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.

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.

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.

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.

Neuronal Sensitization and Synaptic Facilitation in the Superficial Dorsal Horn of a Rat Reserpine-induced Pain Model.

Chronic widespread pain is one of the important issues to be solved in medical practice. Impaired spinal descending pain inhibitory system due to decreased monoamine neurotransmitters is assumed to cause nociceptive hypersensitivities in chronic painful conditions like that described in patients with fibromyalgia (FM). However, response behaviors and synaptic transmission of the spinal dorsal horn neurons in response to reserpine remain to be clarified. Here we examined the activities of superficial dorsal horn (SDH) neurons, as well as excitatory and inhibitory postsynaptic inputs to SDH neurons, using a putative rat model of FM that was established by injecting reserpine. Extracellular recordings in vivo revealed that SDH neurons were sensitized to mechanical stimulation applied to the neurons' receptive fields, and the mechanically sensitized neurons were spontaneously more active. The sensitizing effect was evident 1 day and 3 days after the reserpine treatment, but subsided 5 days after the treatment or later. Using patch-clamp recordings in vivo, spontaneous excitatory postsynaptic currents (sEPSCs) to SDH neurons were found to increase in the pain model, while spontaneous inhibitory postsynaptic currents (sIPSCs) to SDH neurons decreased. These results demonstrate that the SDH neurons were strongly sensitized in response to the reserpine treatment, and that increased excitatory and decreased inhibitory postsynaptic inputs could be responsible for the spinal nociceptive hypersensitivity in the putative FM model.

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

Fibromyalgia (FM) is a debilitating disease characterized by generalized and persistent musculoskeletal pain. Although central mechanisms are strongly implicated in the pathogenesis of FM, the involvement of peripheral mechanisms is poorly understood. To understand the peripheral nociceptive mechanisms, we examined muscular nociceptors in an FM model, which was made by exposing rats to repeated cold stress (RCS). A single muscle C-fiber nociceptors were identified through the teased fiber technique using ex vivo muscle-nerve preparations. Response properties of C-fibers to noxious stimuli were systematically analyzed. Messenger RNA expression of neurotrophic factors and inflammatory mediators were also studied in the muscle. In the RCS group, the mechanical response threshold of C-fibers, measured using a ramp mechanical stimulus, was significantly decreased, and the response magnitude was significantly increased in the RCS group when compared with the SHAM group, where the environmental temperature was not altered. The general characteristics of C-fibers and the responsiveness to noxious cold and heat stimuli were similar between the two groups. Messenger RNAs of neurotrophic factors and inflammatory mediators were not changed in the muscle during and after RCS. These results suggest that augmentation of the mechanical response of muscle C-fiber nociceptors contributes to hyperalgesia in the RCS model.

Mechanism of aspirin-induced inhibition on the secondary hyperalgesia in osteoarthritis model rats.

AIMS: The daily activity of osteoarthritis (OA) patients is limited by chronic pain and central sensitization. Although non-steroidal anti-inflammatory drugs (NSAIDs) and acetaminophen are the first-line drugs for the treatment of OA-related pain, their efficacy on central sensitization remains unclear. In the present study, we evaluated the effect of acetylsalicylic acid (ASA, Aspirin) using an OA model induced by monosodium iodoacetate (MIA), which has a similar disease progression to human OA. MAIN METHODS: Secondary hyperalgesia was assessed at the plantar surface of the hind paw by Von Frey test. We evaluated the expression of acid-sensing ion channel 3 (ASIC3) in dorsal root ganglia and that of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in the spinal cord, which may cause secondary hyperalgesia in OA, by immunohistochemical analysis and real-time qPCR. KEY FINDINGS: The administration of ASA attenuated secondary hyperalgesia at 1-3 weeks after MIA, while celecoxib, a selective cyclooxygenase (COX)-2 inhibitor, failed to attenuate secondary hyperalgesia at week 2 after MIA injection, suggesting that ASA exerts its analgesic effect through a COX-2-independent pathway. Immunohistochemical analysis of the dorsal root ganglia indicated that ASA reduced the expression of ASIC3 during OA progression. Expression of TNF-α mRNA, but not IL-1ß mRNA, in the spinal cord following MIA injection was suppressed by ASA administration. SIGNIFICANCE: These findings suggest that ASA may have the ability to attenuate secondary hyperalgesia through suppression of ASIC3 and/or TNF-α expression. ASA is therefore a clinically useful analgesic drug for treatment of secondary hyperalgesia in OA.

