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Estrogen status is a significant risk factor in the development of temporomandibular joint disorders (TMD). Classically, estrogen status is thought to derive mainly from ovarian sources; however, it is well known that estradiol (E2) also is synthesized by neurons in the brain. This study tested the hypothesis that E2 is produced by neurons in trigeminal subnucleus caudalis (Vc), the principal site of termination for sensory afferents that supply the temporomandibular joint (TMJ), to modify evoked responses in a model of TMJ nociception in male and female rats. Intra-TMJ injection of the small fiber excitant, allyl isothiocyanate (AIC), increased the levels of E2 collected from microdialysis probes sites at Vc of ovariectomized (OvX) female rats, ipsilateral to the stimulus, whereas males displayed no change. Dialysate levels of E2 collected from probe sites in the contralateral Vc or cerebellum in OvX rats were not affected by TMJ stimulation. Reverse dialysis of anastrozole, an aromatase (ARO) inhibitor, via the probe reduced perfusate levels of E2 in Vc. Systemic administration of letrozole, a non-steroid ARO inhibitor, for 4 days prevented TMJ-evoked increases in masseter muscle electromyography (MMemg) activity. ARO-positive neurons were distributed mainly in superficial laminae (I-III) at Vc and cell counts revealed no significant difference between OvX and male rats. Intra-TMJ injection of AIC revealed similar numbers of ARO/Fos dual-labeled neurons in OvX and male rats. By contrast, the percentage of ARO neurons co-labeled for glutamic acid decarboxylase (GAD), the biosynthetic enzyme for GABA, was greater in OvX (35%) than male rats (14%). Few ARO-positive neurons were co-labeled for estrogen receptor alpha. These data indicate that E2 is secreted continuously by Vc neurons and that acute stimulation of TMJ nociceptors evokes further secretion in a sex-dependent manner. Reduced TMJ-evoked MMemg activity after ARO inhibition suggests that locally produced E2 by Vc neurons acts via paracrine mechanisms to modify TMJ nociception in female rats.
Methylglyoxal (MGO), an endogenous reactive carbonyl compound, plays a key role in the pathogenesis of diabetic neuropathy. The aim of this study is to investigate the role of MGO in diabetic itch and hypoalgesia, two common symptoms associated with diabetic neuropathy. : Scratching behavior, mechanical itch (alloknesis), and thermal hypoalgesia were quantified after intradermal (i.d.) injection of MGO in naïve mice or in diabetic mice induced by intraperitoneal (i.p.) injection of streptozotocin (STZ). Behavioral testing, patch-clamp recording, transgenic mice, and gene expression analysis were used to investigate the mechanisms underlying diabetic itch and hypoalgesia in mice. : I.d. injection of MGO evoked dose-dependent scratching in normal mice. Addition of MGO directly activated transient receptor potential ankyrin 1 (TRPA1) to induce inward currents and calcium influx in dorsal root ganglia (DRG) neurons or in TRPA1-expressing HEK293 cells. Mechanical itch, but not spontaneous itch was developed in STZ-induced diabetic mice. Genetic ablation of ( ), pharmacological blockade of TRPA1 and Na1.7, antioxidants, and mitogen-activated protein kinase kinase enzyme (MEK) inhibitor U0126 abrogated itch induced by MGO or in STZ-induced diabetic mice. Thermal hypoalgesia was induced by intrathecal (i.t.) injection of MGO or in STZ-induced diabetic mice, which was abolished by MGO scavengers, intrathecal injection of TRPA1 blockers, and in mice. : This study revealed that Na1.7 and MGO-mediated activation of TRPA1 play key roles in itch and hypoalgesia in a murine model of type 1 diabetes. Thereby, we provide a novel potential therapeutic strategy for the treatment of itch and hypoalgesia induced by diabetic neuropathy.
Migraine is a primary headache disorder which has complex neurogenic pathophysiological mechanisms still requiring full elucidation. The sensory nerves and meningeal mast cell couplings in the migraine target tissue are very effective interfaces between the central nervous system and the immune system. These couplings fall into three categories: intimacy, cross-talk and a shared fate. Acting as the immediate call-center of the neuroimmune system, mast cells play fundamental roles in migraine pathophysiology. Considerable evidence shows that neuroinflammation in the meninges is the key element resulting in the sensitization of trigeminal nociceptors. The successive events such as neuropeptide release, vasodilation, plasma protein extravasation, and mast cell degranulation that form the basic characteristics of the inflammation are believed to occur in this persistent pain state. In this regard, mast cells and sensory neurons represent both the target and source of the neuropeptides that play autocrine, paracrine, and neuro-endocrine roles during this inflammatory process. This review intends to contribute to a better understanding of the meningeal mast cell and sensory neuron bi-directional interactions from molecular, cellular, functional points of view. Considering the fact that mast cells play a role in expanding the opportunities for targeted new migraine therapies, it is of crucial importance to explore these multi-faceted interactions.
