Inflammation repairs tissues in response to stressors. Aging alters circulating pro- and anti-inflammatory markers through immunosenescence, the gradual decline of immune function, and increased cytokine secretion, contributing to chronic inflammation. Exercise can modulate cytokine levels, producing anti-inflammatory effects. However, how age and sex influence these responses remains unclear. Methods: We evaluated blood serum levels of proinflammatory (TNF-α, IL-1β, IL-6) and anti-inflammatory (IL-10) cytokines in young and old male and female Fischer 344 rats before and after a moderate treadmill exercise protocol. Results: TNF-α increased in young rats following both sedentary and exercise treatments, but not in old rats, with no sex differences. Exercise increased IL-1β and IL-6 only in young males, while old males showed elevated levels under sedentary condition that were suppressed by exercise. IL-10 increased exclusively in old females after exercise. Conclusion: These results indicate that exercise does not uniformly suppress inflammation but modulates cytokine activity in an age- and sex-dependent manner. In young rats, proinflammatory responses may support tissue repair and muscle remodeling, whereas in old rats, the attenuation of these markers, along with IL-10 elevation in females, suggests a shift toward anti-inflammatory adaptations that may counteract chronic inflammation.

Temporomandibular disorder (TMD) is the most prevalent painful condition in the craniofacial area. Recent studies have suggested that external or intrinsic trauma to the temporomandibular joint (TMJ) is associated with the onset of painful TMD in patients. Here, we investigated the effects of TMJ trauma through forced-mouth opening (FMO) in mice to determine pain behaviors and peripheral sensitization of trigeminal nociceptors in both sexes. Forced-mouth opening increased mechanical pain as assessed by the von Frey test, with spontaneous pain-like behaviors assessed using the mouse grimace scale and anxiety-like behaviors assessed using the open-field test. Changes in pain-like behaviors were not different between male and female mice. However, in vivo GCaMP Ca2+ imaging of intact trigeminal ganglia (TG) showed modality- and sex-dependent changes. Forced-mouth opening increased spontaneous Ca2+ responses and mechanical hypersensitivity of TG neurons compared to the sham group, which was more pronounced in male mice. Forced-mouth opening also increased Ca2+ responses evoked by cold, heat, and capsaicin stimuli, which was not different between the sexes. In retrogradely labeled trigeminal TMJ afferents, FMO induced an increase in small-sized neuronal proportions with increased colocalization with calcitonin gene-related peptides and transient receptor potential vanilloid subtype 1, which was modestly sex dependent. These results suggest that TMJ injury leads to persistent posttraumatic hyperalgesia associated with peripheral sensitization of trigeminal nociceptors with distinct sex dependency.

Knee osteoarthritis (OA) often causes chronic pain that disproportionately affects females. Proinflammatory cytokines TNF-α, IL-1β, and IL-6 are key effectors of OA pathological changes. Green light shows potential as an alternative intervention for various pain conditions. However, no studies have investigated green light′s analgesic effects in both sexes in chronic knee OA. We induced unilateral knee OA with intra-articular injection of monoiodoacetate (MIA) in male and female Sprague-Dawley rats. Two days post-injection, the rats were exposed to green-light-emitting diodes (GLED) or ambient room light eight hours daily for 24 days. Knee mechanical sensitivity was assessed using a small animal algometer. Blood serum concentrations of TNF-α, IL-1β, IL-6, and IL-10 were quantified at baseline and 23 days post-injection. MIA injection decreased the knee mechanical thresholds of the male and female rats. GLED exposure attenuated mechanical hypersensitivity in both sexes compared to the controls; however, GLED-induced analgesia occurred sooner and with greater magnitude in males than in females. In both sexes, the analgesic effects of green light lasted 5 days after the final GLED session. Finally, GLED exposure reversed the elevation of serum proinflammatory cytokines. These findings suggest that GLED exposure reduces primary hyperalgesia in OA, potentially by lowering proinflammatory cytokines, and indicate sex differences in GLED-induced analgesia.

