Changes in nerve growth factor in vastus lateralis muscle after the first versus second bout of one-leg eccentric cycling.

Delayed onset muscle soreness (DOMS) develops after performing unaccustomed eccentric exercises. Animal studies have shown that DOMS is mechanical hyperalgesia through nociceptor sensitization induced by nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) upregulated by cyclooxygenase-2 (COX-2). However, no previous study has investigated these in relation to DOMS in humans. This study compared the first and second bouts of one-leg eccentric cycling (ECC) for changes in NGF, GDNF, and COX-2 mRNA in the vastus lateralis (VL). Seven healthy adults (18-40 years) performed two bouts of ECC (10 sets of 50 contractions) with 80% maximal voluntary concentric peak torque separated by 2 weeks (ECC1, ECC2). Muscle soreness that was assessed by a visual analog scale and maximal voluntary isometric contraction (MVC) torque of the knee extensors were measured before, immediately after (MVC only), 24 and 48 h post-exercise. Muscle biopsy was taken from the VL before the first bout from nonexercised leg (control) and 24 h after each bout from the exercised leg, and analyzed for NGF, GDNF, and COX-2 mRNA. Peak DOMS was more than two times greater and MVC torque at 48 h post-exercise was approximately 20% smaller after ECC1 than ECC2 (p < 0.05), suggesting the repeated bout effect. NGF mRNA level was higher (p < 0.05) post-ECC1 (0.79 ± 0.68 arbitrary unit) than control (0.06 ± 0.07) and post-ECC2 (0.08 ± 0.10). GDNF and COX-2 mRNA did not show significant differences between control, post-ECC1, and post-ECC2. These results suggest that an increase in NGF is associated with the development of DOMS in humans.

Insulin potentiates the response to capsaicin in dorsal root ganglion neurons in vitro and muscle afferents ex vivo in normal healthy rodents.

Systemic insulin administration evokes sympathoexcitatory actions, but the mechanisms underlying these observations are unknown. We reported that insulin sensitizes the response of thin-fibre primary afferents, as well as the dorsal root ganglion (DRG) that subserves them, to mechanical stimuli. However, little is known about the effects of insulin on primary neuronal responses to chemical stimuli. TRPV1, whose agonist is capsaicin (CAP), is widely expressed on chemically sensitive metaboreceptors and/or nociceptors. The aim of this investigation was to determine the effects of insulin on CAP-activated currents in small DRG neurons and CAP-induced action potentials in thin-fibre muscle afferents of normal healthy rodents. Additionally, we investigated whether insulin potentiates sympathetic nerve activity (SNA) responses to CAP. In whole-cell patch-clamp recordings from cultured mice DRG neurons in vitro, the fold change in CAP-activated current from pre- to post-application of insulin (n = 13) was significantly (P < 0.05) higher than with a vehicle control (n = 14). Similar results were observed in single-fibre recording experiments ex vivo as insulin potentiated CAP-induced action potentials compared to vehicle controls (n = 9 per group, P < 0.05). Furthermore, insulin receptor blockade with GSK1838705 significantly suppressed the insulin-induced augmentation in CAP-activated currents (n = 13) as well as the response magnitude of CAP-induced action potentials (n = 9). Likewise, the renal SNA response to CAP after intramuscular injection of insulin (n = 8) was significantly (P < 0.05) greater compared to vehicle (n = 9). The findings suggest that insulin potentiates TRPV1 responsiveness to CAP at the DRG and muscle tissue levels, possibly contributing to the augmentation in sympathoexcitation during activities such as physical exercise. KEY POINTS: Evidence suggests insulin centrally activates the sympathetic nervous system, and a chemical stimulus to tissues activates the sympathetic nervous system via thin fibre muscle afferents. Insulin is reported to modulate putative chemical-sensitive channels in the dorsal root ganglion neurons of these afferents. In the present study, it is demonstrated that insulin potentiates the responsiveness of thin fibre afferents to capsaicin at muscle tissue levels as well as at the level of dorsal root ganglion neurons. In addition, it is demonstrated that insulin augments the sympathetic nerve activity response to capsaicin in vivo. These data suggest that sympathoexcitation is peripherally mediated via insulin-induced chemical sensitization. The present study proposes a possible physiological role of insulin in the regulation of chemical sensitivity in somatosensory thin fibre muscle afferents.

