Injections Of Acidic Saline Research Articles

Does Fibromyalgia Affect the Structure of the Dentate Gyrus?

Abstract Background The hippocampus is considered an important component of the limbic system of the brain. It is involved in memory and functional diversities. The term hippocampus typically refers to the dentate gyrus and the hippocampus proper, the cornu ammonis (CA). Fibromyalgia is known to cause memory and cognitive function impairment and is characterized by causing chronic widespread muscular pain. Aim of the Work The aim of this work was to study the possible structural and morphometric changes in rats’ hippocampal dentate gyrus induced by repeated intramuscular injections of acidic saline in the left gastrocnemius and to compare it with the control. Material and Methods Fifty-four adult female albino Swiss rats were divided into three equal groups (18 rats each): control group (I), natural saline group (II) and acidic saline group (III). Group I received no medication, group II received two i.m. injections of 100 μl saline (pH 7.4) into the left gastrocnemius muscle at day 0 and 5 respectively under sedation with halothane (2-4%), while group III received two i.m. injections of 100 μl saline (pH 4) into the left gastrocnemius muscle at day 0 and 5 respectively under sedation with halothane (2-4%). Rats of each group were further subdivided into 3 subgroups (a, b, c) 6 rats each. Rats were sacrificed by decapitation at day 7, 14, 21 from the start of experiment and their brains were excised. Results Histological examination of H&E-stained sections of groups I and II showed no difference and were both considered control groups. The dentate region was formed of three laminae: the molecular, granular and polymorphic. The granular cell layer is the principal layer separating the molecular and polymorphic layers and was formed of aggregation of granular cells. They were rounded to oval with large vesicular nuclei and .prominent nucleoli The examination of the H&E-stained sections of rats’ dentate gyrus granular cells (DG) appeared darkly stained with pyknotic nuclei I group III as compared to the control. Glial cells slightly increased especially among molecular and polymorphic layers. Neurofibrillary tangles and vacuolations started to appear in silver-stained sections. The neurodegenerative findings started to be more notable in subgroup (b) then became more prevalent in subgroup(c) together with the appearance of clear areas among the sub granular zone. Morphometric studies detected non-significant difference in mean area percentage of astrocytes between group I and group II subgroups. The statistical analysis demonstrated high significant difference in mean area percentage of astrocytes between control groups and acidic saline groups. Evident gliosis was confirmed in group IIIc when the astrocytes percentage areas become very highly significantly increased compared to groups I and II. Conclusion In conclusion, repeated acidic saline injection induced dentate gyrus alterations obvious but minimal at week 1 and become intense at week 3 after the first injection. This effect ended with neuronal death and gliosis.

Reduced sympathetic activity is associated with the development of pain and muscle atrophy in a female rat model of fibromyalgia

Fibromyalgia (FM) is characterized by chronic widespread musculoskeletal pain accompanied by fatigue and muscle atrophy. Although its etiology is not known, studies have shown that FM patients exhibit altered function of the sympathetic nervous system (SNS), which regulates nociception and muscle plasticity. Nevertheless, the precise SNS-mediated mechanisms governing hyperalgesia and skeletal muscle atrophy in FM remain unclear. Thus, we employed two distinct FM-like pain models, involving intramuscular injections of acidic saline (pH 4.0) or carrageenan in prepubertal female rats, and evaluated the catecholamine content, adrenergic signaling and overall muscle proteolysis. Subsequently, we assessed the contribution of the SNS to the development of hyperalgesia and muscle atrophy in acidic saline-injected rats treated with clenbuterol (a selective β2-adrenergic receptor agonist) and in animals maintained under baseline conditions and subjected to epinephrine depletion through adrenodemedullation (ADM). Seven days after inducing an FM-like model with acidic saline or carrageenan, we observed widespread mechanical hyperalgesia along with loss of strength and/or muscle mass. These changes were associated with reduced catecholamine content, suggesting a common underlying mechanism. Notably, treatment with a β2-agonist alleviated hyperalgesia and prevented muscle atrophy in acidic saline-induced FM-like pain, while epinephrine depletion induced mechanical hyperalgesia and increased muscle proteolysis in animals under baseline conditions. Together, the results suggest that reduced sympathetic activity is involved in the development of pain and muscle atrophy in the murine model of FM analyzed.

Adrenalectomy attenuates hyperalgesia but does not regulate muscle wasting in a female rat model of fibromyalgia.

