Muscle Pain Model Research Articles

Involvement of inflammasome activation via elevation of uric acid level in nociception in a mouse model of muscle pain.

Muscle pain is a common condition in many diseases and is induced by muscle overuse. Muscle overuse induces an increase in uric acid, which stimulates the nucleotide-binding oligomerization domain-like receptor (NLR). This receptor contains the pyrin domain NLRP-3 inflammasome which when activated, results in the secretion of potent pro-inflammatory cytokines such as interleukin-1β (IL-1β). The aim of this study was to investigate the involvement of inflammasome activation via the elevation of uric acid level in nociception in a mouse model of muscle pain. The right hind leg muscles of BALB/c mice were stimulated electrically to induce excessive muscle contraction. The left hind leg muscles were not stimulated as a control. Mechanical withdrawal thresholds, levels of uric acid, IL-1β, and NLRP3, caspase-1 activity, and the number of macrophages were investigated. Furthermore, the effects of xanthine oxidase inhibitors, such as Brilliant Blue G, caspase-1 inhibitor, and clodronate liposome, on pain were investigated. In the stimulated muscles, mechanical withdrawal thresholds decreased, and the levels of uric acid, NLRP3, and IL-1β, caspase-1 activity, and the number of macrophages increased compared to that in the non-stimulated muscles. Administration of the inhibitors attenuated hyperalgesia caused by excessive muscle contraction. These results suggested that IL-1β secretion and NLRP3 inflammasome activation in macrophages produced mechanical hyperalgesia by elevating uric acid level, and xanthine oxidase inhibitors may potentially reduce over-exercised muscle pain.

Left dorsolateral prefrontal cortex repetitive transcranial magnetic stimulation reduces the development of long-term muscle pain.

The left dorsolateral prefrontal cortex (DLPFC) is involved in the experience and modulation of pain, and may be an important node linking pain and cognition. Repetitive transcranial magnetic stimulation (rTMS) to the left DLPFC can reduce chronic and experimental pain. However, whether left DLPFC rTMS can influence the development of chronic pain is unknown. Using repeated intramuscular injection of nerve growth factor to induce the development of sustained muscle pain (lasting weeks), 30 healthy individuals were randomized to receive 5 consecutive daily treatments of active or sham left DLPFC rTMS, starting before the first nerve growth factor injection on day 0. Muscle soreness and pain severity were collected daily for 14 days and disability on every alternate day. Before the first and 1 day after the last rTMS session, anxiety, depression, affect, pain catastrophizing, and cognitive performance on the attention network test were assessed. Left DLPFC rTMS treatment compared with sham was associated with reduced muscle soreness, pain intensity, and painful area (P < 0.05), and a similar trend was observed for disability. These effects were most evident during the days rTMS was applied lasting up to 3 days after intervention. Depression, anxiety, pain catastrophizing, and affect were unchanged. There was a trend toward improved cognitive function with rTMS compared with sham (P = 0.057). These data indicate that repeated left DLPFC rTMS reduces the pain severity in a model of prolonged muscle pain. The findings may have implications for the development of sustained pain in clinical populations.

Aspirin, an ASIC3 inhibitor, blocks the analgesic effect of therapeutic ultrasound in chronic muscle pain

Therapeutic ultrasound is widely used in pain control in physical medicine, and substance P (SubP) is the key pathway. However, the ion channels involving the analgesic process are still not clear. The objective of this study is to prove that the mechanism of the analgesic effects of ultrasound in a rodent model of chronic muscle pain is through acid sensing ion channel 3 (ASIC3). We employed the chronic hyperalgesia mouse model proposed by Sluka et al. and applied therapeutic ultrasound at 3 MHz, 100% duty cycle, 1 W/cm 2 for 3 min. Mice were co-injected with pH 4.0 saline and 100 uM RP-67580 on day 0. From days 4 to 8 after the first acid injection, mice were co-injected with pH 7.4 saline and Aspirin (ASP) 500 uM, 5 mM, or ibuprofen (IBU) 500 uM. The withdrawal response of mouse hind paws was defined as foot lifting when a 0.2-mN von Frey filament was applied. ASP diminished the analgesia induced by ultrasound treatment in mouse chronic muscle pain model. However, IBU, inhibitor of ASIC1a, did not. SubP and pERK content increased after ultrasound in DRG. Two minutes after ultrasound, ipsilateral pERK, subP, and tracer were significantly increased and overlapped each other. However, subP increased to less extent. Therapeutic ultrasound and acid injection could inhibit intramuscular nociceptor activation, and result an anti-nociceptive effect against chronic mechanical hyperalgesia through ASIC3 pathway. In the future, small molecule inducing SubP and ASIC3 can be new candidate to treat chronic diffuse muscle pain.