Masseter muscle contraction and cervical muscle sensitization by nerve growth factor cause mechanical hyperalgesia in masticatory muscle with activation of the trigemino-lateral parabrachial nucleus system in female rats.

OBJECTIVE: To establish a new rat model of craniofacial myalgia, and to clarify which central nervous system pathways are activated in the model. BACKGROUND: Craniofacial myalgia, represented by myogenous temporomandibular disorder and tension-type headache with pericranial tenderness, is more common in female patients. The pain is thought to be a type of multifactorial disorder with several coexisting causes. To our knowledge, there are no models of craniofacial muscle hyperalgesia caused by multiple types of stimuli. METHODS: We injected nerve growth factor into the trapezius muscle of female and male rats and repeatedly stimulated the masseter muscle (MM) electrically for 10 days. We determined the mechanical head-withdrawal threshold of MM and extent of phosphorylated extracellular signal-related kinase 1/2 (pERK) immunoreactivity in various regions of the lower brainstem. We conducted retrograde tract-tracing to determine the projection of mechanosensitive MM-innervating secondary neurons to the lateral parabrachial nucleus. Finally, we administered morphine in rats to determine whether increases of pERK immunoreactivity were dependent on noxious inputs. RESULTS: In female rats, but not male rats, the mechanical head-withdrawal threshold was decreased significantly from days 9 to 12. The number of pERK-immunoreactive neurons in the brainstem was increased significantly in female rats in the group with both stimuli compared to rats in other groups with a single stimulus. Mechanosensitive MM-innervating neurons in the brainstem projected to the parabrachial nucleus. Morphine administration blocked the increase in the number of pERK-immunoreactive neurons in both the brainstem and parabrachial nucleus. CONCLUSIONS: We established a model of craniofacial myalgia by combining trapezius and MM stimuli in female rats. We found mechanical hyperalgesia of the MM and activation of the pain pathway from the brainstem to parabrachial nucleus. The model reflects the characteristics of patients with craniofacial myalgia and might be helpful to clarify the pathogenic mechanisms underlying these disorders.

Synergistic interaction of nerve growth factor and glial cell-line derived neurotrophic factor in muscular mechanical hyperalgesia in rats.

KEY POINTS: Nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) are essential for neuronal development and survival in embryo. However, after birth they play pivotal roles in the generation of hyperalgesia in many painful conditions. Both factors are believed to act on different groups of primary afferents, but interaction between them has not yet been studied. Here we show a synergism between the two factors. Intramuscular injection of a mixture of both factors at a low concentration, each of which alone had no effect, induced a significant muscular mechanical hyperalgesia in rats. We show that synergism occurs in the primary afferent neurons and find that about 25% primary afferents innervating the muscle express both TrkA (NGF receptor) and GFRα1 (GDNF receptor). We show by pharmacological means that afferent neurons with TrkA and GFRα1 express both TRPV1 and ASICs. Our data establish a basis for synergism between NGF and GDNF. In some inflammatory conditions both nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) are upregulated and play pivotal roles in inducing hyperalgesia. However, their interaction has not been studied. We examined whether and where interaction between both neurotrophic factors occurs in SD rats. Intramuscular injection to gastrocnemius muscle (GC) of a mixture of NGF (0.1 µm) and GDNF (0.008 µm), which alone had no effect, induced a significant mechanical hyperalgesia (F(6,30)  = 13.62, P = 0.0001), demonstrating synergism between the two factors. Phosphorylated extracellular signal-regulated kinase (pERK) immunoreactivity in dorsal root ganglia (DRGs) induced by compression of GC increased after injection of the mixture (P = 0.028, compared with PBS); thus the interaction of NGF and GDNF could occur at the primary afferent level. An in situ hybridization study (n = 4) demonstrated that 23.7-29.2% of GC-innervating DRG neurons coexpressed TrkA (NGF receptor) and GFRα1 (GDNF receptor). The cell size of the coexpressing GC DRG neurons showed no skewing towards the small size range but was distributed widely from the small to the large size ranges. Therefore, some of the coexpressing neurons with thin axons are thought to contribute to this mechanical hyperalgesia. The hyperalgesia was reversed by both amiloride (F(1,13)  = 5.056, P = 0.0425, compared with PBS) and capsazepine (F(1,10)  = 8.402, P = 0.0159, compared with DMSO), suggesting that the primary afferents sensitized by the mixture express both TRPV1 and ASICs. These results showed a basis of synergism between NGF and GDNF.