This study investigates the impact of combining psychophysical stress, induced by forced swim (FSS), with masseter inflammation on reactive oxygen species (ROS) production in trigeminal ganglia (TG), TRPA1 upregulation in TG, and mechanical hyperalgesia. In a rat model, we demonstrate that FSS potentiates and prolongs CFA-induced ROS upregulation within TG. The ROS levels in CFA combined with FSS group surpass those in the CFA-only group on days 4 and 28 post-treatment. FSS also enhances TRPA1 upregulation in TG, with prolonged expression compared to CFA alone. Furthermore, CFA-induced mechanical hyperalgesia is significantly prolonged by FSS, persisting up to day 28. PCR array analyses reveal distinct alterations in oxidative stress genes under CFA and CFA combined with FSS conditions, suggesting an intricate regulation of ROS within TG. Notably, genes like Nox4, Hba1, Gpx3, and Duox1 exhibit significant changes, providing potential targets for managing oxidative stress and inflammatory pain. Western blot and immunohistochemistry confirm DUOX1 protein upregulation and localization in TG neurons, indicating a role in ROS generation under inflammatory and stress conditions. This study underscores the complex interplay between psychophysical stress, inflammation, and oxidative stress in the trigeminal system, offering insights into novel therapeutic targets for pain management.

ABSTRACTTemporomandibular disorder (TMD) is the most prevalent painful condition in the craniofacial area. The pathophysiology of TMD is not fully understood, and it is necessary to understand pathophysiology underlying painful TMD conditions to develop more effective treatment methods. Recent studies suggested that external or intrinsic trauma to TMJ is associated with chronic TMD in patients. Here, we investigated the effects of the TMJ trauma through forced-mouth opening (FMO) in mice to determine pain behaviors and peripheral sensitization of trigeminal nociceptors. FMO increased mechanical hyperalgesia assessed by von Frey test, spontaneous pain-like behaviors assessed by mouse grimace scale, and anxiety-like behaviors assessed by open-field test. In vivo GCaMP Ca2+imaging of intact trigeminal ganglia (TG) showed increased spontaneous Ca2+activity and mechanical hypersensitivity of TG neurons in the FMO compared to the sham group. Ca2+responses evoked by cold, heat, and capsaicin stimuli were also increased. FMO-induced hyperalgesia and neuronal hyperactivities were not sex dependent. TG neurons sensitized following FMO were primarily small to medium-sized nociceptive afferents. Consistently, most TMJ afferents in the TG were small-sized peptidergic neurons expressing calcitonin gene-related peptides, whereas nonpeptidergic TMJ afferents were relatively low. FMO-induced intraneural inflammation in the surrounding tissues of the TMJ indicates potentially novel mechanisms of peripheral sensitization following TMJ injury. These results suggest that the TMJ injury leads to persistent post-traumatic hyperalgesia associated with peripheral sensitization of trigeminal nociceptors.

Diffuse noxious inhibitory controls (DNICs), or the pain inhibits pain phenomenon, refer to reduced pain-like behaviors that are displayed following a noxious conditioning stimulus located far from the test stimulus and have also been referred to as “descending control of nociception” when measured in awake-behaving animals. In this study, we sought to determine the impact of moderate long-term exercise on the DCN response and determine if this effect differed across age and sex. After a six-week exercise program consisting of 30 min of moderate treadmill running 5 days a week, the animals’ forepaws were injected with capsaicin, and DCN responses were assessed using thermal withdrawal latencies of the hind paw. Young, exercised male and female rats displayed prolonged DCN responses relative to their sedentary counterparts, with the young exercised male group displaying longer-lasting DCN facilitation than the young exercised females. Exercise did not impact DCN responses in either male or female aged rats. Additionally, the serum testosterone levels did not change following exercise in any group. Importantly, the levels of corticosterone did not change following the exercise program, indicating that changes in the DCN response are not due to stress-induced analgesia. Our findings suggest that moderate exercise can facilitate the DCN response in young animals, even when this exercise does not change the levels of serum testosterone.