Evidence for the involvement of TNF-α, IL-1β and IL-10 in the antinociceptive and anti-inflammatory effects of indole-3-guanylhydrazone hydrochloride, an aromatic aminoguanidine, in rodents

BackgroundIndole-3-guanylhydrazone hydrochloride (LQM01) is a new derivative of aminoguanidine hydrochloride, an aromatic aminoguanidine. MethodsMice were treated with LQM01 (5, 10, 25 or 50 mg/kg, i.p.), vehicle (0.9% saline i.p.) or a standard drug. The mice were subjected to carrageenan-induced pleurisy, abdominal writhing induced by acetic acid, the formalin test and the hot-plate test. The model of non-inflammatory chronic muscle pain induced by saline acid was also used. Mice from the chronic protocol were assessed for withdrawal threshold, muscle strength and motor coordination. LQM01 or vehicle treated mice were evaluated for Fos protein. ResultsLQM01 inhibits TNF-α and IL-1β production, as well as leukocyte recruitment during inflammation process. The level of IL-10 in LQM01-treated mice increased in pleural fluid. In addition, LQM01 decreased the nociceptive behavior in the acetic acid induced writhing test, the formalin test (both phases) and increased latency time on the hot-plate. LQM01 treatment also decreased mechanical hyperalgesia in mice with chronic muscle pain, with no changes in muscle strength and motor coordination. LQM01 reduced the number of Fos positive cells in the superficial dorsal horn. This compound exhibited antioxidant properties in in vitro assays. ConclusionsLQM01 has an outstanding anti-inflammatory and analgesic profile, probably mediated through a reduction in proinflammatory cytokines release, increase in IL-10 production and reduction in neuron activity in the dorsal horn of the spinal cord in mice. General significanceBeneficial effects of LQM01 suggest that it has some important clinical features and can play a role in the management of ‘dysfunctional pain’ and inflammatory diseases.

(144) - Ischemia and reperfusion injury induces acute and chronic muscle pain-related behaviors through increased expression of the GFRα1 receptor

Muscle pain is a common complication of conditions that produce ischemia/reperfusion injuries (I/R), like peripheral artery disease (PAD) or sickle cell anemia. Previously, we have found that I/R injury in mouse induces increased pain-related behaviors that correlate with group III/IV muscle afferent sensitization and increased gene expression in the affected muscles and the dorsal root ganglia (DRG). In particular, glial cell line-derived neurotrophic factor (GDNF) expression was increased in the injured muscle tissue, while its receptor, GDNF family receptor α1 (GFRα1) was upregulated in DRGs. Both GDNF and GFRα1 have been linked to behavioral changes as well as afferent sensitization in other models of muscle pain. We hypothesized that enhanced expression of GFRα1 in sensory neurons regulates the development of pain-related behaviors after I/R. To test this, we selectively knocked down GFRα1 in mice by median and ulnar nerve injection of Penetratin1-linked GFRα1-targeting siRNAs in vivo prior to unilateral I/R injury of the forelimb. We then performed pain-related behavioral tests and single unit recordings of group III/IV muscle afferents along with real-time PCR on the I/R affected DRGs. Selective knockdown of GFRα1 expression in the DRGs effectively decreased the I/R-induced pain related behaviors and the observed reduction in mechanical thresholds in group III/IV afferents. GFRα1 inhibition also specifically prevented I/R-induced upregulation of the purinergic receptor, P2X5 and acid sensing ion channel 3 (ASIC3). Finally, repetitive I/R injury induced prolonged spontaneous pain-like behaviors (paw guarding) compared to animals with a single injury. This also correlated with increased GFRα1 expression in DRGs. These results suggest that GFRα1 upregulation may play an important role in the development of pain-related behaviors via group III/IV muscle afferent sensitization. GFRα1 and the receptors downstream of its signaling, may provide potential targets for therapies aimed at patients experiencing acute or chronic pain from ischemia/reperfusion injuries.

Spinal protein kinase C/extracellular signal-regulated kinase signal pathway mediates hyperalgesia priming.