Insulin potentiates the response to mechanical stimuli in small dorsal root ganglion neurons and thin fibre muscle afferents in vitro.

KEY POINTS: Insulin is known to activate the sympathetic nervous system centrally. A mechanical stimulus to tissues activates the sympathetic nervous system via thin fibre afferents. Evidence suggests that insulin modulates putative mechanosensitive channels in the dorsal root ganglion neurons of these afferents. In the present study, we report the novel finding that insulin augments the mechanical responsiveness of thin fibre afferents not only at dorsal root ganglion, but also at muscle tissue levels. Our data suggest that sympathoexcitation is mediated via the insulin-induced mechanical sensitization peripherally. The present study proposes a novel physiological role of insulin in the regulation of mechanical sensitivity in somatosensory thin fibre afferents. ABSTRACT: Insulin activates the sympathetic nervous system, although the mechanism underlying insulin-induced sympathoexcitation remains to be determined. A mechanical stimulus to tissues such as skin and/or skeletal muscle, no matter whether the stimulation is noxious or not, activates the sympathetic nervous system via thin fibre afferents. Evidence suggests that insulin modulates putative mechanosensitive channels in the dorsal root ganglion (DRG) neurons of these afferents. Accordingly, we investigated whether insulin augments whole-cell current responses to mechanical stimuli in small DRG neurons of normal healthy mice. We performed whole-cell patch clamp recordings using cultured DRG neurons and observed mechanically-activated (MA) currents induced by mechanical stimuli applied to the cell surface. Local application of vehicle solution did not change MA currents or mechanical threshold in cultured DRG neurons. Insulin (500 mU mL-1 ) significantly augmented the amplitude of MA currents (P < 0.05) and decreased the mechanical threshold (P < 0.05). Importantly, pretreatment with the insulin receptor antagonist, GSK1838705, significantly suppressed the insulin-induced potentiation of the mechanical response. We further examined the impact of insulin on thin fibre muscle afferent activity in response to mechanical stimuli in normal healthy rats in vitro. Using a muscle-nerve preparation, we recorded single group IV fibre activity to a ramp-shaped mechanical stimulation. Insulin significantly decreased mechanical threshold (P < 0.05), although it did not significantly increase the response magnitude to the mechanical stimulus. In conclusion, these data suggest that insulin augments the mechanical responsiveness of small DRG neurons and potentially sensitizes group IV afferents to mechanical stimuli at the muscle tissue level, possibly contributing to insulin-induced sympathoexcitation.

A single administration of Neurotropin reduced the elongated immobility time in the forced swimming test of rats exposed to repeated cold stress.

Many people suffer from a major depressive disorder, and chronic pain conditions are often associated with depressive symptoms. Neurotropin, an extract from the inflamed skin of rabbits inoculated with vaccinia virus, has been used for pain relief. Decrease of brain-derived neurotrophic factor (BDNF) in the brain is one of the proposed mechanisms for the major depressive disorders, and Neurotropin has been reported to restore the decreased BDNF in the hippocampus. In this experiment, we examined whether Neurotropin had an antidepressant-like effect in a model of fibromyalgia and whether BDNF in the brain was altered after repeated cold stress (RCS) and Neurotropin treatment. Rats were exposed to RCS because these animals have been used as a model for fibromyalgia syndrome. Depression-like behavior was evaluated using elongation of immobility time in a forced swimming test. Change in expression of BDNF in the brain was also examined by western blot analysis of several brain areas. Depression-like behavior in the forced swimming test was significantly increased 10-14 days after RCS, and this increase was reversed by a single injection of an antidepressant, imipramine, but not by PBS. Increased depression-like behavior was also dose-dependently suppressed by a single administration of Neurotropin (50-200 NU/kg, subcutaneously). BDNF expression was not changed in the brain areas examined (hippocampus, amygdala, prefrontal cortex, and striatum) either after RCS or by Neurotropin injected after RCS. These results suggest that RCS induced a depression-like state in rats, and Neurotropin reversed this state. However, we did not observe a BDNF-related mechanism for these effects.

