreactivity in the dorsal horn (examined 2 days after ECC/SHAM exercise) was not changed by either ECC or compression (1568 mN) to the EDL muscle by itself, but it was significantly increased by applying compression to the EDL muscle 2 days after ECC. This increase was observed in the superficial dorsal horn of the L4 segment of the ipsilateral side, and was clearly suppressed by morphine treatment (10 mg kg−1, i.p.). These results demonstrated the existence of mechanical hyperalgesia in the muscle subjected to ECC. This model could be used for future study of the neural mechanism of muscle soreness.

Influence of surface anesthesia on the pressure pain threshold measured with different-sized probes

Transcutaneous pressure with pressure probes of arbitrary diameters have been commonly used for measuring the threshold and magnitude of muscle pain, yet this procedure lacks scientific validation. To examine the valid probe dimensions, we conducted physiological experiments using 34 human subjects. Pin-prick pain, pressure pain threshold (PPT) to pressure probes of various diameters, heat pain threshold, and electrical pain threshold of deep tissues were measured before and after application of surface lidocaine anesthesia to the skin surface over the brachioradial muscle in a double-blinded manner. The anesthesia neither affected PPT with larger probes (diameters: 1.6 and 15 mm) nor increased electric pain threshold of deep structures, whereas it diminished pain count in pin-prick test and PPT with a 1.0 mm diameter probe, suggesting that mechanical pain thresholds measured with 1.6 and 15 mm probes reflect the pain threshold of deep tissues, possibly muscle. Pain thresholds to heat did not change after application of the anesthesia. These results suggest that larger pressure probes can give a better estimation of muscular pain threshold.

Persistent deep mechanical hyperalgesia induced by repeated cold stress in rats

Chronic muscle pain of the neck, shoulder and low back is quite common and often related to a stressed condition. In this study we tried to make a model of long‐lasting muscle mechanical hyperalgesia based on one type of stress, repeated cold stress (RCS) (Kita T, Hata T, Yoneda R, Okage T. Stress state caused by alternation of rhythm in environmental temperature, and the functional disorders in mice and rats. Folia Pharmacol Jpn 1975;71:195–210). We first validated a method of measuring the muscle mechanical nociceptive threshold through skin, with surface anesthesia of the skin covering the muscle. We found that a pressure test using a Randall–Selitto analgesiometer equipped with a larger probe (φ 2.6 mm) can measure the deep mechanical withdrawal threshold even under the presence of cutaneous punctuate hyperalgesia. RCS was performed by changing the temperature from 22 °C to either 4 °C (RCS at 4 °C) or −3 °C (RCS at −3 °C) every 30 min, and then maintained at 4 °C/−3 °C from 17:30 to 10:00 the next day. RCS at 4 °C for 5 days induced bilateral deep mechanical hyperalgesia lasting 2–3 weeks without cutaneous punctuate hyperalgesia. Deep mechanical hyperalgesia observed after RCS at −3 °C lasted longer (∼6 weeks) and was severer than RCS at 4 °C. Bilateral cutaneous punctuate hyperalgesia was also observed with RCS at −3 °C. Intramuscular injection of lidocaine confirmed that the muscle was hyperalgesic. RCS might serve as a useful model for study of the mechanism of chronic muscle pain and its treatment.

Dorsal horn neurons having input from low back structures in rats.

&NA; The mechanisms of nociception in the low back are poorly understood, partly because systematic recordings from dorsal horn neurons with input from the low back are largely missing. The purpose of this investigation was to (1) identify spinal segments and dorsal horn neurons receiving input from the low back, (2) test the effect of nerve growth factor (NGF) injected into the multifidus muscle (MF) on the neurons’ responsiveness, and (3) study the influence of a chronic MF inflammation on the responses. In rats, microelectrode recordings were made in the segments L2, L3, and L5 to find dorsal horn neurons having input from the low back (LB neurons). In control animals, the proportion of LB neurons in L2 was larger than in L3 and L5. Most LB neurons had a convergent input from several tissues. Injections of NGF into MF increased the proportion of LB neurons significantly. A chronic MF inflammation likewise increased the proportion of LB neurons and the input convergence. The centers of the neurons’ receptive fields (RFs) were consistently located 2–3 segments caudally relative to their recording site. The results show that (1) input convergence from various tissues is common for LB neurons, (2) the input from structures of the low back is processed 2–3 segments cranially relative to the vertebral level of the RFs, and (3) the responsiveness of LB neurons is increased during a pathologic alteration of the MF. The above findings may be relevant for some cases of chronic low back pain in patients.

Cutaneous C-fiber nociceptor responses and nociceptive behaviors in aged Sprague-Dawley rats.