Chronic pain can be initiated by one or more acute stimulations to sensitize neurons into the primed state. In the primed state, the basal nociceptive thresholds of the animal are normal, but, in response to another hyperalgesic stimulus, the animal develops enhanced and prolonged hyperalgesia. The exact mechanism of how primed state is formed is not completely understood. Here, we showed that spinal protein kinase C (PKC)/extracellular signal-regulated kinase (ERK) signal pathway is required for neuronal plasticity change, hyperalgesic priming formation, and the development of chronic hyperalgesia using acid-induced muscle pain model in mice. We discovered that phosphorylated extracellular signal-regulated kinase-positive neurons in the amygdala, spinal cord, and dorsal root ganglion were significantly increased after first acid injection. Inhibition of the phosphorylated extracellular signal-regulated kinase activity intrathecally, but not intracerebroventricularly or intramuscularly before first acid injection, prevented the development of chronic pain induced by second acid injection, which suggests that hyperalgesic priming signal is stored at spinal cord level. Furthermore, intrathecal injection of PKC but not protein kinase A blocker prevented the development of chronic pain, and PKC agonist was sufficient to induce prolonged hyperalgesia response after acid injection. We also found that mammalian target of rapamycin-dependent protein synthesis was required for the priming establishment. To test whether hyperalgesic priming leads to synaptic plasticity change, we recorded field excitatory postsynaptic potentials from spinal cord slices and found enhanced long-term potentiation in mice that received one acid injection. This long-term potentiation enhancement was prevented by inhibition of extracellular signal-regulated kinase. These findings show that the activation of PKC/ERK signal pathway and downstream protein synthesis is required for hyperalgesic priming and the consolidation of pain singling.

Involvement of neutrophils and interleukin-18 in nociception in a mouse model of muscle pain.

Muscle pain is a common condition that relates to various pathologies. Muscle overuse induces muscle pain, and neutrophils are key players in pain production. Neutrophils also play a central role in chronic pain by secreting interleukin (IL)-18. The aim of this study was to investigate the involvement of neutrophils and IL-18 in a mouse model of muscle pain. The right hind leg muscles of BALB/c mice were stimulated electrically to induce excessive muscle contraction. The left hind leg muscles were not stimulated. The pressure pain threshold, number of neutrophils, and IL-18 levels were investigated. Furthermore, the effects of the IL-18-binding protein and Brilliant Blue G on pain were investigated. In stimulated muscles, pressure pain thresholds decreased, and neutrophil and IL-18 levels increased compared with that in non-stimulated muscles. The administration of IL-18-binding protein and Brilliant Blue G attenuated hyperalgesia caused by excessive muscle contraction. These results suggest that increased IL-18 secretion from larger numbers of neutrophils elicits mechanical hyperalgesia.

Sensory processing of deep tissue nociception in the rat spinal cord and thalamic ventrobasal complex.

Sensory processing of deep somatic tissue constitutes an important component of the nociceptive system, yet associated central processing pathways remain poorly understood. Here, we provide a novel electrophysiological characterization and immunohistochemical analysis of neural activation in the lateral spinal nucleus (LSN). These neurons show evoked activity to deep, but not cutaneous, stimulation. The evoked responses of neurons in the LSN can be sensitized to somatosensory stimulation following intramuscular hypertonic saline, an acute model of muscle pain, suggesting this is an important spinal relay site for the processing of deep tissue nociceptive inputs. Neurons of the thalamic ventrobasal complex (VBC) mediate both cutaneous and deep tissue sensory processing, but in contrast to the lateral spinal nucleus our electrophysiological studies do not suggest the existence of a subgroup of cells that selectively process deep tissue inputs. The sensitization of polymodal and thermospecific VBC neurons to mechanical somatosensory stimulation following acute muscle stimulation with hypertonic saline suggests differential roles of thalamic subpopulations in mediating cutaneous and deep tissue nociception in pathological states. Overall, our studies at both the spinal (lateral spinal nucleus) and supraspinal (thalamic ventrobasal complex) levels suggest a convergence of cutaneous and deep somatosensory inputs onto spinothalamic pathways, which are unmasked by activation of muscle nociceptive afferents to produce consequent phenotypic alterations in spinal and thalamic neural coding of somatosensory stimulation. A better understanding of the sensory pathways involved in deep tissue nociception, as well as the degree of labeled line and convergent pathways for cutaneous and deep somatosensory inputs, is fundamental to developing targeted analgesic therapies for deep pain syndromes.