Intramuscularly injected neurotropin reduced muscular mechanical hyperalgesia induced by repeated cold stress in rats.

An extract of rabbit skin inflamed by inoculation with the vaccinia virus, neurotropin [by intravenous, oral, and intramuscular (i.m.) administration], has been used in China and Japan for the treatment of chronic pain. In this study, we investigated the analgesic mechanism of i.m. neurotropin. Rats were exposed to repeated cold stress, and muscular mechanical hyperalgesia was evaluated by measuring the withdrawal threshold of the gastrocnemius muscle using Randall-Selitto apparatus. I.m. but not subcutaneous, neurotropin dose dependently reduced the repeated cold stress-induced muscular mechanical hyperalgesia for 3 h, but it had no effect in normal rats. Injections of neurotropin into the right gastrocnemius, quadriceps femoris, biceps brachii, and trapezius muscles reduced the muscular mechanical hyperalgesia of the gastrocnemius muscle bilaterally. Intrathecal administration of antagonists to GABAergic, serotonergic, and cholinergic receptors, but not α2-adrenergic receptors, and intraperitoneal administration of opioid receptor antagonist inhibited the analgesic effect of neurotropin. These results indicated that an i.m. injection of neurotropin induced long-lasting wide-spread bilateral muscular analgesia by activating spinal serotonergic and GABAergic receptors. As distinct from analgesia by systemic administration, spinal cholinergic and opioidergic, but not adrenergic receptors, are also involved. The present study supports the effectiveness of neurotropin treatment for muscular mechanical hyperalgesia.

Role of nerve growth factor in pain.

Nerve growth factor (NGF) was first identified as a substance that is essential for the development of nociceptive primary neurons and later found to have a role in inflammatory hyperalgesia in adults. Involvement of NGF in conditions with no apparent inflammatory signs has also been demonstrated. In this review we look at the hyperalgesic effects of exogenously injected NGF into different tissues, both human and animal, with special emphasis on the time course of these effects. The roles of NGF in inflammatory and neuropathic conditions as well as cancer pain are then reviewed. The role of NGF in delayed onset muscle soreness is described in more detail than its other roles based on the authors' recent observations. Acute effects are considered to be peripherally mediated, and accordingly, sensitization of nociceptors by NGF to heat and mechanical stimulation has been reported. Changes in the conductive properties of axons have also been reported. The intracellular mechanisms so far proposed for heat sensitization are direct phosphorylation and membrane trafficking of TRPV1 by TrkA. Little investigation has been done on the mechanism of mechanical sensitization, and it is still unclear whether mechanisms similar to those for heat sensitization work in mechanical sensitization. Long-lasting sensitizing effects are mediated both by changed expression of neuropeptides and ion channels (Na channels, ASIC, TRPV1) in primary afferents and by spinal NMDA receptors. Therapeutic perspectives are briefly discussed at the end of the chapter.

Interferon regulatory factor 8 expressed in microglia contributes to tactile allodynia induced by repeated cold stress in rodents.

We investigated the role of interferon regulatory factor 8 (IRF8) in a model of chronic pain in which repeated cold stress (RCS) exposure produces tactile allodynia. RCS exposure produced a decrease in paw withdrawal threshold (PWT) to mechanical stimulation. Spinal microglia of RCS-exposed mice were morphologically activated. Expression of IRF8 was significantly increased in the spinal cord of RCS-exposed mice and was localized in microglia. IRF8-knockout mice failed to show the RCS-induced decrease in PWT. Thus, RCS exposure activates spinal microglia and upregulation of IRF8 in these cells is involved in the development of tactile allodynia after RCS exposure.