Reactive oxygen species (ROS) are generated in nociceptive pathways in response to inflammation and injury. ROS are accumulated within the sensory ganglia following peripheral inflammation, but the functional role of intraganlionic ROS in inflammatory pain is not clearly understood. The aims of this study were to investigate whether peripheral inflammation leads to prolonged ROS accumulation within the trigeminal ganglia (TG), whether intraganglionic ROS mediate pain hypersensitivity via activation of TRPA1, and whether TRPA1 expression is upregulated in TG during inflammatory conditions by ROS. We demonstrated that peripheral inflammation causes excess ROS production within TG during the period when inflammatory mechanical hyperalgesia is most prominent. Additionally, scavenging intraganglionic ROS attenuated inflammatory mechanical hyperalgesia and a pharmacological blockade of TRPA1 localized within TG also mitigated inflammatory mechanical hyperalgesia. Interestingly, exogenous administration of ROS into TG elicited mechanical hyperalgesia and spontaneous pain-like responses via TRPA1, and intraganglionic ROS induced TRPA1 upregulation in TG. These results collectively suggest that ROS accumulation in TG during peripheral inflammation contributes to pain and hyperalgesia in a TRPA1 dependent manner, and that ROS further exacerbate pathological pain responses by upregulating TRPA1 expression. Therefore, any conditions that exacerbate ROS accumulation within somatic sensory ganglia can aggravate pain responses and treatments reducing ganglionic ROS may help alleviate inflammatory pain.

Temporomandibular disorders (TMD) represent a group of musculoskeletal conditions involving the temporomandibular joints (TMJ), the masticatory muscles and associated structures. Painful TMD are highly prevalent and conditions afflict 4% of US adults annually. TMD include heterogenous musculoskeletal pain conditions, such as myalgia, arthralgia, and myofascial pain. A subpopulations of TMD patients show structural changes in TMJ, including disc displacement or degenerative joint diseases (DJD). DJD is a slowly progressing, degenerative disease of the TMJ characterized by cartilage degradation and subchondral bone remodeling. Patients with DJD often develop pain (TMJ osteoarthritis; TMJ OA), but do not always have pain (TMJ osteoarthrosis). Therefore, pain symptoms are not always associated with altered TMJ structures, which suggests that a causal relationship between TMJ degeneration and pain is unclear. Multiple animal models have been developed for determining altered joint structure and pain phenotypes in response to various TMJ injuries. Rodent models of TMJOA and pain include injections to induce inflammation or cartilage destruction, sustained opening of the oral cavity, surgical resection of the articular disc, transgenic approaches to knockout or overexpress key genes, and an integrative approach with superimposed emotional stress or comorbidities. In rodents, TMJ pain and degeneration occur during partially overlapping time periods in these models, which suggests that common biological factors may mediate TMJ pain and degeneration over different time courses. While substances such as intra-articular pro-inflammatory cytokines commonly cause pain and joint degeneration, it remains unclear whether pain or nociceptive activities are causally associated with structural degeneration of TMJ and whether structural degeneration of TMJ is necessary for producing persistent pain. A thorough understanding of the determining factors of pain-structure relationships of TMJ during the onset, progression, and chronification by adopting novel approaches and models should improve the ability to simultaneously treat TMJ pain and TMJ degeneration.

In this study, we investigated age and sex differences in acute and chronic pain in rats. Groups of young (3-6 months) and aged (20-24 months) male and female Fischer 344 rats were used to assess basal thermal and mechanical thresholds, capsaicin-induced acute nocifensive responses and c-Fos expression in the spinal cord, and monoiodoacetate (MIA)-induced knee osteoarthritis (OA)-like pain responses. There was a significant sex, but not age, effect on thermal threshold on the hindpaw and mechanical threshold on the knee joint. No significant age and sex differences in capsaicin-induced nocifensive and c-Fos responses were observed. MIA induced a greater peak reduction of weight-bearing responses in aged males than young rats. Aged females developed the most profound weight-bearing deficit. With knee joint sensitivity as a primary outcome measure, MIA induced more pronounced and longer-lasting hyperalgesia in older rats, with aged female rats showing the worst effect. These data suggest that age may not have significant effect on acute nociceptive processing, but it significantly impacts OA-like pain, making aged rats, especially females, more vulnerable to chronic pain conditions. These preclinical models should provide important tools to investigate basic mechanisms underlying the impact of age and sex in chronic pain conditions.