Optopharmacology is a very promising approach based on the use of light-deliverable drugs, which allows manipulating physiological processes with high spatiotemporal resolution. Light-dependent drugs (i.e. caged-compounds) targeting G protein-coupled receptors (GPCRs) have been developed to provide great pharmacological precision on the control of pain. Metabotropic glutamate type 5 (mGlu) receptors are widely expressed through the pain neuraxis and play a key role in pain transmission. In line with this, selective mGlu receptor negative allosteric modulators (NAMs) have consistently shown analgesic activity in experimental animal models of inflammatory pain. Accordingly, we synthesized a light-deliverable drug (i.e. caged compound) using the chemical structure of raseglurant, a mGlu receptor NAM, as a molecular scaffold. And thereafter, we evaluated the analgesic activity of the caged compound in formalin-injected (hind paw) mice upon light irradiation (405 nm). Of note, light was both delivered at the peripheral (i.e. hind paw) and central level (i.e. thalamus), by means of brain-implanted fiber-optics. The novel light-deliverable drug, JF-NP-26, showed antinociceptive activity upon violet light irradiation either of the hind paw or the thalamus, demonstrating the ability of precisely activating, in time and space, the caged compound. Here, we describe in detail the protocol used to perform a reliable and reproducible formalin nociception test in mice using an optopharmacology approach (i.e. light-deliverable compounds).
The negative side effects of opioid-based narcotics underscore the search for alternative non-opioid bioactive compounds that act on the peripheral nervous system to avoid central nervous system-mediated side effects. The transient receptor potential V1 ion channel (TRPV1) is a peripheral pain generator activated and sensitized by heat, capsaicin, and a variety of endogenous ligands. TRPV1 contributes to peripheral sensitization and hyperalgesia, in part, triggering the release of proinflammatory peptides, such as calcitonin gene-related peptide (CGRP), both locally and at the dorsal horn of the spinal cord. Ultrapotent exogenous TRPV1 agonists, such as resiniferatoxin identified in the latex of the exotic , trigger hyperalgesia followed by long lasting, peripheral analgesia. The present study reports on the analgesic properties of , a relative of , native to the Southern United States. The study hypothesized that latex extract induces long-lasting, non-opioid peripheral analgesia in a rat model of inflammatory pain. Both inflamed and non-inflamed adult male and female rats were injected with the methanolic extract of latex into the hindpaw and changes in pain behaviors were reassessed at various time points up to 4 weeks. Primary sensory neuron cultures also were treated with the latex extract or vehicle for 15 min followed by stimulation with the TRPV1 agonist capsaicin. Results showed that latex extract evoked significant pain behaviors in both male and female rats at 20 min post-injection and lasting around 1-2 h. At 6 h post-injection, analgesia was observed in male rats that lasted up to 4 weeks, whereas in females the onset of analgesia was delayed to 72 h post-injection. In sensory neurons, latex extract significantly reduced capsaicin-evoked CGRP release. Blocking TRPV1, but not opioid receptors, attenuated the onset of analgesia and capsaicin-induced CGRP release. Latex was analyzed by mass spectrometry and eleven candidate compounds were identified and reported here. These findings indicate that phytochemicals in the latex induce hyperalgesia followed by peripheral, non-opioid analgesia in both male and female rats, which occurs in part TRPV1 and may provide novel, non-opioid peripheral analgesics that warrant further examination.
Mast cells (MCs) are present in the painful degenerate human intervertebral disc (IVD) and are associated with disease pathogenesis. MCs release granules containing enzymatic and inflammatory factors in response to stimulants or allergens. The serine protease, tryptase, is unique to MCs and its activation of the G-protein coupled receptor, Protease Activated Receptor 2 (PAR2), induces inflammation and degradation in osteoarthritic cartilage. Our previously published work has demonstrated increased levels of MC marker tryptase in IVD samples from discogenic back pain patients compared to healthy control IVD samples including expression of chemotactic agents that may facilitate MC migration into the IVD. To further elucidate MCs' role in the IVD and mechanisms underlying its effects, we investigated whether (1) human IVD cells can promote MC migration, (2) MC tryptase can mediate up-regulation of inflammatory/catabolic process in human IVD cells and tissue, and (3) the potential of PAR2 antagonist to function as a therapeutic drug in human and bovine pilot models of disease. MC migration was quantitatively assessed using conditioned media from primary human IVD cells and MC migration examined through Matrigel. Exposure to soluble IVD factors significantly enhanced MC migration, suggesting IVD cells can recruit MCs. We also demonstrated significant upregulation of MC chemokine SCF and angiogenic factor VEGFA gene expression in human IVD cells in response to recombinant human tryptase, suggesting tryptase can enhance recruitment of MCs and promotion of angiogenesis into the usually avascular IVD. Furthermore, tryptase can degrade proteoglycans in IVD tissue as demonstrated by significant increases in glycosaminoglycans released into surrounding media. This can create a catabolic microenvironment compromising structural integrity and facilitating vascular migration usually inhibited by the anti-angiogenic IVD matrix. Finally, as a "proof of concept" study, we examined the therapeutic potential of PAR2 antagonist (PAR2A) on human IVD cells and bovine organ culture IVD model. While preliminary data shows promise and points toward structural restoration of the bovine IVD including down-regulation of VEGFA, effects of PAR2 antagonist on human IVD cells differ between gender and donors suggesting that further validation is required with larger cohorts of human specimens.