Neurochemical mechanism of muscular pain: Insight from the study on delayed onset muscle soreness.

We reviewed fundamental studies on muscular pain, encompassing the characteristics of primary afferent fibers and neurons, spinal and thalamic projections, several muscular pain models, and possible neurochemical mechanisms of muscle pain. Most parts of this review were based on data obtained from animal experiments, and some researches on humans were also introduced. We focused on delayed-onset muscle soreness (DOMS) induced by lengthening contractions (LC), suitable for studying myofascial pain syndromes. The muscular mechanical withdrawal threshold (MMWT) decreased 1-3 days after LC in rats. Changing the speed and range of stretching showed that muscle injury seldom occurred, except in extreme conditions, and that DOMS occurred in parameters without muscle damage. The B2 bradykinin receptor-nerve growth factor (NGF) route and COX-2-glial cell line-derived neurotrophic factor (GDNF) route were involved in the development of DOMS. The interactions between these routes occurred at two levels. A repeated-bout effect was observed in MMWT and NGF upregulation, and this study showed that adaptation possibly occurred before B2 bradykinin receptor activation. We have also briefly discussed the prevention and treatment of DOMS.

Increased nociceptive behaviors and spinal c-Fos expression in the formalin test in a rat repeated cold stress model.

Repeated cold stress (RCS) can trigger the development of fibromyalgia (FM)-like symptoms, including persistent deep-tissue pain, although nociceptive changes to the skin have not been fully characterized. Using a rat RCS model, we investigated nociceptive behaviors induced by noxious mechanical, thermal, and chemical stimuli applied to plantar skin. Neuronal activation in the spinal dorsal horn was examined using the formalin pain test. In rats exposed to RCS, nociceptive behavioral hypersensitivity was observed in all modalities of cutaneous noxious stimuli: the mechanical withdrawal threshold was decreased, and the heat withdrawal latency was shortened one day after the cessation of stress. The duration of nocifensive behaviors in the formalin test was prolonged in phase II but not in phase I. The number of c-Fos-positive neurons increased in the entire dorsal horn laminae I-VI, ipsilateral, but not contralateral, to formalin injection at the L3-L5 segments. The duration of nocifensive behavior in phase II was significantly and positively correlated with the number of c-Fos-positive neurons in laminae I-II. These results demonstrate that cutaneous nociception is facilitated in rats exposed to RCS for a short time and that the spinal dorsal horn neurons are hyperactivated by cutaneous formalin in the RCS model.

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 (LC) often causes delayed onset muscle soreness (DOMS), characterised as muscular mechanical hyperalgesia. Previously we reported that a bradykinin-like substance released from the muscle during exercise plays a pivotal role in triggering the process of muscular mechanical hyperalgesia by upregulating nerve growth factor (NGF) in exercised muscle of rats. We show here that cyclooxygenase (COX)-2 and glial cell line-derived neurotrophic factor (GDNF) are also involved in DOMS. COX-2 inhibitors but not COX-1 inhibitors given orally before LC completely suppressed the development of DOMS, but when given 2 days after LC they failed to reverse the mechanical hyperalgesia. COX-2 mRNA and protein in exercised muscle increased six- to 13-fold in mRNA and 1.7-2-fold in protein 0-12 h after LC. COX-2 inhibitors did not suppress NGF upregulation after LC. Instead, we found GDNF mRNA was upregulated seven- to eight-fold in the exercised muscle 12 h-1 day after LC and blocked by pretreatment of COX-2 inhibitors. In situ hybridisation studies revealed that both COX-2 and GDNF mRNA signals increased at the periphery of skeletal muscle cells 12 h after LC. The accumulation of COX-2 mRNA signals was also observed in small blood vessels. Intramuscular injection of anti-GDNF antibody 2 days after LC partly reversed DOMS. Based on these findings, we conclude that GDNF upregulation through COX-2 activation is essential to mechanical hyperalgesia after exercise.