Craniofacial muscle pain is highly prevalent in temporomandibular disorders but is difficult to treat. Enhanced understanding of neurobiology unique to craniofacial muscle pain should lead to the development of novel mechanism-based treatments. Herein, we review recent studies to summarize neural pathways of craniofacial muscle pain. Nociceptive afferents in craniofacial muscles are predominantly peptidergic afferents enriched with TRPV1. Signals from peripheral glutamate receptors converge onto TRPV1, leading to mechanical hyperalgesia. Further studies are needed to clarify whether hyperalgesic priming in nonpeptidergic afferents or repeated acid injections also affect craniofacial muscle pain. Within trigeminal ganglia, afferents innervating craniofacial muscles interact with surrounding satellite glia, which enhances the sensitivity of the inflamed neurons as well as nearby uninjured afferents, resulting in hyperalgesia and ectopic pain originating from adjacent orofacial tissues. Craniofacial muscle afferents project to a wide area within the trigeminal nucleus complex, and central sensitization of medullary dorsal horn neurons is a critical factor in muscle hyperalgesia related to ectopic pain and emotional stress. Second-order neurons project rostrally to pathways associated with affective pain, such as parabrachial nucleus and medial thalamic nucleus, as well as sensory-discriminative pain, such as ventral posteromedial thalamic nuclei. Abnormal endogenous pain modulation can also contribute to chronic muscle pain. Descending serotonergic circuits from the rostral ventromedial medulla facilitate activation of second-order neurons in the trigeminal nucleus complex, which leads to the maintenance of mechanical hyperalgesia of inflamed masseter muscle. Patients with temporomandibular disorders exhibit altered brain networks in widespread cortical and subcortical regions. Recent development of methods for neural circuit manipulation allows silencing of specific hyperactive neural circuits. Chemogenetic silencing of TRPV1-expressing afferents or rostral ventromedial medulla neurons attenuates hyperalgesia during masseter inflammation. It is likely, therefore, that further delineation of neural circuits mediating craniofacial muscle hyperalgesia potentially enhances treatment of chronic muscle pain conditions.

Endogenous pain inhibition is less efficient in chronic pain patients. Diffuse noxious inhibitory control (DNIC), a form of endogenous pain inhibition, is compromised in women and older people, making them more vulnerable to chronic pain. However, the underlying mechanisms remain unclear. Here, we used a capsaicin-induced DNIC test and resting-state functional MRI to investigate the impact of aging and sex on endogenous pain inhibition and associated brain circuitries in healthy rats. We found that DNIC was less efficient in young females compared with young males. Diffuse noxious inhibitory control response was lost in old rats of both sexes, but the brain networks engaged during DNIC differed in a sex-dependent manner. Young males had the most efficient analgesia with the strongest connectivity between anterior cingulate cortex (ACC) and periaqueductal gray (PAG). The reduced efficiency of DNIC in young females seemed to be driven by widespread brain connectivity. Old males showed increased connectivity between PAG, raphe nuclei, pontine reticular nucleus, and hippocampus, which may not be dependent on connections to ACC, whereas old females showed increased connectivity between ACC, PAG, and more limbic regions. These findings suggest that distinct brain circuitries including the limbic system may contribute to higher susceptibility to pain modulatory deficits in the elderly population, and sex may be a risk factor for developing age-related chronic pain.