Although it is well established that fibromyalgia (FM) syndrome is characterized by chronic diffuse musculoskeletal hyperalgesia, very little is known about the effect of this pathology on muscle tissue plasticity. Therefore, the present study aimed to characterize the putative alterations in skeletal muscle mass in female rats subjected to a FM model by inducing chronic diffuse hyperalgesia (CDH) through double injections of acidic saline (pH 4.0) into the left gastrocnemius muscle at 5-day intervals. To determine protein turnover, the total proteolysis, proteolytic system activities and protein synthesis were evaluated in oxidative soleus muscles of pH 7.2 (control) and pH 4.0 groups at 7 days after CDH induction. All animals underwent behavioural analyses of mechanical hyperalgesia, strength and motor performance. Our results demonstrated that, in addition to hyperalgesia, rats injected with acidic saline exhibited skeletal muscle loss, as evidenced by a decrease in the soleus fibre cross-sectional area. This muscle loss was associated with increased proteasomal proteolysis and expression of the atrophy-related gene (muscle RING-finger protein-1), as well as reduced protein synthesis and decreased protein kinase B/S6 pathway activity. Although the plasma corticosterone concentration did not differ between the control and pH 4.0 groups, the removal of the adrenal glands attenuated hyperalgesia, but it did not prevent the increase in muscle protein loss in acidic saline-injected animals. The data suggests that the stress-related hypothalamic-pituitary-adrenal axis is involved in the development of hyperalgesia, but is not responsible for muscle atrophy observed in the FM model induced by intramuscular administration of acidic saline. Although the mechanisms involved in the attenuation of hyperalgesia in rats injected with acidic saline and subjected to adrenalectomy still need to be elucidated, the results found in this study suggest that glucocorticoids may not represent an effective therapeutic approach to alleviate FM symptoms.

Antifibromyalgic Activity of Phytomolecule Niranthin: In-Vivo Analysis, Molecular Docking, Dynamics and DFT

Fibromyalgia (FM) is characterized by chronic pain and heightened sensitivity to painful stimuli. This study investigated the potential of Niranthin (NR), a natural compound derived from Phyllanthus species, in a rat model of FM. We employed a multifaceted approach to comprehensively assess the potential benefits of NR as a treatment for FM, including in-vivo analysis, molecular docking studies, and molecular dynamics (MD) simulations, and Density Functional Theory (DFT) calculations. Three doses of NR (5 mg/kg, 10 mg/kg and 20 mg/kg) significantly reduced mechanical allodynia induced by acidic saline injections. In an acute study, NR dosing increased the mechanical threshold in both ipsilateral and contralateral paws. In a four-day study, twice-daily NR treatment further elevated the mechanical threshold. Temporary treatment interruption led to a re-establishment of allodynia, but subsequent reinitiation of NR treatment remained effective, ruling out tolerance development. MD simulations were conducted to investigate the stability and dynamics of NR-target complexes, revealing stable binding interactions and conformational changes associated with NR binding. Docking calculations indicated that the NR molecule had a similar binding affinity to the drug Amiloride towards Acid-sensing ion channels (ASICs). NR showed interactions with amino acid residues, including LYS 373, ARG 370, GLU 374 and GLN 277. DFT calculations provided insights into the electronic and structural properties of NR and its interactions with FM-related molecular target ASICs. We propose that NR may modulate ASICs, leading to decreased pain sensitivity in FM rats. However, further research is required to fully understand the mechanism of action and explore NRs therapeutic potential for the treatment of FM.

RNA Sequencing Reveals Transcriptional Changes in Muscle Tissue in Response to Muscle Pain in Exercised and Sedentary Mice

Exercise produces analgesia through multiple peripheral, spinal, and supraspinal pathways. However, uncovering these pathways one by one is time consuming, biased based on prior literature, and requires significant number animals to be tested. RNA sequencing allows for discovery of numerous transcriptional differences and can lead to the discovery of novel nociceptive pathways. The purpose of this study was to measure transcriptional differences in response to muscle pain between sedentary and exercised animals. Male and female C57/Black6 mice were grouped as pain free, sedentary with muscle pain model, and exercised with muscle pain model. The muscle pain model was induced with 2 intramuscular injections of acidic saline (20μl, pH 5.0±0.1) combined with 6 minutes of fatiguing muscle contractions delivered to the left gastrocnemius muscle. For exercise, animals were individually housed with 24hr access to a running wheel for 8 weeks prior to induction of the pain model. The left gastrocnemius muscle was harvested 24hrs after induction of the pain model and RNA was extracted. Bulk RNA sequencing was performed on an Illumina NovaSeq 6000. Differentially expressed genes (DEGs) were generated based on raw counts at the gene and transcript level using DESeq2. Within each sex, DEGs were determined between the pain free and sedentary groups and between exercised and sedentary with pain model groups. DEGs were then imported into iPathwayGuide commercial software and results were compared between sexes. Between pain free and sedentary in pain animals 160 DEGs were discovered in males and 661 were discovered in females. Between exercised and sedentary groups with the muscle pain model, 13 DEGs were discovered in males and 195 in females. iPathwayGuide analysis for all data sets revealed activation of immune system pathways including chemokine signaling, cytokine-cytokine receptor interaction, and antigen processing and presentation. Future work will confirm results with qPCR and behavioral pharmacology. Grant support from NIH AR073187.

