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.

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.

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.

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.

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.

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 (varphi 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 degrees C to either 4 degrees C (RCS at 4 degrees C) or -3 degrees C (RCS at -3 degrees C) every 30 min, and then maintained at 4 degrees C/-3 degrees C from 17:30 to 10:00 the next day. RCS at 4 degrees 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 degrees C lasted longer ( approximately 6 weeks) and was severer than RCS at 4 degrees C. Bilateral cutaneous punctuate hyperalgesia was also observed with RCS at -3 degrees 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.