Acid-sensing ion channels (ASICs) are important acid sensors involved in neural modulation in the central nervous system and pain-associated tissue acidosis in the peripheral system. Among ASIC subtypes, ASIC1b is the most selectively expressed in peripheral sensory neurons. However, the role of ASIC1b is still elusive in terms of its functions and expression profile. In this study, we probed the role of ASIC1b in acid-induced muscle pain in -knockout () and transgenic ( ) mice. We tested the effect of ASIC1b knockout in a mouse model of fibromyalgia induced by dual intramuscular acid injections. In this model, a unilateral acid injection to the gastrocnemius muscle induced transient bilateral hyperalgesia in wild-type () but not mice; a second acid injection, spaced 1 or 5 days apart, to the same muscle induced chronic hyperalgesia lasting for 4 weeks in mice, but the duration of hyperalgesia was significantly shortened in mice. Mambalgin-1, an ASIC1b-containing channel inhibitor that was mixed with acid saline at the first injection, dose-dependently blocked the acid-induced transient and chronic hyperalgesia in mice. In contrast, psalmotoxin 1 (PcTx1), an ASIC1a-selective antagonist, had no effect on acid-induced transient or chronic hyperalgesia. We used whole-cell patch clamp recording to study the properties of acid-induced currents in ASIC1b-expressing dorsal root ganglia (DRG) neurons from -TdTomato reporter mice. Medium- to large-sized ASIC1b-expressing DRG neurons mainly exhibited an amiloride-sensitive ASIC-like biphasic current () in response to acid stimulation, whereas small- to medium-sized ASIC1b-expressing DRG neurons predominantly exhibited an amiloride-insensitive sustained current. Specifically, mambalgin-1 selectively inhibited the in most ASIC1b-expressing DRG neurons. However, PcTx1 or APETx2 (an ASIC3-selective antagonist) had only a mild inhibitory effect on in about half of the ASIC1b-expressing DRG neurons. hybridization revealed that ASIC1b-positive DRG neurons co-expressed highly with ASIC1a and ASIC2a mRNA and partially with ASIC3 and ASIC2b. Thus, ASIC1b might form a wide variety of heteromeric channels. ASIC1b-containing heteromeric channels might be promising targets for the therapeutic treatment of acid-induced chronic muscle pain.
17β-estradiol plays a role in pain sensitivity, analgesic drug efficacy, and neuropathic pain prevalence, but the underlying mechanisms remain unclear. Here, we investigated whether voltage-gated chloride channel-3 (ClC-3) impacts the effects of 17β-estradiol (E2) on spared nerve injury (SNI)-induced neuropathic pain in ovariectomized (OVX) female Sprague Dawley rats that were divided into OVX, OVX + SNI, OVX + SNI + E2, OVX + SNI + E2 + DMSO (vehicle, dimethyl sulfoxide), or OVX + SNI + E2+Cltx (ClC-3-blocker chlorotoxin) groups. Changes in ClC-3 protein expression were monitored by western blot analysis. Behavioral testing used the paw withdrawal threshold to acetone irritation and paw withdrawal thermal latency (PWTL) to thermal stimulation. Immunofluorescence indicated the localization and protein expression levels of ClC-3. OVX + SNI + E2 rats were subcutaneously injected with 17β-estradiol once daily for 7 days; a sheathed tube was implanted, and chlorotoxin was injected for 4 days. Intrathecal Cltx to OVX and OVX + SNI rats was administered for 4 consecutive days (days 7-10 after SNI) to further determine the contribution of ClC-3 to neuropathic pain. Patch clamp technology in current clamp mode was used to measure the current threshold (rheobase) dorsal root ganglion (DRG) neurons and the minimal current that evoked action potentials (APs) as excitability parameters. The mean number of APs at double-strength rheobase verified neuronal excitability. There was no difference in behaviors and ClC-3 expression after OVX. Compared with OVX + SNI rats, OVX + SNI + E2 rats showed a lower paw withdrawal threshold to the acetone stimulus, but the PWTL was not significantly different, indicating increased sensitivity to cold but not to thermal pain. Co-immunofluorescent data revealed that ClC-3 was mainly distributed in A- and C-type nociceptive neurons, especially in medium/small-sized neurons. 17β-estradiol administration was associated with increased expression of ClC-3. 17β-estradiol-induced increase in ClC-3 expression was blocked by co-administration of Cltx. Cltx causes hyperalgesia and decreased expression of ClC-3 in OVX rats. Patch clamp results suggested that 17β-estradiol attenuated the excitability of neurons induced by SNI by up-regulating the expression of ClC-3 in the DRG of OVX rats. 17β-estradiol administration significantly improved cold allodynia thresholds in OVX rats with SNI. The mechanism for this decreased sensitivity may be related to the upregulation of ClC-3 expression in the DRG.