Muscle pain induced by static contraction in rats is modulated by peripheral inflammatory mechanisms

Muscle pain is an important health issue and frequently related to static force exertion. The aim of this study is to evaluate whether peripheral inflammatory mechanisms are involved with static contraction-induced muscle pain in rats. To this end, we developed a model of muscle pain induced by static contraction performed by applying electrical pulses through electrodes inserted into muscle. We also evaluated the involvement of neutrophil migration, bradykinin, sympathetic amines and prostanoids. A single session of sustained static contraction of gastrocnemius muscle induced acute mechanical muscle hyperalgesia without affecting locomotor activity and with no evidence of structural damage in muscle tissue. Static contraction increased levels of creatine kinase but not lactate dehydrogenase, and induced neutrophil migration. Dexamethasone (glucocorticoid anti-inflammatory agent), DALBK (bradykinin B1 antagonist), Atenolol (β1 adrenoceptor antagonist), ICI 118,551 (β2 adrenoceptor antagonist), indomethacin (cyclooxygenase inhibitor), and fucoidan (non-specific selectin inhibitor) all reduced static contraction-induced muscle hyperalgesia; however, the bradykinin B2 antagonist, bradyzide, did not have an effect on static contraction-induced muscle hyperalgesia. Furthermore, an increased hyperalgesic response was observed when the selective bradykinin B1 agonist des-Arg9-bradykinin was injected into the previously stimulated muscle. Together, these findings demonstrate that static contraction induced mechanical muscle hyperalgesia in gastrocnemius muscle of rats is modulated through peripheral inflammatory mechanisms that are dependent on neutrophil migration, bradykinin, sympathetic amines and prostanoids. Considering the clinical relevance of muscle pain, we propose the present model of static contraction-induced mechanical muscle hyperalgesia as a useful tool for the study of mechanisms underlying static contraction-induced muscle pain.

Modulation of opioid and serotonergic systems by alpha‐terpineol complexed in beta‐cyclodextrin reduces hyperalgesia in a chronic muscle pain model

The aim of this study was to evaluate the effect of α‐terpineol, an alcohol monoterpene found in the essential oil of aromatic species, complexed in β‐cyclodextrin (TP‐βCD) in chronic muscle pain noninflammatory model and its mechanism of action. The model of chronic muscle pain was induced by two injections of pH 4.0 saline (20 μl) into the left gastrocnemius 5 days apart. After confirming hyperalgesia, male mice were treated with α‐terpineol (αTPN 100 mg/kg, p.o.), TP‐βCD (25, 50 or 100 mg/kg; p.o.) or vehicle (saline 0.9%, p.o.) daily for 10 days. The possible involvement of the opioid and serotonergic systems were evaluated using formalin test (1%; 20 μL; i.pl.), which animals were pretreated with αTPN‐βCD (p.o.) or vehicle (p.o.), naloxone (an opioid antagonist, 5 mg/kg; i.p.) or ondansetron (an 5HT3 antagonist, 0.5 mg/kg; i.p.). The experimental protocols were approved for the UFS Ethic Committee (CEPA: 08/11). TP‐βCD reduced significantively (p&lt;0.001) the mechanical hyperalgesia when compared with vehicle. Besides, the complexation with βCD increased the effect time of TP of 3 hrs to 7 hrs. Naloxone and ondansetron were able to reverse this analgesic effect. These results demonstrate that TP‐βCD reduced the hyperalgesia followed by the chronic muscle pain model, probably evoked by the descending inhibitory pain system, specifically by opioid and serotoninergic receptors.Support or Funding InformationFAPITEC/SE, CAPES, CNPq (Brazil).

Transcutaneous electrical nerve stimulator of 5000 Hz frequency provides better analgesia than that of 100 Hz frequency in mice muscle pain model

Transcutaneous electrical nerve stimulators (TENSs) have been proved to be effective in muscle pain management for several decades. However, there is no consensus for the optimal TENS program. Previous research demonstrated that a 100 Hz TENS (L-TENS) provided better analgesia than a conventional TENS (< 5 Hz). However, no research compared a higher-frequency (> 100 Hz) TENS with a 100 Hz TENS. We used a 5000 Hz (5 kHz) frequency TENS (M-TENS) and an L-TENS to compare analgesic effect on a mice skin/muscle incision retraction model. Three groups of mice were used (sham, L-TENS, and M-TENS) and applied with different TENS programs on Day 4 after the mice skin/muscle incision retraction model; TENS therapy was continued as 20 min/d for 3 days. Mice analgesic effects were measured via Von Frey microfilaments with the up–down method. After therapy, mice spinal cord dorsal horn and dorsal root ganglion (DRG) were harvested for cytokine evaluation (tumor necrosis factor-α and interleukin-1β) with the Western blotting method. Our data demonstrated that the M-TENS produced better analgesia than the L-TENS. Cytokine in the spinal cord or DRG all expressed lower than that of the sham group. However, there is no difference in both cytokine levels between TENSs of different frequencies in the spinal cord and DRG. We concluded that the M-TENS produced faster and better mechanical analgesia than the L-TENS in the mice skin/muscle incision retraction model. Those behavior differences were not in accordance with cytokine changes in the spinal cord or DRG.