&NA; The change with age in pain perception in humans and the nociceptive behaviors in animals elicited by noxious stimuli to the skin are not well understood, and little is known about the peripheral neural mechanisms of cutaneous nociception in the aged. We systematically examined cutaneous nociceptor responses and nociceptive behaviors in young (9–14 w) and in aged (127–138 w) Sprague–Dawley rats. C‐fiber nociceptors in the skin were identified by mechanical and electrical stimulation, and extracellularly recorded from hind paw skin‐saphenous nerve preparations in vitro. In the aged rats, the proportions of mechano‐responsive and/or heat‐responsive C‐nociceptors were significantly lower. The proportion of mechano‐ and thermo‐insensitive units, on the other hand, was significantly increased. In addition, the response threshold to mechanical stimulus tended to be higher and the magnitude of the response tended to be smaller. There were no differences between the two age groups in the response magnitudes of mechano‐responsive C‐nociceptors to bradykinin, cold or heat. Repetitive electrical stimulation of afferent fibers revealed exaggerated slowing of conduction velocity in mechano‐responsive C‐fibers in the aged. This showed for the first time that not only receptive properties of afferent terminals but also membrane properties of conducting axons are changed in aged rats. Nociceptive behaviors in response to noxious levels of cold (cold plate test) and heat (Hargreaves’ radiant heat test) were facilitated in aged animals, while mechanical sensitivity measured by von Frey hairs remained unchanged. These discrepancies between the changes in peripheral afferents and the behavioral outcomes might be explained by facilitatory changes in the central nervous system.

Upregulated glial cell line‐derived neurotrophic factor through cyclooxygenase‐2 activation in the muscle is required for mechanical hyperalgesia after exercise in rats

•  Unaccustomed strenuous exercise that includes lengthening contraction often causes delayed onset muscle soreness (DOMS), characterised as muscular mechanical hyperalgesia. •  It has been reported that bradykinin triggers upregulation of nerve growth factor in exercised muscle, sensitizing nociceptors and resulting in DOMS, but additional mechanism(s) may be involved. •  We showed that pretreatment with cyclooxygenase (COX)‐2 inhibitors completely suppressed the development of DOMS, but treatment 2 days after lengthening contraction failed to reverse existing mechanical hyperalgesia. •  We demonstrated that COX‐2 induced upregulation of glial cell line‐derived neurotrophic factor (GDNF) and that intramuscularly injected anti‐GDNF antibody reduced muscle mechanical hyperalgesia after exercise. •  These results suggest that upregulation of GDNF through COX‐2 activation is essential to mechanical hyperalgesia after exercise, and is another pathway alongside the bradykinin–nerve growth factor pathway that is involved in DOMS development.

Nociception originating from the crural fascia in rats.

&NA; Peripheral mechanisms of fascial nociception and spinal projections were revealed, strengthening the supposition that the muscle fascia is a nociceptive sensory tissue/organ. &NA; Little is documented in the literature as to the function of muscle fascia in nociception and pain. The aim of this study was to examine the distribution of presumptive nociceptive nerve fibers, to characterize fascial thin‐fiber sensory receptors, and to examine the spinal projection of nociceptive input from the rat crural fascia (CF). Nerve fibers labeled with specific antibodies to calcitonin gene‐related peptide (CGRP) and peripherin were found to be densely distributed in the distal third of the CF. Thin‐fiber receptors (A&dgr;‐ and C‐fibers) responding to pinching stimuli to the CF with sharpened watchmaker’s forceps, identified in vivo with the teased fiber technique from the common peroneal nerve, exist in the CF. Forty‐three percent of the mechano‐responsive fascial C‐fibers were polymodal receptors (nociceptors) responding to mechanical, chemical (bradykinin), and heat stimuli, whereas almost all A&dgr;‐fibers were responsive only to mechanical stimuli. Repetitive pinching stimulus to the CF induced c‐Fos protein expression in the middle to medial part of superficial layers ie, laminae I–II of the spinal dorsal horn at segments L2 to L4, peaking at L3. These results clearly demonstrate the following: 1) peptidergic and non‐peptidergic axons of unmyelinated C‐fibers with nerve terminals are distributed in the CF; 2) peripheral afferents responding to noxious stimuli exist in the fascia, and 3) nociceptive information from the CF is mainly processed in the spinal dorsal horn at the segments L2 to L4. These results together indicate that the “muscle fascia,” a tissue often overlooked in pain research, can be an important source of nociception under normal conditions.

Neuroanatomical pathway of nociception originating in a low back muscle (multifidus) in the rat

The neural mechanisms of low back pain (LBP) are still enigmatic. Presently, low back muscles are being discussed as an important source of LBP. Here, the neuroanatomical pathway of the nociceptive information from the caudal multifidus muscle (MF) was studied. True blue was injected into the MF at the level L5 to visualize the dorsal root ganglion (DRG) cells that supply this muscle. The distribution of the stained cells had a maximum in the DRG L3, not in L5. Injection of 5% formalin into the MF at levels L4 and L5 induced a significant increase in the number of c-Fos-immunoreactive (-ir) nuclei in the dorsal horn in many lumbar segments. Cells expressing c-Fos were particularly numerous in the most lateral part of the ipsilateral laminae I-II. The number of c-Fos-ir nuclei in the dorsal horn of segment L3 was significantly higher than that in segment L5. To visualize supraspinal projections, fluorogold (FG) was injected into the contralateral ventrolateral periaqueductal gray (vlPAG) 6 days prior to formalin or saline injection into the MF. The number of double-labeled dorsal horn neurons (FG-positive plus c-Fos-ir) in all lumbar segments was significantly higher in the formalin group than in the saline group. These results show that (1) the origin of the sensory supply of the MF is shifted two segments cranially relative to the location of the muscle, (2) the spinal cells processing nociceptive input from the caudal MF are widely distributed, and (3) the vlPAG is a supraspinal center of nociception from the MF.