Select Androgen Receptor Modulator Microparticle Formulation Reverses Muscle Hyperalgesia in Mouse Model of Widespread Muscle Pain

There is a need for the generation of non-opioid analgesics for chronic pain. The analgesic effects of testosterone have been demonstrated in preclinical and clinical studies. However, treatment with testosterone is not clinically feasible due to adverse effects. Select androgen receptor modulators (SARMs) were developed to overcome adverse effects of testosterone by selectively activating androgen receptors associated with anabolic effects while minimizing activation of androgenic effects. The purpose of this study was to develop a SARM-loaded microparticle formulation with a long-term release profile and test its efficacy in a preclinical model of chronic muscle pain. Chronic muscle pain was induced by 2 intramuscular injections of acidic saline (20μl, pH 4.0±0.1) spaced 5 days apart were delivered into the left gastrocnemius muscle of C57/BL6J male and female mice. Muscle withdrawal thresholds (MWT) were assessed with force sensitive tweezers applied to the gastrocnemius muscle before and weekly for 4 weeks after induction of the model. Efficacy of SARM ((s)-3-(4-cyanophenoxy)-n-(4-cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide) was tested through daily systemic injection of the drug (25mg/kg, s.c.) or two injections of SARM loaded poly(lactic-co-glycolic acid) (PLGA) microparticles (200mg, s.c.; 24hr, 1wk). Drug release profiles from the SARM microparticle formulation was assessed both in vitro and in vivo via HPLC-UV. Liver and cardiac toxicity was analyzed from serum samples from animals receiving SARM microparticles. Statistical analysis for MWT was performed with repeated measures ANOVA compared with vehicle treatment. Both daily administration of SARMs and two injections of SARM microparticles alleviated decreased MWT bilaterally in both sexes (p<0.01). In vitro and in vivo release studies showed SARM was steadily released from microparticles for 4 weeks. Toxicity panels revealed no adverse effects of SARM microparticle treatment. The current study shows SARMs can alleviate muscle pain and SARM loaded microparticles increase clinical utility of administration due to fewer injections and long-term release profiles. Grant support from NIH AR073187 and P30CA086862.

Translating Outcomes from the Clinical Setting to Preclinical Models: Chronic Pain and Functionality in Chronic Musculoskeletal Pain.

Fibromyalgia (FM) is a chronic pain disorder characterized by chronic widespread musculoskeletal pain (CWP), resting pain, movement-evoked pain (MEP), and other somatic symptoms that interfere with daily functioning and quality of life. In clinical studies, this symptomology is assessed, while preclinical models of CWP are limited to nociceptive assays. The aim of the study was to investigate the human-to-model translatability of clinical behavioral assessments for spontaneous (or resting) pain and MEP in a preclinical model of CWP. For preclinical measures, the acidic saline model of FM was used to induce widespread muscle pain in adult female mice. Two intramuscular injections of acidic or neutral pH saline were administered following baseline measures, 5 days apart. An array of adapted evoked and spontaneous pain measures and functional assays were assessed for 3 weeks. A novel paradigm for MEP assessment showed increased spontaneous pain following activity. For clinical measures, resting and movement-evoked pain and function were assessed in adult women with FM. Moreover, we assessed correlations between the preclinical model of CWP and in women with fibromyalgia to examine whether similar relationships between pain assays that comprise resting and MEP existed in both settings. For both preclinical and clinical outcomes, MEP was significantly associated with mechanical pain sensitivity. Preclinically, it is imperative to expand how the field assesses spontaneous pain and MEP when studying multi-symptom disorders like FM. Targeted pain assessments to match those performed clinically is an important aspect of improving preclinical to clinical translatability of animal models.

Effectiveness of maturity of Rubus occidentalis on hyperalgesia induced by acidic saline injection in rats