Chronic Sciatic Neuropathy in Rat Reduces Voluntary Wheel-Running Activity With Concurrent Chronic Mechanical Allodynia

Animal models of peripheral neuropathy produced by a number of manipulations are assessed for the presence of pathologic pain states such as allodynia. Although stimulus-induced behavioral assays are frequently used and important to examine allodynia (ie, sensitivity to light mechanical touch; von Frey fiber test), other measures of behavior that reflect overall function are not only complementary to stimulus-induced responsive measures, but are also critical to gain a complete understanding of the effects of the pain model on quality of life, a clinically relevant aspect of pain on general function. Voluntary wheel-running activity in rodent models of inflammatory and muscle pain is emerging as a reliable index of general function that extends beyond stimulus-induced behavioral assays. Clinically, reports of increased pain intensity occur at night, a period typically characterized with reduced activity during the diurnal cycle. We therefore examined in rats whether alterations in wheel-running activity were more robust during the inactive phase compared with the active phase of their diurnal cycle in a widely used rodent model of chronic peripheral neuropathic pain, the sciatic nerve chronic constriction injury (CCI) model. In adult male Sprague Dawley rats, baseline (BL) hindpaw threshold responses to light mechanical touch were assessed using the von Frey test before measuring BL activity levels using freely accessible running wheels (1 hour/day for 7 sequential days) to quantify the distance traveled. Running wheel activity BL values are expressed as total distance traveled (m). The overall experimental design was after BL measures, rats underwent either sham or CCI surgery followed by repeated behavioral reassessment of hindpaw thresholds and wheel-running activity levels for up to 18 days after surgery. Specifically, separate groups of rats were assessed for wheel-running activity levels (1 hour total/trial) during the onset (within first 2 hours) of either the (1) inactive (n = 8/group) or (2) active (n = 8/group) phase of the diurnal cycle. An additional group of CCI-treated rats (n = 8/group) was exposed to a locked running wheel to control for the potential effects of wheel-running exercise on allodynia. The 1-hour running wheel trial period was further examined at discrete 20-minute intervals to identify possible pattern differences in activity during the first, middle, and last portions of the 1-hour trial. The effect of neuropathy on activity levels was assessed by measuring the change from their respective BLs to distance traveled in the running wheels. Although wheel-running distances between groups were not different at BL from rats examined during either the inactive phase of the diurnal cycle or active phase of the diurnal cycle, sciatic nerve CCI reduced running wheel activity levels compared with sham-operated controls during the inactive phase. In addition, compared with sham controls, bilateral low-threshold mechanical allodynia was observed at all time points after surgical induction of neuropathy in rats with free-wheel and locked-wheel access. Allodynia in CCI compared with shams was replicated in rats whose running wheel activity was examined during the active phase of the diurnal cycle. Conversely, no significant reduction in wheel-running activity was observed in CCI-treated rats compared with sham controls at any time point when activity levels were examined during the active diurnal phase. Finally, running wheel activity patterns within the 1-hour trial period during the inactive phase of the diurnal cycle were relatively consistent throughout each 20-minute phase. Compared with nonneuropathic sham controls, a profound and stable reduction of running wheel activity was observed in CCI rats during the inactive phase of the diurnal cycle. A concurrent robust allodynia persisted in all rats regardless of when wheel-running activity was examined or whether they ran on wheels, suggesting that acute wheel-running activity does not alter chronic low-intensity mechanical allodynia as measured using the von Frey fiber test. Overall, these data support that acute wheel-running exercise with limited repeated exposures does not itself alter allodynia and offers a behavioral assay complementary to stimulus-induced measures of neuropathic pain.