Temporomandibular joint disorder is a common chronic craniofacial pain condition, often involving persistent, widespread craniofacial muscle pain. Although the etiology of chronic muscle pain is not well known, sufficient clinical and preclinical information supports a contribution of trigeminal nociceptors to craniofacial muscle pain processing under various experimental and pathological conditions. Here, we review cellular and molecular mechanisms underlying sensitization of muscle nociceptive afferents. In particular, we summarize findings on pronociceptive roles of peripheral glutamate in humans, and we discuss mechanistic contributions of glutamate receptors, including N-methyl-D-aspartate receptors and metabotropic glutamate receptors, which have considerably increased our understanding of peripheral mechanisms of craniofacial muscle pain. Several members of the transient receptor potential (TRP) family, such as transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1, also play essential roles in the development of spontaneous pain and mechanical hypersensitivity in craniofacial muscles. Furthermore, glutamate receptors and TRP channels functionally and bi-directionally interact to modulate trigeminal nociceptors. Activation of glutamate receptors invokes protein kinase C, which leads to the phosphorylation of TRPV1. Sensitization of TRPV1 by inflammatory mediators and glutamate receptors in combination with endogenous ligands contributes to masseter hyperalgesia. The distinct intracellular signaling pathways through which both receptor systems engage and specific molecular regions of TRPV1 are offered as novel targets for the development of mechanism-based treatment strategies for myogenous craniofacial pain conditions.

Epigenetic modulation by DNA methylation is associated with aberrant gene expression in sensory neurons, which consequently leads to pathological pain responses. In this study, we sought to investigate whether peripheral inflammation alters global DNA methylation in trigeminal ganglia (TG) and results in abnormal expression of pro-nociceptive genes. Our results show that peripheral inflammation remotely reduced the level of global DNA methylation in rat TG with a concurrent reduction in DNMT1 and DNMT3a expression. Using unbiased steps, we selected the following pro-nociceptive candidate genes that are potentially regulated by DNA methylation: TRPV1, TRPA1, P2X3, and PIEZO2. Inhibition of DNMT with 5-Aza-dC in dissociated TG cells produced dose-dependent upregulation of TRPV1, TRPA1, and P2X3. Systemic treatment of animals with 5-Aza-dC significantly increased the expression of TRPV1, TRPA1, and PIEZO2 in TG. Furthermore, the overexpression of DNMT3a, as delivered by a lentiviral vector, significantly downregulated TRPV1 and PIEZO2 expression and also reliably decreased TRPA1 and P2X3 transcripts. MeDIP revealed that this overexpression also significantly enhanced methylation of CGIs associated with TRPV1 and TRPA1. In addition, bisulfite sequencing data indicated that the CGI associated with TRPA1 was methylated in a pattern catalyzed by DNMT3a. Taken together, our results show that all 4 pro-nociceptive genes are subject to epigenetic modulation via DNA methylation, likely via DNMT3a under inflammatory conditions. These findings provide the first evidence for the functional importance of DNA methylation as an epigenetic factor in the transcription of pro-nociceptive genes in TG that are implicated in pathological orofacial pain responses.

Introduction: Pain-related behavior secondary to masticatory function can be assessed with the rodent bite force model. A reduction of the bite force has been shown to be related to pain associated with the masseter muscle and jaw activity, while an increase in bite force suggests improvement of muscle function and less pain, To evaluate the usefulness of the bite force measure in studying long-lasting orofacial pain we analyzed biting parameters during prolonged myofascial pain induced by ligation injury of the masseter muscle tendon (TL) in mice. Methods: C57Bl/6 mice were habituated to bite at a pair of aluminum plates attached to a force displacement transducer. The transduced voltage signals were amplified and converted to force through calibration with a standard weight set. Voluntary biting behavior was recorded for 100 s/session and those with bite forces >= 980 mN were analyzed. Nociception was also verified with von Frey, conditioned place avoidance (CPA) tests and mouse grimace scale. Persistent orofacial pain was induced with unilateral ligation of one tendon of the masseter muscle (TL). Results: To reduce interference of random bites of smaller forces, the top 5 or 15 bite forces (BF5/15) were chosen as a measure of masticatory function and related to pain behavior. Both male and female mice exhibited similar BF5/15. For the first nascent test of all mice, mean bite force was significantly and positively correlated with the body weight. However, this correlation was less clear in the latter tests (2-8 w). TL induced a reduction of BF5/15 that peaked at 1 w and returned to the baseline within 3 w. The von Frey and CPA tests indicated that mechanical allodynia/hyperalgesia persisted at the time when the BF had returned to the pre-injury level. Infusion of pain-relieving bone marrow stromal cells improved biting behavior in both male and female mice as shown by significantly increased BF5/15, compared to vehicle-treated mice. Conclusions: Mouse voluntary biting behavior can be reliably measured and quantified with a simplified setup. The bite force showed an inverse relationship with the level of pain after TL and was improved by pain-relieving manipulations. However, the injury-induced reduction of bite force peaked early and did not parallel with other measures of nociception in the later phase of hyperalgesia. The results suggest that multiple factors such as the level of habituation, cognitive motive, physical status, and feeding drive may affect random voluntary biting and confound the biting parameters related to maintained hyperalgesia.