BackgroundRubus occidentalis, also known as black raspberry, contains several bioactive components that vary depending on the maturity of the fruit. The goal of this study was to evaluate the efficacy of immature Rubus occidentalis extract(iROE) on acid-induced hyperalgesia, investigate the mechanism involved, and compare the antihyperalgesic effect of immature and mature ROEs.MethodsIn adult male Sprague-Dawley rats, chronic muscle pain was induced via two injections of acidic saline into one gastrocnemius muscle. To evaluate the dose response, the rats were injected intraperitoneally with 0.9% saline or iROE (10, 30, 100, or 300 mg/kg) following hyperalgesia development. To evaluate the mechanism underlying iROE-induced analgesia, the rats were injected intraperitoneally with saline, yohimbine 2 mg/kg, dexmedetomidine 50 μg/kg, prazosin 1 mg/kg, atropine 5 mg/kg, mecamylamine 1 mg/kg, or naloxone 5 mg/kg 24 h after hyperalgesia development, followed by iROE 300 mg/kg administration. To compare immature versus mature ROE, the rats were injected with mature ROE 300 mg/kg and immature ROE 300 mg/kg after hyperalgesia development. For all experiments, the mechanical withdrawal threshold(MWT) was evaluated using von Frey filaments before the first acidic saline injection, 24 h after the second injection, and at various time points after drug administration. Data were analysed using multivariate analysis of variance(MANOVA) and the linear mixed-effects model(LMEM). We compared the MWT at each time point using analysis of variance with the Bonferroni correction.ResultsThe iROE 300 mg/kg injection resulted in a significant increase in MWT compared with the control, iROE 30 mg/kg, and iROE 100 mg/kg injections at ipsilateral and contralateral sites. The iROE injection together with yohimbine, mecamylamine, or naloxone significantly decreased the MWT compared with iROE alone, whereas ROE together with dexmedetomidine significantly increased the MWT. According to MANOVA, the effects of immature and mature ROEs were not significantly different; however, the LMEM presented a significant difference between the two groups.ConclusionsImmature R. occidentalis showed antihyperalgesic activity against acid-induced chronic muscle pain, which may be mediated by the α2-adrenergic, nicotinic cholinergic, and opioid receptors. The iROE displayed superior tendency regarding analgesic effect compared to mature ROE.

Mirogabalin, a novel ligand for α2δ subunit of voltage-gated calcium channels, improves cognitive impairments in repeated intramuscular acidic saline injection model rats, an experimental model of fibromyalgia

Mirogabalin is a novel potent and selective ligand for the α2δ subunit of voltage-gated calcium channels, and shows potent and sustained analgesic effects in neuropathic pain and fibromyalgia models. Fibromyalgia is often associated with multiple comorbid symptoms, such as anxiety, depression and cognitive impairment. In the present study, we investigated the effects of mirogabalin on cognitive impairments in an experimental animal model for fibromyalgia, repeated intramuscular acidic saline injection model (Sluka model) rats. Male rats received two repeated intramuscular injections of pH 4 acidic saline into their gastrocnemius muscle. After developing mechanical hypersensitivity as identified in the von Frey test, the animals received the test substance orally once daily for 13 days and were subjected to four cognitive function tests, (Y-maze, novel object recognition, Morris water maze and step-through passive avoidance). Sluka model rats showed cognitive impairments in all four tests. Oral administration of mirogabalin (3 and 10 mg/kg) improved the cognitive impairments in these rats. In conclusion, mirogabalin improved the impaired cognitive function in Sluka model rats. It may thus also alleviate cognitive impairments as well as painful symptoms in fibromyalgia patients.

Electroacupuncture reduces chronic fibromyalgia pain through attenuation of transient receptor potential vanilloid 1 signaling pathway in mouse brains.

Objective(s):Fibromyalgia pain is a mysterious clinical pain syndrome, characterized by inflammation in the brain, whose molecular mechanisms are still unknown. Females are more commonly affected by fibromyalgia, exhibiting symptoms such as widespread mechanical pain, immune dysfunction, sleep disturbances, and poor quality of life. Electroacupuncture (EA) has been used to relieve several types of pain, including fibromyalgia pain.Materials and Methods:In the present study, we used dual injections of acidic saline into the gastrocnemius muscle to initiate a neural activation that resulted in fibromyalgia pain in mice. We used the Von Frey test to measure mechanical hyperalgesia and Western blot to measure protein levels. Results:Results indicated that mechanical hyperalgesia can be induced in mice for 4 weeks, suggesting the induction of chronic fibromyalgia (CFM). Furthermore, continuous EA treatment reliably attenuated the mechanical hyperalgesia, but not in the sham control group. Results also suggested that the mechanical hyperalgesia can be prevented in mice with TRPV1 gene deletion. Mice with CFM showed increased expressions of TRPV1, Nav1.7, and Nav1.8 in the dorsal root ganglion (DRG) and the spinal cord (SC). The expression of TRPV1-associated molecules such as pPKA, pERK, and pCREB was also increased in the thalamus and somatosensory cortex (SSC) of the mice. All the aforementioned mechanisms were reversed by EA treatment and TRPV1 gene deletion. Conclusion:Altogether, our results implied significant mechanisms of CFM and EA-analgesia that involve the regulation of the TRPV1 signaling pathway. These findings may be relevant to the evaluation and treatment of CFM.

Anxiolytic-like effects of mirogabalin, a novel ligand for α2δ ligand of voltage-gated calcium channels, in rats repeatedly injected with acidic saline intramuscularly, as an experimental model of fibromyalgia.