Vacuolar-ATPase-mediated muscle acidification caused muscular mechanical nociceptive hypersensitivity after chronic stress in rats, which involved extracellular matrix proteoglycan and ASIC3.

Although widespread pain, such as fibromyalgia, is considered to have a central cause, peripheral input is important. We used a rat repeated cold stress (RCS) model with many characteristics common to fibromyalgia and studied the possible involvement of decreased muscle pH in muscle mechanical hyperalgesia. After a 5-day RCS, the muscle pH and the muscular mechanical withdrawal threshold (MMWT) decreased significantly. Subcutaneously injected specific inhibitor of vacuolar ATPase (V-ATPase), bafilomycin A1, reversed both changes almost completely. It also reversed the increased mechanical response of muscle thin-fibre afferents after RCS. These results show that V-ATPase activation caused muscle pH drop, which led to mechanical hypersensitivity after RCS. Since extracellular matrix proteoglycan and acid sensitive ion channels (TRPV1 and ASIC3) have been considered as possible mechanisms for sensitizing/activating nociceptors by protons, we investigated their involvement. Manipulating the extracellular matrix proteoglycan with chondroitin sulfate and chondroitinase ABC reversed the MMWT decrease after RCS, supporting the involvement of the extracellular mechanism. Inhibiting ASIC3, but not TRPV1, reversed the decreased MMWT after RCS, and ASIC3 mRNA and protein in the dorsal root ganglia were upregulated, indicating ASIC3 involvement. These findings suggest that extracellular mechanism and ASIC3 play essential roles in proton-induced mechanical hyperalgesia after RCS.

Extracellular matrix proteoglycan plays a pivotal role in sensitization by low pH of mechanosensitive currents in nociceptive sensory neurones.

Ischaemia, inflammation, and exercise lead to tissue acidosis, which induces pain and mechanical hyperalgesia. Corresponding to this, enhanced thin-fibre afferent responses to mechanical stimulation have been recorded in vitro at low pH. However, knowledge about how this sensitization by low pH occurs is lacking. In this study, we found that all three types (rapidly adapting (RA), intermediately adapting and slowly adapting) of mechanically activated currents recorded with the whole cell patch-clamp method were sensitized by low pH in rat cultured dorsal root ganglion neurones. This sensitization was mainly observed in neurones positively labelled with isolectin B4 (IB4), which binds to versican, a chondroitin sulfate proteoglycan. Inhibitors of acid-sensitive channels (amiloride and capsazepine) did not block sensitization by low pH except in RA neurones, and extracellular calcium was not involved even in the sensitization of this type of neurone. A broad spectrum kinase inhibitor and a phospholipase C inhibitor (staurosporine and U73122) failed to block pH-induced sensitization in IB4-positive neurones, suggesting that these intracellular signalling pathways are not involved. Notably, both excess chondroitin sulfate in the extracellular solution and pretreatment of the neurone culture with chondroitinase ABC attenuated this low pH-induced sensitization in IB4-positive neurones. These findings suggest that a change in interaction between mechanosensitive channels and/or their auxiliary molecules and the side chain of versican on the cell surface causes this sensitization, at least in IB4-positive neurones. This report proposes a novel mechanism for sensitization that involves extracellular proteoglycans (versican).

Pressure pain threshold map of thoracolumbar paraspinal muscles after lengthening contractions in young male asymptomatic volunteers.

This study aimed to characterise topographic distribution of pressure pain thresholds (PPTs) of thoracolumbar paraspinal muscles and its change after lengthening contractions (LCs) of the back muscles. Using young male asymptomatic participants in Experiment 1, we systematically examined the distribution of PPTs bilaterally in the range of Th1-L5 at measurement points 2 and 4 cm from the midline. PPTs were found to be higher in the lumbar segments of the paraspinal muscles than in the thoracic segments, and in muscles closer to the vertebrae (2 vs. 4 cm from the midline). The PPTs did not differ between the left and right sides in each segment. In Experiment 2, LC was applied by asking a part of participants recruited in Experiment 1 to fall their trunk from a starting position (parallel to the floor) to 40° flexed position, and then made it back as quickly as possible to the starting position. This cycle was repeated until participants could not keep contractions (30 times/set, 25.4 ± 10.6 sets). PPTs of the LC group decreased prominently in the lower thoracic and lumbar segments, and the decrease was more evident 24 h after LC compared to that 48 h after. In contrast, PPTs in the control group without LC remained unchanged. These results provided broad topographic images of PPTs in the thoracolumbar paraspinal muscles of young male participants with and without LC, and the obtained PPT maps could be a useful guide for better treatment of exercise-induced myofascial pain in the lower back.