Diffuse noxious inhibitory control (DNIC), which involves endogenous pain modulation, has been investigated as a potential mechanism for the differences in pain modulation observed between men and women, though the literature shows contradictory findings. We used a capsaicin-induced DNIC behavioral assay and resting state functional magnetic resonance imaging (rsfMRI) to assess the effect of testosterone on pain modulation and related brain circuitry in rats. We hypothesized that testosterone is required for DNIC that leads to efficient pain inhibition by increasing descending pain modulation. Male, female, and orchidectomized (GDX) male rats had a capsaicin injection into the forepaw to induce DNIC and mechanical thresholds were observed on the hindpaw. rsfMRI scans were acquired before and after capsaicin injection to analyze the effects of DNIC on periaqueductal gray (PAG), anterior cingulate cortex (ACC) and nucleus accumbens (NAc) connectivity to the whole brain. The strength of DNIC was higher in males compared to females and GDX males. PAG connectivity with prelimbic cortex (PrL), ACC and insula was stronger in males compared to females and GDX males, whereas females and GDX males had increased connectivity between the right ACC, hippocampus and thalamus. GDX males also showed a stronger connectivity between right ACC and NAc, and right NAc with PrL, ACC, insula and thalamus. Our findings suggest that testosterone plays a key role in reinforcing the endogenous pain inhibitory system, while circuitries related to reward and emotion are more strongly recruited in the absence of testosterone.

OBJECTIVES:This placebo-controlled randomized double-blinded clinical study assessed the analgesic efficacy of intramuscular morphine in TMD patients with myofascial pain and sex-dependent responses of the morphine treatment. SUBJECTS AND METHODS:Men and women with TMD were treated with morphine (1.5 or 5 mg), lidocaine, or saline in the masseter muscle. VAS of pain intensity, PPT, and PPtol were compared between treatment groups and gender. An additional group was treated with morphine in the trapezius muscle to evaluate the systemic effect of morphine that may reduce pain in the masseter muscle. RESULTS:There was a significant difference in VAS scores between the morphine 5 mg group and the saline group favoring morphine, but not between the morphine 5 mg and lidocaine. Morphine 1.5 and 5 mg treatments led to consistently and significantly elevated PPT and PPtol measures in men, but not in women. Morphine administered in the trapezius muscle did not affect the outcome measures. CONCLUSION:A single dose intramuscular morphine produced analgesic effects up to 48 hr in patients with myofascial pain. Intramuscular morphine elevated mechanical pain threshold and tolerance in the masseter only in male patients, suggesting sex differences in local morphine effects. No systemic effect of intramuscular morphine was detected.

Spontaneous pain and function-associated pain are prevalent symptoms of multiple acute and chronic muscle pathologies. We established mouse models for evaluating spontaneous pain and bite-evoked pain from masseter muscle, and determined the roles of transient receptor potential cation channel subfamily V member 1 (TRPV1) and the contribution of TRPV1- or neurokinin 1 (NK1)-dependent nociceptive pathways. Masseter muscle inflammation increased Mouse Grimace Scale scores and face-wiping behavior, which were attenuated by pharmacological or genetic inhibition of TRPV1. Masseter inflammation led to a significant reduction in bite force. Inhibition of TRPV1 only marginally relieved the inflammation-induced reduction of bite force. These results suggest a differential extent of contribution of TRPV1 to the 2 types of muscle pain. However, chemical ablation of TRPV1-expressing nociceptors or chemogenetic silencing of TRPV1-lineage nerve terminals in masseter muscle attenuated inflammation-induced changes in Mouse Grimace Scale scores as well as bite force. Furthermore, ablation of neurons expressing NK1 receptor in trigeminal subnucleus caudalis also prevented both types of muscle pain. Our results suggest that TRPV1 differentially contributes to spontaneous pain and bite-evoked muscle pain, but TRPV1-expressing afferents and NK1-expressing second order neurons commonly mediate both types of muscle pain. Therefore, manipulation of the nociceptive circuit may provide a novel approach for management of acute or chronic craniofacial muscle pain. Perspective: We report the profound contribution of TRPV1 to spontaneous muscle pain but not to bite-evoked muscle pain. These 2 types of muscle pain are transmitted through a common nociceptive pathway. These results may help to develop new strategies to manage multiple modes of muscle pain simultaneously by manipulating pain circuits. (C) 2017 by the American Pain Society