Mental disorders including anxiety and depression are common comorbidities in fibromyalgia patients, and exert a profound impact on their quality of life. Mirogabalin, a novel ligand for the α2δ-subunit of voltage-gated calcium channels, shows analgesic effects in fibromyalgia and neuropathic pain models. To provide additional information regarding its potential utility for treating chronic pain, we examined its anxiolytic-like effects in rats repeatedly injected with acidic saline intramuscularly (Sluka model), as an experimental fibromyalgia model. Male Sprague-Dawley rats received two intramuscular injections of acidic saline (pH 4.0) into the gastrocnemius muscle. After the development of tactile allodynia demonstrated by decreased paw withdrawal threshold to von Frey filaments, anxiety-like behaviours were evaluated using the open field test and the elevated plus maze test. Sluka model rats exhibited anxiety-like behaviours in the open field test (significant decreases in distance travelled and time spent in the central area, and significant increases in time spent in the wall area) and the elevated plus maze test (significant decreases in time spent in the open arms and significant increases in time spent in the closed arms). A single oral dose of mirogabalin (3 or 10mg/kg) significantly alleviated and normalised these anxiety-like behaviours. Sluka model rats exhibited anxiety-like behaviours in the open field test and the elevated plus maze test, but mirogabalin alleviated these behaviours. Mirogabalin might thus have the potential to relieve anxiety in fibromyalgia patients.

Inorganic phosphate and lactate potentiate the pressor response to acidic stimuli in rats.

What is the central question of this study? What is the contribution of the main acidic compounds accumulated during contractions, namely H+ , lactic acid and inorganic phosphate, to evoke the metabolic component of the exercise pressor reflex? What is the main finding and its importance? We found that the pressor response to acidic stimuli is driven by the concentration of hydrogen ions and that lactate and inorganic phosphate act as potentiating agents. H+ ions, lactate and inorganic phosphate are produced by contracting skeletal muscles and evoke, in part, the metabolic component of the exercise pressor reflex. Owing to their disparate dissociation constants (i.e. pKa ), the contribution of each acid to the muscle metaboreflex is unclear. This lack of information prompted us to determine the reflex pressor responses to injection of acidic saline, lactate (24mm)and inorganic phosphate(86mm)at various values of pH (from 2.66 to 7.5), alone or in combination, into the arterial supply of hindlimb skeletal muscle of decerebrate rats. In particular, we tested the hypothesis that the pressor response to an injection of a combination of lactate and phosphate at an acidic pH is greater than that evoked by injection of either phosphate or lactate alone at the same pH. We found that injection of acidic saline produced a pressor response only at a pH of 2.66 (7±4mmHg), an effect that was potentiated when the solution contained lactate (50±20mmHg). At a pH of 6.0, however, this effect was lost. At a pH of 6.0, only the injection of inorganic phosphate produced a significant pressor response (23±12mmHg). A large potentiating effect was found when lactate was added to the inorganic phosphate solution (39±18mmHg), an effect that was lost at a pH>7.0. Our findings led to the conclusion that the pressor response to injection of acidic solutions was driven by H+ ions and that inorganic phosphate and lactate functioned as sensitizing agents.

Does aerobic exercise associated with tryptophan supplementation attenuates hyperalgesia and inflammation in female rats with experimental fibromyalgia?

The objective of this study was to verify the effects of aerobic exercise associated with tryptophan (TRP) supplementation on hyperalgesia, as well as on cortisol, IL-6 and TNF concentrations in female rats with experimental fibromyalgia (FM). Female Wistar rats (initial body weight: ~ 350 g; age: 12 months) were randomly divided into 5 groups: CON (Control); F (Fibromyalgia induced); FE (Fibromyalgia induced plus exercise); FES (Fibromyalgia induced plus exercise and TRP supplementation) and FS (Fibromyalgia induced plus TRP supplementation). Fibromyalgia was induced with two injections (20 μL) of acidic saline (pH 4.0) into the right gastrocnemius muscle with a 3-day interval. Control animals received the same doses of neutral saline (pH 7.4). The exercised animals underwent progressive low-intensity aerobic exercise (LIAE) on a treadmill (10–12 m/min, 30–45 min/day, 5 days/week) for three weeks. During this period, the supplemented animals received a TRP supplemented diet (210 g/week), while the others received a control diet. Mechanical hyperalgesia was evaluated weekly and serum cortisol and muscle IL-6 and TNF concentrations were assessed after three weeks of interventions. Experimental FM caused bilateral hind paw hyperalgesia and augmented serum cortisol and muscle IL-6 concentrations. After 3 weeks of interventions, LIAE alone reduced hyperalgesia (151%) and reduced serum cortisol concentrations (72%). Tryptophan supplementation itself diminished hyperalgesia (57%) and reduced serum cortisol concentrations (67%). Adding TRP supplementation to LIAE did not further reduce hyperalgesia significantly (11%), which was followed by an important decrease in muscle IL-6 concentrations (68%), though reduction in serum cortisol pulled back to 45%. Muscle TNF concentrations were not affected. In conclusion, the association of TRP supplementation to LIAE does not potentiate significantly the reduction of bilateral mechanical hyperalgesia promoted by LIAE in female rats with experimental FM, however an important decrease in IL-6 is evident.