Characterisation of Delayed Onset of Muscle Soreness (DOMS) in the hand, wrist and forearm using a finger dynamometer: A pilot study

Background: Experimentally-induced delayed-onset muscle soreness of large muscle groups is frequently used in as an injurious model of muscle pain. We wanted to develop an experimental model of DOMS to mimic overuse injuries from sports where repeated finger flexion activity is vital such as rock climbing. The aim of this pilot study was to evaluate the utility of a ‘finger trigger device’ to induce DOMS in the fingers, hands, wrists and lower arms. Methods: A convenient sample of six participants completed an experiment in which they undertook finger exercises to exhaustion after which measurements of pain, skin sensitivity to fi ne touch, forearm circumference and grip strength in the hand, wrist and forearm were taken from the experimental and contralateral none-exercised (control) arms. Results: Pain intensity was greater in the experimental arm at rest and on movement when compared with the control arm up to 24 hours after exercise, although the location of pain varied between participants. Pressure pain threshold was significantly lower in the experimental arm compared with the control arm immediately after exercises locations close to the medial epicondyle but not at other locations. There were no statistical significant differences between affected and non-affected limbs for mechanical detection threshold, forearm circumference or grip strength. Conclusion: Repetitive finger flexion exercises of the index finger by pulling a trigger against a resistance can induced DOMS. We are currently undertaking a more detailed characterization of sensory and motor changes following repetitive finger flexion activity using a larger sample.

The interaction between NGF-induced hyperalgesia and acid-provoked pain in the infrapatellar fat pad and tibialis anterior muscle of healthy volunteers.

Tissue pH is lowered in inflamed tissues, and the increased proton concentration activates acid-sensing ion channels (ASICs), contributing to pain and hyperalgesia. ASICs can be upregulated by nerve growth factor (NGF). The aim of this study was to investigate two new human experimental pain models combining NGF- and acid-induced pain in a randomized, controlled, double-blind study. In experiment 1, volunteers (N=16) received an injection of either NGF or isotonic saline in each infrapatellar fat pad (IFP). One day after 5mL of phosphate-buffered acidic saline was infused into each IFP at a rate of 20mL/h. In experiment 2, the tibialis anterior (TA) muscle of additional volunteers (N=16) was examined, following the same procedure except that the volume and infusion rate of acid were different (10mL, 30mL/h). Continuous pain ratings were recorded during and after acid infusions. In addition, soreness scores on a Likert scale and pressure pain thresholds (PPTs) were assessed. The PPT of the IFP was significantly decreased at the NGF injection site on day 1, but acid-provoked pain ratings and the change in PPT from pre- to postinfusion between the knees were similar. In the muscle pain model, local mechanical hyperalgesia developed 3h after the NGF injection and a significant additional decrease in PPT was found after acid infusion compared to preinfusion. NGF sensitization in the IFP was not facilitated by acid, whereas an acid-provoked enhancement of muscle hyperalgesia was found. NGF sensitization of adipose tissue responds differently to acid provocation compared to muscle tissue. Quantification of two novel pain models combining NGF and acid. Hyperalgesia developed after NGF injection in the infrapatellar fat pad, but it was not facilitated by acid provocation. Contrary, NGF-induced hyperalgesia in muscle tissue was enhanced by acid.

Quercetin Inhibits Peripheral and Spinal Cord Nociceptive Mechanisms to Reduce Intense Acute Swimming-Induced Muscle Pain in Mice.

The present study aimed to evaluate the effects of the flavonoid quercetin (3,3´,4´,5,7-pentahydroxyflavone) in a mice model of intense acute swimming-induced muscle pain, which resembles delayed onset muscle soreness. Quercetin intraperitoneal (i.p.) treatment dose-dependently reduced muscle mechanical hyperalgesia. Quercetin inhibited myeloperoxidase (MPO) and N-acetyl-β-D- glucosaminidase (NAG) activities, cytokine production, oxidative stress, cyclooxygenase-2 (COX-2) and gp91phox mRNA expression and muscle injury (creatinine kinase [CK] blood levels and myoblast determination protein [MyoD] mRNA expression) as well as inhibited NFκB activation and induced Nrf2 and HO-1 mRNA expression in the soleus muscle. Beyond inhibiting those peripheral effects, quercetin also inhibited spinal cord cytokine production, oxidative stress and glial cells activation (glial fibrillary acidic protein [GFAP] and ionized calcium-binding adapter molecule 1 [Iba-1] mRNA expression). Concluding, the present data demonstrate that quercetin is a potential molecule for the treatment of muscle pain conditions related to unaccustomed exercise.

Rubus occidentalis alleviates hyperalgesia induced by repeated intramuscular injection of acidic saline in rats.