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.

ATP decreases mechanical sensitivity of muscle thin-fiber afferents in rats.

ATP is an energy rich substance contained in cells in the order of mM. It is released when cells are damaged and when muscle is compressed or contracted. Subcutaneous injection of ATP induces pain-related behavior and hyperalgesia to mechanical and heat stimulation in rats. However, the effects of ATP in muscle have not been fully studied. In the present study we examined the effects of ATP on muscle C-fiber afferent activities using single fiber recordings, and on nociceptive behavior. Muscle C-fiber activities were recorded in vitro using extensor digitorum longus muscle-common peroneal nerve preparations excised from rats deeply anesthetized with pentobarbital. ATP (100 µM and 1 mM, but not 1 µM) superfused for 5 min before the mechanical stimulation suppressed the mechanical responses of muscle thin fibers irrespective of whether they excited the fiber. This suppressive effect was reversed by P2X receptor antagonists PPADS (100 µM) and suramin (300 µM). We also found that subcutaneous injection of ATP (10 mM) induced nociceptive behavior, whereas intramuscular injection had no effect. These findings showed that effects of ATP on muscle afferents differ from those on cutaneous afferents.

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.

Peripheral and spinal mechanisms of nociception in a rat reserpine-induced pain model.

Chronic widespread pain is a serious medical problem, yet the mechanisms of nociception and pain are poorly understood. Using a reserpine-induced pain model originally reported as a putative animal model for fibromyalgia, this study was undertaken to examine the following: (1) expression of several ion channels responsible for pain, mechanotransduction, and generation/propagation of action potentials in the dorsal root ganglion (DRG), (2) activities of peripheral nociceptive afferents, and (3) alterations in spinal microglial cells. A significant increase in mRNA expression of the acid-sensing ion channel (ASIC)-3 was detected in the DRG, and the behavioral mechanical hyperalgesia was significantly reversed by subcutaneous injection of APETx2, a selective blocker of ASIC3. Single-fiber recordings in vitro revealed facilitated mechanical responses of mechanoresponsive C-fibers both in the skin and muscle although the proportion of mechanoresponsive C-nociceptors was paradoxically decreased. In the spinal dorsal horn, microglial cells labeled with Iba1 immunoreactivity was activated, especially in laminae I-II where the nociceptive input is mainly processed compared with the other laminae. The activated microglia and behavioral hyperalgesia were significantly tranquilized by intraperitoneal injection of minocycline. These results suggest that the increase in ASIC3 in the DRG facilitated mechanical response of the remaining C-nociceptors and that activated spinal microglia may direct to intensify pain in this model. Pain may be further amplified by reserpine-induced dysfunction of the descending pain inhibitory system and by the decrease in peripheral drive to this system resulting from a reduced proportion of mechanoresponsive C-nociceptors.

Monocyte chemoattractant protein-1-induced excitation and sensitization to mechanical stimulation of mechanosensitive C-fiber afferents in rat skin.

It has been previously demonstrated that chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) increases the excitability of nociceptive neurons after peripheral nerve injury or inflammation. Moreover, decreased nocifensive mechanical threshold in behavioral tests and increased calcium influx in cultured dorsal root ganglion neurons by MCP-1 application have been reported. However, the effects of MCP-1 on peripheral afferent terminals have not been studied yet. The present study aimed to examine the effect of MCP-1 on the response of cutaneous unmyelinated afferents. For this purpose, single fiber recordings of mechanosensitive C-afferents were made in vitro from skin-saphenous nerve preparations excised from rats euthanized by CO2. Since IB4-positive neurons were previously implicated in MCP-1 induced mechanical hyperalgesia, sensitivity to α,ß-methylene ATP (metATP), an indicator of IB4-positive neurons, was also studied. Application of MCP-1 100 ng/ml to the receptive field elicited excitation in one half of mechanosensitive unmyelinated afferents in the skin. MCP-1 also sensitized metATP insensitive fibers to mechanical stimulation, but not metATP sensitive fibers. The incidence of heat sensitive fibers was decreased in the MCP-1 treated group with a decrease in the response threshold. These results demonstrate MCP-1 is an effective stimulant of mechanosensitive unmyelinated peripheral afferents in the rat skin.