Analgesic effects of mirogabalin, a novel ligand for α2δ subunit of voltage-gated calcium channels, in experimental animal models of fibromyalgia.

Mirogabalin, a novel ligand for the α2δ subunit of voltage-gated calcium channels, is under the development for the treatment of neuropathic pain. Mirogabalin specifically and potently binds to α2δ subunits, and it shows analgesic effects in both peripheral and central neuropathic pain models in rats. To expand pharmacological findings on mirogabalin and provide additional information of its potential for chronic pain therapy, we examined the effects of mirogabalin in 2 experimental models of fibromyalgia, namely, the intermittent cold stress model (ICS model) and the unilateral intramuscular acidic saline injection model (Sluka model). To induce chronic mechanical hypersensitivity, mice were placed under ICS conditions for 3days, whereas rats were injected twice with acidic saline (pH4) into the gastrocnemius muscle in a 4-day interval. The pain sensitivity was evaluated by the von Frey test. Long-lasting increases in pain response score or decreases in pain threshold to the von Frey stimulation were observed in both the ICS and Sluka models. Mirogabalin (1, 3, or 10mg/kg, p.o.) dose-dependently alleviated the mechanical hypersensitivity, with significant effects persisting at 6 or 8h following administration. The standard α2δ ligand, pregabalin (30mg/kg, p.o.), also significantly reduced the mechanical hypersensitivity. In summary, mirogabalin showed analgesic effects in the ICS model mice and in the Sluka model rats. Therefore, mirogabalin may have the potential to provide effective pain relief in patients with fibromyalgia.

Sex differences in the contributions of spinal atypical PKCs and downstream targets to the maintenance of nociceptive sensitization.

BackgroundChronic pain has been shown to depend on nociceptive sensitization in the spinal cord, and while multiple mechanisms involved in the initiation of plastic changes have been established, the molecular targets which maintain spinal nociceptive sensitization are still largely unknown. Building upon the established neurobiology underlying the maintenance of long-term potentiation in the hippocampus, this present study investigated the contributions of spinal atypical protein kinase C (PKC) isoforms PKCι/λ and PKMζ and their downstream targets (p62/GluA1 and NSF/GluA2 interactions, respectively) to the maintenance of spinal nociceptive sensitization in male and female rats.ResultsPharmacological inhibition of atypical PKCs by ZIP reversed established allodynia produced by repeated intramuscular acidic saline injections in male animals only, replicating previously demonstrated sex differences. Inhibition of both PKCι/λ and downstream substrates p62/GluA1 resulted in male-specific reversals of intramuscular acidic saline-induced allodynia, while female animals continued to display allodynia. Inhibition of NSF/GluA2, the downstream target to PKMζ, reversed allodynia induced by intramuscular acidic saline in both sexes. Neither PKCι/λ, p62/GluA1 or NSF/GluA2 inhibition had any effect on formalin response for either sex.ConclusionThis study provides novel behavioural evidence for the male-specific role of PKCι/λ and downstream target p62/GluA1, highlighting the potential influence of ongoing afferent input. The sexually divergent pathways underlying persistent pain are shown here to converge at the interaction between NSF and the GluA2 subunit of the AMPA receptor. Although this interaction is thought to be downstream of PKMζ in males, these findings and previous work suggest that females may rely on a factor independent of atypical PKCs for the maintenance of spinal nociceptive sensitization.