BackgroundThe purpose of this study was to evaluate the antinociceptive effect of black raspberry (Rubus occidentalis) fruit extract (ROE) in a rat model of chronic muscle pain and examine the mechanisms involved.MethodsAdult male Sprague–Dawley rats were used, and chronic muscle pain was induced by two injections of acidic saline into one gastrocnemius muscle. For the first experiment, 50 rats were randomly assigned to five groups. After the development of hyperalgesia, rats were injected intraperitoneally with 0.9 % saline or ROE (10, 30, 100, or 300 mg/kg). For the second experiment, 70 rats were randomly assigned to seven groups. Rats were injected intraperitoneally with saline, yohimbine, dexmedetomidine, prazosin, atropine, mecamylamine, or naloxone after the development of hyperalgesia. Ten minutes later, ROE (300 mg/kg) was administered intraperitoneally. For both experiments, the mechanical withdrawal threshold (MWT) was evaluated with von Frey filaments before the first acidic saline injection, 24 h after the second injection, and at 15, 30, 45, 60, 80, 100, and 120 min, 24 and 48 h after the drug administration.ResultsCompared with the control group, the MWT significantly increased up to 45 min after injection of ROE 100 mg/kg and up to 60 min after injection of ROE 300 mg/kg, respectively. Injection of ROE together with yohimbine or mecamylamine significantly decreased the MWT compared with the effect of ROE alone, while ROE together with dexmedetomidine significantly increased the MWT.ConclusionsROE showed antinociceptive activity against induced chronic muscle pain, which may be mediated by α2-adrenergic and nicotinic cholinergic receptors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-016-1192-z) contains supplementary material, which is available to authorized users.

Resident Macrophages in Muscle Contribute to Development of Hyperalgesia in a Mouse Model of Noninflammatory Muscle Pain.

Macrophages play a role in innate immunity within the body, are located in muscle tissue, and can release inflammatory cytokines that sensitize local nociceptors. In this study we investigate the role of resident macrophages in the noninflammatory muscle pain model induced by 2 pH 4.0 preservative-free sterile saline (pH 4.0) injections 5days apart in the gastrocnemius muscle. We showed that injecting 2 pH 4.0 injections into the gastrocnemius muscle increased the number of local muscle macrophages, and depleting muscle macrophages with clodronate liposomes before acid injections attenuated the hyperalgesia produced by this model. To further examine the contribution of local macrophages to this hyperalgesia, we injected mice intramuscularly with C34, a toll-like receptor 4 (TLR4) antagonist. When given before the first pH 4.0 injection, C34 attenuated the muscle and tactile hyperalgesia produced by the model. However, when given before the second injection C34 had no effect on the development of hyperalgesia. Then to test whether activation of local macrophages sensitizes nociceptors to normally non-nociceptive stimuli we replaced either the first or second acid injection with the immune cell activator lipopolysaccharide, or the inflammatory cytokine interleukin (IL)-6. Injecting LPS or IL-6 instead of the either the first or second pH 4.0 injection resulted in a dose-dependent increase in paw withdrawal responses and decrease in muscle withdrawal thresholds. The highest doses of LPS and IL-6 resulted in development of hyperalgesia bilaterally. The present study showed that resident macrophages in muscle are key to development of chronic muscle pain. This article presents evidence for the role of macrophages in the development of chronic muscle pain using a mouse model. These data suggest that macrophages could be a potential therapeutic target to prevent transition of acute to chronic muscle pain particularly in tissue acidosis conditions.

Docking, characterization and investigation of β-cyclodextrin complexed with citronellal, a monoterpene present in the essential oil of Cymbopogon species, as an anti-hyperalgesic agent in chronic muscle pain model

BackgroundCitronellal (CT) is a monoterpene with antinociceptive acute effect. β-Cyclodextrin (βCD) has enhanced the analgesic effect of various substances. Hypothesis/PurposeTo evaluate the effect of CT both complexed in β-cyclodextrin (CT-βCD) and non-complexed, in a chronic muscle pain model (CMP) in mice. Study DesignThe complex containing CT in βCD was obtained and characterized in the laboratory. The anti-hyperalgesic effect of CT and CT-βCD was evaluated in a pre-clinical in vivo study in a murine CMP. MethodsThe complex was characterized through differential scanning calorimetry, derivative thermogravimetry, moisture determination, infrared spectroscopy and scanning electron microscopy. Male Swiss mice were pre-treated with CT (50mg/kg, po), CT-βCD (50mg/kg, po), vehicle (isotonic saline, po) or standard drug (tramadol4 mg/kg, ip). 60 min after the treatment and then each 1h, the mechanic hyperalgesia was evaluated to obtain the time effect. In addition, the muscle strength using grip strength meter and hyperalgesia were also performed daily, for 7 days. We assessed by immunofluorescence for Fos protein on brains and spinal cords of mice. The involvement of the CT with the glutamatergic system was studied with molecular docking. ResultsAll characterization methods showed the CT-βCD complexation. CT-induced anti-hyperalgesic effect lasted until 6h (p <0.001) while CT-βCD lasted until 8h (p <0.001vs vehicle and p <0.001vs CT from the 6th h). CT-βCD reduced mechanical hyperalgesia on all days of treatment (p <0.05), without changing muscle strength. Periaqueductal gray (p <0.01) and rostroventromedular area (p <0.05) showed significant increase in the Fos protein expression while in the spinal cord, there was a reduction (p <0.001). CT showed favorable energy binding (–5.6 and –6.1) to GluR2-S1S2J protein based in the docking score function. ConclusionWe can suggest that βCD improved the anti-hyperalgesic effect of CT, and that effect seems to involve the descending pain-inhibitory mechanisms, with a possible interaction of the glutamate receptors, which are considered as promising molecules for the management of chronic pain such as CMP.