Augmented mechanical response of muscular thin-fiber receptors in aged rats recorded in vitro.

Musculoskeletal pain deteriorates quality of life by disrupting daily activities and is a considerable economic burden to many countries because of the large number of patients. Little is known about the peripheral neural mechanisms of muscular nociception in the aged, although structural and functional changes in the muscle are apparent as a function of age. The aim of the present study was to investigate the activities of aged muscle nociceptors systematically to mechanical, chemical and thermal stimuli, and to compare with the data from young animals. Activities of single C-fibers were recorded from in vitro preparations of extensor digitorum longus muscle-nerve excised from hind legs of aged rats (125-133 weeks). Mechanical threshold measured by a ramp mechanical stimulus in the aged muscle (median; 45.2 mN (IQR; 38.1-59.1 mN), n=29) was significantly lower than that in the younger muscle (median; 65.4 mN (IQR; 46.6-122.0 mN), n=33, p<0.01, Mann-Whitney U-test) reported in our previous study (Taguchi et al., 2005). In addition, the magnitude of the mechanical response during the first 5s of the 10s stimulus was significantly greater in the aged muscle (11.0 spikes (IQR; 6.5-20.5 spikes)) than in the young (7.0 spikes (IQR; 4.0-11.5 spikes), p<0.05, Mann-Whitney U-test). In contrast, the numbers of discharges induced by chemical (pH 5.5, ATP and bradykinin) and thermal (cold and heat) stimuli were not different with the different ages. These results showed an augmented mechanical response in muscle C-afferents in the aged rats.

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.

The inner ear is involved in the aggravation of nociceptive behavior induced by lowering barometric pressure of nerve injured rats.

Patients suffering from neuropathic pain often complain of pain aggravation when the weather is changing. The exact mechanism for weather change-induced pain has not been clarified. We have previously demonstrated that experimentally lowering barometric pressure (LP) intensifies pain-related behaviors in rats with chronic constriction injury (CCI). In the present experiment we examined whether this pain aggravating effect of LP exposure in nerve injured rats is still present after lesioning of the inner ear. We used both CCI and spinal nerve ligation (SNL) models for this study. We injected into the middle ear sodium arsanilate solution (100mg/ml, 50microl/ear), which is known to degenerate vestibular hair cells, under anesthesia the day before surgery. Rats were exposed to LP (27hPa decrease over 8min) 7-9 days after CCI or 5-8 days after SNL surgery, and pain-related behavior (number of paw lifts induced by von Frey hair stimuli) was measured. When the inner ear lesioned SNL or CCI rats were exposed to LP, they showed no augmentation of pain-related behavior. On the other hand, the pain aggravating effect of a temperature decrease (from 24 to 17 degrees C) was maintained in both SNL and CCI rats. These results suggest that the barometric sensor/sensing system influencing nociceptive behavior during LP in rats is located in the inner ear.

TRPV3 in keratinocytes transmits temperature information to sensory neurons via ATP.

Transient receptor potential V3 (TRPV3) and TRPV4 are heat-activated cation channels expressed in keratinocytes. It has been proposed that heat-activation of TRPV3 and/or TRPV4 in the skin may release diffusible molecules which would then activate termini of neighboring dorsal root ganglion (DRG) neurons. Here we show that adenosine triphosphate (ATP) is such a candidate molecule released from keratinocytes upon heating in the co-culture systems. Using TRPV1-deficient DRG neurons, we found that increase in cytosolic Ca(2+)-concentration in DRG neurons upon heating was observed only when neurons were co-cultured with keratinocytes, and this increase was blocked by P2 purinoreceptor antagonists, PPADS and suramin. In a co-culture of keratinocytes with HEK293 cells (transfected with P2X(2) cDNA to serve as a bio-sensor), we observed that heat-activated keratinocytes secretes ATP, and that ATP release is compromised in keratinocytes from TRPV3-deficient mice. This study provides evidence that ATP is a messenger molecule for mainly TRPV3-mediated thermotransduction in skin.

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

The roles of ion channels in sensory neurons were examined in experimental models of muscle pain in the rat. Rats were injected with 50 microl 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 6-24h 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.