Mouse incising central pattern generator: Characteristics and modulation by pain

IntroductionVertebrate incising and chewing are controlled by a set of neurons comprising the central pattern generator (CPG) for mastication. Mandibular positioning and force generation to perform these tasks is complex and requires coordination of multiple jaw opening and closing muscle compartments located in muscles on both sides of the jaw. The purpose of this study was to determine the characteristics of the CPG by recording mouse incising forces in the home cage environment to evaluate changes in force characteristics with incising frequency and force direction. A second purpose was to evaluate the effects of jaw closing muscle pain on CPG output parameters. MethodsDigitized incising forces were recorded for approximately 24 h using a 3-dimensional force transducer attached to solid food chow. Male and female CD-1 mice were evaluated during their last (fourth) baseline assessment and seven days after a second acidic saline injection into the left masseter muscle when maximum pain was experienced. Incising force resultants were calculated from the three axes data and force parameters were assessed including inter-peak intervals (IPI), peak amplitude, load time and unload time. Multiple regression analyses were conducted to identify incising episodes that had parameters of force that were significantly correlated (p < 0.001). These incising episodes were considered to represent the output of the CPG with a steady state of incoming sensory afferent inputs. Incising parameters were evaluated for each of the discrete incising frequencies (4.6, 5.3, 6.2, 7.6 Hz) and the predominant force directions: jaw closing (−Z), jaw retrusion (+X) and jaw protrusion (−X). ResultsA significant correlation between incising frequency (IPI) and the load time was observed. A significant decrease in peak amplitude was observed with higher incising frequency while the load rate significantly increased. The force peak amplitude and load rates were found to be statistically different when the force direction was considered, with smaller peak amplitudes and smaller load rates found in the jaw closing direction. The effect of pain on incising was to reduce the peak amplitude and load rate of incising compared to the baseline condition at lower incising frequencies. ConclusionsLike the central pattern generator for locomotion, the CPG for incising controls rhythmicity, peak amplitude and force load duration/rate. However, unlike the CPG for locomotion, the amplitude of incising force decreases as the frequency increases. During incising, load rate increases with faster rhythm and is consistent with the recruitment of larger motor units. Muscle pain reduced the excitatory drive of the CPG on motoneurons and provides further support of the Pain Adaptation Model.

Stimulation of Peripheral Opioid Receptors during Acute Exercise Decreases Anxiety‐Like Behaviors in a Rat Model of Fibromyalgia

Patients with fibromyalgia often experience comorbid anxiety. Studies show exercise training can decrease pain in patients with fibromyalgia as well as decrease anxiety in healthy populations. Evidence suggests endorphins acting on opioid receptors contribute to exercise‐induced analgesia, but the role of these receptors on anxiety has not been determined in patients with fibromyalgia. The purpose of this study was to compare the effects of sedentary behavior and exercise training on anxiety in a rat model of fibromyalgia and to determine the contribution of peripheral opioid receptors to the exercise‐induced response. Female Sprague Dawley rats were induced with fibromyalgia by acidic saline injections (0.1 mL; pH 4). To assess anxiety‐like behaviors, rats were placed in an open field arena (90 × 90 cm) for two minutes. The arena floor was marked with 20.5 cm grids and the number of grids crossed during the observation period was recorded: a decrease in grids crossed indicated increased anxiety. A group of sedentary rats (SED; n=8) were tested for anxiety 5 days/week for 4 weeks. Another group of treadmill‐trained rats (NAL; n=5) received injections of the nonspecific opioid receptor antagonist naloxone (120 μg in 0.1 mL saline) in both gastrocnemius muscles ten minutes prior to moderate‐intensity (15 m/sec) treadmill running (4 weeks; 5 days/week; 30 min/day). NAL rats were observed in the open field arena before (PRE) and after (POST) each running bout. Mean grids crossed for SED rats in the open field were 21.3 ± 2.4 for week 1, 23.7 ± 2.9 for week 2, 29.7 ± 2.9 for week 3, and 28.0 ± 3.0 for week 4. While NAL PRE grids crossed were not different from SED grids crossed, the POST grids crossed for NAL rats decreased for all weeks: 16.5 ± 2.0 (PRE) and 10.1 ± 1.9 (POST) for week 1, 24.0 ± 1.9 (PRE) and 12.4 ± 1.4 (POST) for week 2, 21.8 ± 1.9 (PRE) and 10.4 ± 1.7 (POST) for week 3, and 23.6 ± 2.2 (PRE) and 13.2 ± 1.8 (POST) for week 4 (all p&lt;0.05). Further, NAL POST grids crossed were significantly different from SED grids crossed for weeks 1, 2, 3 and 4 (p&lt;0.05). While our data does not show a long‐term effect of exercise training on anxiety, it does suggest that stimulation of peripheral opioid receptors during acute exercise decreases anxiety‐like behaviors in the rat model. In conclusion, this study provides evidence that peripheral, in addition to central, opioid receptors contribute to an exercise‐induced decrease in anxiety and that even one exercise bout is beneficial when compared to sedentary behavior.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of ASIC3, Nav1.7 and Nav1.8 in electroacupuncture-induced analgesia in a mouse model of fibromyalgia pain.

The mechanisms underlying fibromyalgia (FM) pain are not understood. The US Food and Drug Administration has recommended three drugs for treating FM-namely, pregabalin, duloxetine and milnacipran; however, these medications are associated with severe side effects. To create a mouse model of FM pain using dual injections of acidic saline to cause mechanical hyperalgesia and test whether ASIC3, Nav1.7 and Nav1.8 are involved in this process and whether electroacupuncture (EA) can reverse these phenomena. The FM model was established by injecting acidic saline twice into 40 ICR mice. The mice were assigned to subgroups (n=8 each) treated with different EA frequencies (2, 15 and 50 Hz). ASIC3, Nav1.7 and Nav1.8 expression levels were measured by Western blotting and immunohistochemistry. Significant mechanical hyperalgesia was induced on day 8 in FM mice, which was reversed by 2, 15 and 50 Hz EA. ASIC3, Nav1.7 and Nav1.8 protein levels increased significantly in both the dorsal root ganglion and in the spinal cord of FM model mice. These changes were further attenuated by 2, 15 and 50 Hz EA. Reduced nociceptive ASIC3, Nav1.7 and Nav1.8 proteins are involved in the preventive effects of EA against FM, and this series of molecules may represent targets for FM treatment.