Growing evidence indicates cannabinoid receptors as potential therapeutic targets for chronic pain. Consequently, there is an increasing interest in developing cannabinoid receptor agonists for treating human and veterinary pain. To better understand the actions of a drug, it is of paramount importance to know the cellular distribution of its specific receptor(s). The distribution of canonical and putative cannabinoid receptors in the peripheral and central nervous system of dogs is still in its infancy. In order to help fill this anatomical gap, the present study has been designed to identify the cellular sites of cannabinoid and cannabinoid-related receptors in canine spinal ganglia. In particular, the cellular distribution of the cannabinoid receptors type 1 and 2 (CB and CB) and putative cannabinoid receptors G protein-coupled receptor 55 (GPR55), nuclear peroxisome proliferator-activated receptor alpha (PPARα), and transient receptor potential vanilloid type 1 (TRPV1) have been immunohistochemically investigated in the C6-C8 cervical ganglia of dogs. About 50% of the neuronal population displayed weak to moderate CB receptor and TRPV1 immunoreactivity, while all of them were CB-positive and nearly 40% also expressed GPR55 immunolabeling. Schwann cells, blood vessel smooth muscle cells, and pericyte-like cells all expressed CB receptor immunoreactivity, endothelial cell being also PPARα-positive. All the satellite glial cells (SGCs) displayed bright GPR55 receptor immunoreactivity. In half of the study dogs, SGCs were also PPARα-positive, and limited to older dogs displayed TRPV1 immunoreactivity. The present study may represent a morphological substrate to consider in order to develop therapeutic strategies against chronic pain.
Traumatic spinal cord injury (SCI) has devastating implications for patients, including a high predisposition for developing chronic pain distal to the site of injury. Chronic pain develops weeks to months after injury, consequently, patients are treated after irreparable changes have occurred. Nociceptors are central to chronic pain; however, the diversity of this cellular population presents challenges to understanding mechanisms and attributing pain modalities to specific cell types. To begin to address how peripheral sensory neurons below the injury level may contribute to the below-level pain reported by SCI patients, we examined SCI-induced changes in gene expression in lumbar dorsal root ganglia (DRG) below the site of injury. SCI was performed at the T10 vertebral level, with injury produced by a vessel clip with a closing pressure of 15 for 1 min. Alterations in gene expression produce long-term sensory changes, therefore, we were interested in studying SCI-induced transcripts before the onset of chronic pain, which may trigger changes in downstream signaling pathways and ultimately facilitate the transmission of pain. To examine changes in the nociceptor subpopulation in DRG distal to the site of injury, we retrograde labeled sensory neurons projecting to the hairy hindpaw skin with fluorescent dye and collected the corresponding lumbar (L2-L6) DRG 4 days post-injury. Following dissociation, labeled neurons were purified by fluorescence-activated cell sorting (FACS). RNA was extracted from sorted sensory neurons of naïve, sham, or SCI mice and sequenced. Transcript abundances validated that the desired population of nociceptors were isolated. Cross-comparisons to data sets from similar studies confirmed, we were able to isolate our cells of interest and identify a unique pattern of gene expression within a subpopulation of neurons projecting to the hairy hindpaw skin. Differential gene expression analysis showed high expression levels and significant transcript changes 4 days post-injury in SCI cell populations relevant to the onset of chronic pain. Regulatory interrelationships predicted by pathway analysis implicated changes within the synaptogenesis signaling pathway as well as networks related to inflammatory signaling mechanisms, suggesting a role for synaptic plasticity and a correlation with pro-inflammatory signaling in the transition from acute to chronic pain.