Compression-induced ATP release from rat skeletal muscle with and without lengthening contraction.

Adenosine triphosphate (ATP) is well known to be released from injured or inflamed tissues, and to excite/sensitize nociceptors in response to heat and mechanical stimulation. To determine whether muscle releases ATP when it is compressed, we measured ATP release from the extensor digitorum longus muscle (EDL). In addition, we investigated whether there is any difference in ATP release from the EDL of rats 2 days after lengthening contraction (LC), since the condition of the muscle is different, i.e., mechanically hyperalgesic and swollen. The EDL was put in a small chamber and superfused with Krebs-Henseleit solution equilibrated with a gas mixture of 95% oxygen and 5% carbon dioxide. The muscle was quantitatively stimulated with a servo-controlled mechanical stimulator. Reproducibility of ATP release was examined with stimulation using a 20 g force. Stimulus intensity-dependency of ATP release was also examined with 5 time compression with intensities of 5, 10, 20 and 40 g force. Bioluminescent determination by the luciferin-luciferase method was used to quantify ATP in the sample. The ATP release was decreased by repetitive mechanical stimulation of the EDL with 30 min intervals, and it was stimulus intensity (5-40 g force)-dependent. The amount of ATP released from the muscle preparations was not different between the non-treated control and the LC group. These results provide clear evidence that ATP is released from rat skeletal muscle by compression.

Changes in cardiovascular parameters and plasma norepinephrine level in rats after chronic constriction injury on the sciatic nerve.

To evaluate whether neuropathic pain affects autonomic nervous activities, we investigated daily change in cardiovascular parameters and plasma norepinephrine (NE) in free-moving rats after chronic constriction injury (CCI) on the sciatic nerve. Arterial blood pressure (BP), heart rate (HR), and the power spectrum of pulse interval variability were analyzed. Daily change in motor activity and nociceptive behavior was also measured from some CCI rats. In others, NE from daily blood samples was quantified and spontaneous pain was evaluated by daily monitoring of foot guarding behavior. We identified three stages in the daily change of cardiovascular parameters and plasma NE level over 3 weeks following CCI. The first stage (up to 3 days after the surgery) was characterized by increased MAP and HR, especially in the daytime, even though plasma NE was unchanged and motor activity decreased. The second stage (mid first to mid second postoperative weeks) was characterized by increased daytime MAP and HR, and the animals developed punctate hyperalgesia in the affected hindpaw. An NE surge that may have been related to spontaneous pain was present 3-5 days after CCI. The third stage, which appeared after the second postoperative week, was characterized by normalized MAP and decreased HR, and increased high-frequency (0.8-3.0Hz) power in pulse interval variability, which is an index of cardiac parasympathetic tone. These results demonstrated that cardiovascular function was kept high through sympathetic and non-sympathetic activity for 2 weeks after CCI, followed by a predominance of parasympathetic tone.

Change with age in muscular mechanical hyperalgesia after lengthening contraction in rats.

To determine whether there is any change by aging in mechanical hyperalgesia (delayed onset muscle soreness) after lengthening contraction (LC, also termed as eccentric contraction), we applied LC to the dorsi-flexors of the hind legs in young (7-week-old) and aged (130-week-old) rats and examined the change in mechanical withdrawal threshold of the exercised muscle with a Randall-Selitto apparatus and by c-Fos expression in the dorsal horn. The baseline mechanical withdrawal threshold did not differ among two age groups. One day after LC the withdrawal threshold started to decrease in both age groups, however, the duration of decreased withdrawal threshold was different: young rats had their withdrawal threshold lowered only for 3 days after LC while that of aged rats remained lowered two more days, showing delayed recovery in aged rats. Induction of c-Fos expression in the spinal dorsal horn by compression of the muscle was examined in aged animals 3 days after LC. Significantly larger numbers of c-Fos positive neurons was observed in the superficial dorsal horn than the control animals (no treatment). This increase was observed not only in L4 but also in L5, a wider distribution than in young animals (L4 only) in our previous report [Taguchi, T., Matsuda, T., Tamura, R., Sato, J., Mizumura, K., 2005a. Muscular mechanical hyperalgesia revealed by behavioural pain test and c-Fos expression in the spinal dorsal horn after eccentric contraction in rats.