Effects of Exercise and Peripheral Opioid Receptor Stimulation on Anxiety‐Like Behaviors in a Rat Model of Chronic Pain

Patients with fibromyalgia are more likely to suffer from anxiety. In the acidic saline‐induced chronic pain model, rats exhibit long‐lasting, wide‐spread pain along with anxious behaviors. Exercise training has been shown to decrease pain in both patients with fibromyalgia and in this rat model of chronic pain; however the effect of exercise on anxiety and the contribution of peripheral opioid receptors to this effect are unknown. All experiments were performed in female Sprague‐Dawley rats that demonstrated chronic widespread pain due to acidic saline injections and all rats underwent moderate‐intensity treadmill training (4 weeks; 5 days/week; 30 min/day at 15 m/sec). Anxiety‐like behaviors were determined using the open field test. Control rats (n=4) were observed in the open field (90 × 90 cm) for two minutes before (PRE) and after (POST) each exercise bout. Time spent moving was recorded: decreased motility demonstrated anxiety. A separate group of rats (NAL; n=5) received injections of the nonspecific opioid receptor antagonist naloxone (120 μg in 100 μL saline) in both gastrocnemius muscles ten minutes prior to treadmill running in addition to open field testing. Results showed that treadmill training alone had no effect on control rats' average time spent moving in the open field: 32.5 ± 4.2 s (PRE) and 31.2 ± 4.4 s (POST) for week 1; 35.1 ± 4.2 s (PRE) and 31.7 ± 3.8 s (POST) for week 2; 32.7 ± 4.3 s (PRE) and 38.3 ± 4.8 s (POST) for week 3; and 35.6 ± 4.6 s (PRE) and 34.5 ± 3.1 s (POST) for week 4. However, NAL rats demonstrated increased anxiety‐like behaviors indicated by decreased average time spent moving for all weeks tested: 25 ± 3.9 s (PRE) and 14.2 ± 3.2 s (POST) for week 1; 34.6 ± 3.4 s (PRE) and 18.9 ± 2.8 s (POST) for week 2; 35 ± 4.1 s (PRE) and 16.5 ± 3.2 s (POST) for week 3; and 38 ± 4.5 s (PRE) and 17.9 ± 2.9 s (POST) for week 4. In NAL rats, PRE and POST times were significantly different for all four weeks (p&lt;0.05). PRE times were not different between control and NAL groups, however, POST times were significantly different between control and NAL groups for all weeks. Our data suggests that endorphins released during exercise act on peripheral opioid receptors to reduce anxiety‐like behaviors associated with the exercise bout in a rat model of chronic pain. These findings support the use of exercise to reduce anxiety and chronic pain in people with fibromyalgia.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Acid-induced experimental muscle pain and hyperalgesia with single and repeated infusion in human forearm

Background and purposeAcid has long been thought to play an important role in the pain process. Animal study showed that repeated acid stimulation induced central sensitization. The purpose of the study is to investigate muscle pain and hyperalgesia evoked by intramuscular infusion of saline at different pH levels, and to compare the effect of a single versus repeated acid infusions. MethodsTwenty healthy subjects received infusions of buffered saline (pH 5.0, 6.0, and 7.4) into the brachioradialis muscle in a randomized order. Twelve of the subjects received repeated infusions. The subjects rated the pain intensity on visual analogue scale (VAS). Thermal pain sensitivity, and pressure pain threshold (PPT) were assessed in both arm before, during, immediately after, one hour after, and one day after the infusion. A McGill Pain Questionnaire and pain mapping were completed after each infusion. ResultsThe pH 5 solution caused significantly higher pain and larger areas than pH 6.0 or 7.4. The local PPTs were significantly decreased (hyperalgesia) during and immediately after infusion of all three solutions. No significant differences were detected between the first and second infusion. ConclusionsThe intensity of acid-induced muscle pain is pH-dependent. All three solutions induced pressure hyperalgesia at the infusion site. Repeated infusions did not induce increased pain or prolonged hyperalgesia as compared with a single injection. Human intramuscular acidic saline infusion could not produce chronic pain model. ImplicationsThe acid-induced pain model may reflect the early stage responses to tissue injury of clinical conditions. Repeated intramuscular acidic saline injection model of prolonged hyperalgesia in rodents could not be translated into a human for modelling chronic musculoskeletal pain.