Capsaicin is an ingredient in spicy peppers that produces burning pain by activating transient receptor potential vanilloid 1 (TRPV1), a Ca2+-permeable ion channel in nociceptors. Capsaicin has also been used as an analgesic, and its topical administration is approved for the treatment of certain pain conditions. The mechanisms underlying capsaicin-induced analgesia likely involve reversible ablation of nociceptor terminals. However, the mechanisms underlying these effects are not well understood. To visualize TRPV1-lineage axons, a genetically engineered mouse model was used in which a fluorophore is expressed under the TRPV1 promoter. Using a combination of these TRPV1-lineage reporter mice and primary afferent cultures, we monitored capsaicin-induced effects on afferent terminals in real time. We found that Ca2+ influx through TRPV1 is necessary for capsaicin-induced ablation of nociceptive terminals. Although capsaicin-induced mitochondrial Ca2+ uptake was TRPV1-dependent, dissipation of the mitochondrial membrane potential, inhibition of the mitochondrial transition permeability pore, and scavengers of reactive oxygen species did not attenuate capsaicin-induced ablation. In contrast, MDL28170, an inhibitor of the Ca2+-dependent protease calpain, diminished ablation. Furthermore, overexpression of calpastatin, an endogenous inhibitor of calpain, or knockdown of calpain 2 also decreased ablation. Quantitative assessment of TRPV1-lineage afferents in the epidermis of the hind paws of the reporter mice showed that EGTA and MDL28170 diminished capsaicin-induced ablation. Moreover, MDL28170 prevented capsaicin-induced thermal hypoalgesia. These results suggest that TRPV1/Ca2+/calpain-dependent signaling plays a dominant role in capsaicin-induced ablation of nociceptive terminals and further our understanding of the molecular mechanisms underlying the effects of capsaicin on nociceptors.

The involvement of testosterone in pain, inflammation, and analgesia has been reported, but the role of androgen receptor (AR), a steroid receptor for testosterone, is not well understood. We have previously shown that peripheral inflammation upregulates μ-opioid receptor (MOR) in rat trigeminal ganglia (TG) in a testosterone-dependent manner. In this study, we hypothesized that testosterone regulates MOR expression via transcriptional activities of AR in TG. We first examined whether AR is co-expressed with MOR in TG neurons. Our immunohistochemical experiment revealed that AR staining is detected in neurons of all sizes in TG and that a subset of AR is expressed in MOR as well as in TRPV1-positive neurons. We identified the promoter region of the rat MOR gene contains putative AR binding sites. Using chromatin immunoprecipitation assay, we demonstrated that AR directly binds to these sites in TG extracts. We confirmed with luciferase reporter assay that AR activated the MOR promoter in response to androgens in a human neuroblastoma cell line (5H-5YSY). These data demonstrated that AR functions as a transcriptional regulator of the MOR gene activity. Finally, we showed that flutamide, a specific AR antagonist, prevents complete Freund's adjuvant (CFA)-induced upregulation of MOR mRNA in TG, and that flutamide dose-dependently blocks the efficacy of DAMGO, a specific MOR agonist, on CFA-induced mechanical hypersensitivity. Our results expand the knowledge regarding the role of androgens and their receptor in pain and analgesia and have important clinical implications, particularly for inflammatory pain patients with low or compromised plasma testosterone levels.