α-Terpineol, a monoterpene alcohol, complexed with β-cyclodextrin exerts antihyperalgesic effect in animal model for fibromyalgia aided with docking study

The anti-hyperalgesic effect of the complex containing α-terpineol (αTPN) and β-cyclodextrin (βCD) was analyzed in a non-inflammatory chronic muscle pain model, as well as its mechanism of action through docking study for a possible interaction with receptors. The αTPN-βCD complex was prepared and characterized through the thermogravimetry/derivate thermogravimetry (TG/DTG), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM). The model of chronic muscle pain was induced by two injections of pH 4.0 saline (20 μl) into the left gastrocnemius 5 days apart. After confirming hyperalgesia, male mice were treated with αTPN-βCD (25, 50 or 100 mg/kg; p.o.) or vehicle (saline 0.9%, p.o.) daily for 10 days. 1 h after the mechanical hyperalgesia, motor performance was evaluated. In addition, the systemic administration of naloxone and ondansetron tested the analgesic effect on the active opioid and serotonin receptors, respectively. The characterization tests indicated that αTPN was efficiently incorporated into βCD. The oral treatment with αTPN-βCD, at all doses tested, produced a significant (p < 0.001) decrease in the mechanical hyperalgesia, without causing any alteration in the force and in motor performance. This analgesic effect was reversed by the systemic administration of naloxone or ondansetron. These findings are corroborated by the docking study described in the present study, which verified a possible interaction of αTPN-βCD with opioid (MU, Kappa, Delta) and 5-HT receptors. Thus, it can be concluded that αTPN-βCD reduced the hyperalgesia followed by the chronic muscle pain model, probably evoked by the descending inhibitory pain system, specifically by opioid and serotoninergic receptors.

(373) Testosterone protect against development of chronic widespread fatigue-induced muscle pain

Chronic widespread pain conditions such as fibromyalgia are more common in women than men. We discovered previously that female mice develop bilateral, more severe and longer duration of hyperalgesia than male mice in a model of fatigue-induced muscle pain. We hypnotized that gonadal hormones might related to the development of hyperalgesia, but ovariectomy had no effect on the development of hyperalgesia in the model. The object of this study was to determine if testosterone protect against development of widespread fatigue-induced muscle pain. C57BL6/J mice (n=49) were used in this study. For inducing muscle hyperalgesia, we combined the localized muscle fatigue with two sub-threshold muscle insults (20μl normal saline (pH 5.0) into the gastrocnemius muscle 5 days apart) and muscle fatigue contractions (6 minutes electrically-induced isometric contractions ), neither of which produced muscle hyperalgesia when given alone. Hyperalgesia was assessed by pinching the belly of the muscle with force-sensitive tweezers and was measured before and up to 6 weeks after induction. Male mice were orchiectomized (Orchi) 2 weeks before induction of the model, and compared to sham, and to female mice. Orchidectomized mice and females developed bilateral hyperalgesia 6 days after induction of the model that persists for at least 6 weeks. In comparison, intact males only develop hyperalgesia unilaterally and for only 2 weeks. 6. Replacement of testosterone (Add dose here) in Orchi-male mice protected the animals from developing long-lasting bilateral hyperalgesia with a similar short-duration and unilateral hyperalgesia to intact male mice. Treatment of female mice with testosterone (add dose here) prevented the bilateral hyperalgesia and resulted in a shorter duration of hyperalgesia and were significantly different from female mice treated with vehicle. These results suggest that testosterone protects male and female mice from developing long-lasting widespread mechanical hyperalgesia in a mouse model of chronic muscle pain.