Nociceptive chemical hypersensitivity in the spinal cord of a rat reserpine-induced fibromyalgia model.

The pathological mechanisms of fibromyalgia (FM) are largely unknown. Recently, a rat reserpine-induced pain model showing exaggerated pain-related behaviors to mechanical and thermal stimuli has been used in FM research. However, the model has not been fully characterized. Here, we investigated nociceptive hypersensitivity to chemical stimuli and its spinal mechanisms to further characterize the model. The rat model was induced by administering reserpine to the nervous system. Nociceptive behaviors to chemical stimuli were quantified using the formalin pain test, and neuronal activation of the stimuli was examined using spinal c-Fos immunohistochemistry and electrophysiological recordings of superficial dorsal horn (SDH) neurons. The duration of pain-related behaviors was prolonged in both phases I (0-5 min) and II (10-60 min) and the interphase; and the number of c-Fos-immunoreactive nuclei increased in laminae I-II, III-IV, and V-VI at the spinal segments L3-L5 on the side ipsilateral to the formalin injection, and these factors were significantly and positively correlated. The action potentials of SDH neurons induced by formalin injection were markedly increased in rats treated with reserpine. These results demonstrate that pain-related behaviors are facilitated by noxious chemical stimuli in a rat reserpine-induced FM model, and that the behavioral hypersensitivity is associated with hyperactivation of SDH neurons.

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 approximately 1 d. Using B(2) 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. B(1) 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 B(2) 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.

The rate and magnitude of atmospheric pressure change that aggravate pain-related behavior of nerve injured rats.

Complaints of patients with chronic pain may increase when the weather changes. The exact mechanism for weather change-induced pain has not been clarified. We have previously demonstrated that artificially lowering barometric pressure (LP) intensifies pain-related behaviors in rats with neuropathic pain [chronic constriction injury (CCI) and spinal nerve ligation (SNL)]. In the present study, we examined the rate and magnitude of LP that aggravates neuropathic pain. We measured pain-related behaviors [number of paw lifts to von Frey hair (VFH) stimulation] in awake rats after SNL or CCI surgery, and found that rates of decompression ≥5 hPa/h and ≥10 hPa/h and magnitudes of decompression ≥5 hPa and ≥10 hPa augmented pain-related behaviors in SNL and CCI rats, respectively. These results indicate that LP within the range of natural weather patterns augments neuropathic pain in rats, and that SNL rats are more sensitive to LP than CCI rats.

Responses of cutaneous C-fiber afferents and spinal microglia after hindlimb cast immobilization in rats.

Previous studies have shown that persistent limb immobilization using a cast increases nociceptive behavior to somatic stimuli in rats. However, the peripheral neural mechanisms of nociception remain unclear. Using single-fiber electrophysiological recordings in vitro, we examined the general characteristics of cutaneous C-fiber afferents in the saphenous nerve and their responsiveness to mechanical and heat stimuli in a rat model of immobilization-induced pain by subjecting the rats to hindlimb cast immobilization for 4 weeks. The mechanical response of C-fibers appeared to increase in the model; however, statistical analysis revealed that neither the response threshold nor the response magnitude was altered. The general characteristics and heat responses of the C-fibers were not altered. The number of microglia and cell diameters significantly increased in the superficial dorsal horn of the lumbar spinal cord. Thus, activated microglia-mediated spinal mechanisms are associated with the induction of nociceptive hypersensitivity in rats after persistent cast immobilization.