In vitro effects of photobiomodulation therapy on 50B11 sensory neurons: evaluation of cell metabolism,oxidative stress,mitochondrial membrane potential (MMP), and capsaicin-induced calcium flow

The analgesic properties of photobiomodulation therapy (PBMT) have been raising increasing interest in the clinical community due to the positive effects observed on patients, nevertheless the mechanistic basis of its action on peripheral sensory neurons remains still elusive. In this study, the effect of near-infrared (NIR) PBMT at 800 and 970 nm of wavelength was investigated on the 50B11 immortalized nociceptive sensory neuronal cell line by evaluating capsaicin-induced calcium flow and different markers correlated to mitochondria, that is, ATP, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP). Calcium peak stimulated by capsaicin, the ligand of TRPV1 channel, was decreased in neurons pre-irradiated with the combination of the two wavelengths. Furthermore, delivering the 800 and 970 nm separately an increment of ATP, as well as MMP hyperpolarization were detected; notably, the 800 nm wavelength also increased ROS and O₂⁻ levels. Our findings, obtained on an in vitro model of nociception, show the positive effect of PBMT on two potential photo-targets of NIR light, namely the TRPV1 channel and the mitochondria.     

Pain management using photobiomodulation: Mechanisms,location, and repeatability quantified by pain threshold and neural biomarkers in mice

Photobiomodulation (PBM) is a simple, efficient and cost‐effective treatment for both acute and chronic pain. We previously showed that PBM applied to the mouse head inhibited nociception in the foot. Nevertheless, the optimum parameters, location for irradiation, duration of the effect and the mechanisms of action remain unclear. In the present study, the pain threshold in the right hind paw of mice was studied, after PBM (810 nm CW laser, spot size 1 or 6 cm², 1.2–36 J/cm²) applied to various anatomical locations. The pain threshold, measured with von Frey filaments, was increased more than 3‐fold by PBM to the lower back (dorsal root ganglion, DRG), as well as to other neural structures along the pathway such as the head, neck and ipsilateral (right) paw. On the other hand, application of PBM to the contralateral (left) paw, abdomen and tail had no effect. The optimal effect occurred 2 to 3 hours post‐PBM and disappeared by 24 hours. Seven daily irradiations showed no development of tolerance. Type 1 metabotropic glutamate receptors decreased, and prostatic acid phosphatase and tubulin‐positive varicosities were increased as shown by immunofluorescence of DRG samples. These findings elucidate the mechanisms of PBM for pain and provide insights for clinical practice.      

1064 nm Nd:YAG laser light affects transmembrane mitochondria respiratory chain complexes

Photobiomodulation (PBM) is a non‐plant‐cell manipulation through a transfer of energy by means of light sources at the non‐ablative or thermal intensity. Authors showed that cytochrome‐c‐oxidase (complex IV) is the specific chromophore's target of PBM at the red (600‐700 nm) and NIR (760‐900 nm) wavelength regions. Recently, it was suggested that the infrared region of the spectrum could influence other chromospheres, despite the interaction by wavelengths higher than 900 nm with mitochondrial chromophores was not clearly demonstrated. We characterized the interaction between mitochondria respiratory chain, malate dehydrogenase, a key enzyme of Krebs cycle, and 3‐hydroxyacyl‐CoA dehydrogenase, an enzyme involved in the β‐oxidation (two mitochondrial matrix enzymes) with the 1064 nm Nd:YAG (100mps and 10 Hz frequency mode) irradiated at the average power density of 0.50, 0.75, 1.00, 1.25 and 1.50 W/cm² to generate the respective fluences of 30, 45, 60, 75 and 90 J/cm². Our results show the effect of laser light on the transmembrane mitochondrial complexes I, III, IV and V (adenosine triphosphate synthase) (window effects), but not on the extrinsic mitochondrial membrane complex II and mitochondria matrix enzymes. The effect is not due to macroscopical thermal change. An interaction of this wavelength with the Fe‐S proteins and Cu‐centers of respiratory complexes and with the water molecules could be supposed.      

Regulation of galanin and galanin receptor 2 expression by capsaicin in primary cultured dorsal root ganglion neurons

Galanin is a 29-amino-acid neuropeptide expressed in dorsal root ganglion (DRG) neurons which is thought to play a role in modulation of nociception in neuropathic states. Activation of galanin receptor 2 (GalR2) plays a pronociceptive role and enhances capsaicin-induced nociception in the periphery. GalR2 and vanilloid receptor 1 (VR1) are co-expressed in DRG neurons. Capsaicin evokes acute pain via activation of VR1 expressed in primary sensory neurons. It is not known to what extent galanin and its receptor GalR2 expression is regulated by capsaicin in DRG neurons. Effects of acute (4 h) or chronic (4 d) treatment with capsaicin at different concentrations (0.01, 0.1, 1 micromol/L) on galanin and GalR2 expression in primary cultured DRG neurons were investigated in the present study. Our results showed that acute exposure of high concentration capsaicin (1 micromol/L) increased galanin expression, whereas chronic exposure of low concentration capsaicin (0.01, 0.1 micromol/L) promoted galanin expression. Only chronic exposure of 0.1 micromol/L concentration capsaicin could elevate GalR2 expression, whereas capsaicin did not have this effect at any other conditions in this experiment. These results indicated that certain concentrations or exposure time of capsaicin stimulation may be relevant to upregulation of galanin and its receptor GalR2 expression in DRG cultures suggesting a response to peripheral neuronal stimulation. And also, capsaicin-induced GalR2 expression may be also modulated by capsaicin-induced galanin expression. The possible significance of the neurotransmission of nociceptive information involved in galanin or GalR2 expression caused by capsaicin is still to be clarified.     

Photobiomodulation preconditioning prevents cognitive impairment in a neonatal rat model of hypoxia‐ischemia

Neonatal hypoxia‐ischemia (HI) injury caused by oxygen deprivation is the most common cause of mortality and severe neurologic deficits in neonates. The present work evaluated the preventative effect of photobiomodulation (PBM) preconditioning, and its underlying mechanism of action on brain damage in an HI model in neonatal rats. According to the optimal time response of ATP levels in brain samples removed from normal rats, a PBM preconditioning (PBM‐P) regimen (808 nm CW laser, 1 cm² spot, 100 mW/cm², 12 J/cm²) was delivered to the scalp 6 hours before HI. PBM‐P significantly attenuated cognitive impairment, volume shrinkage in the brain, neuron loss, dendritic and synaptic injury after HI. Further mechanistic investigation found that PBM‐P could restore HI‐induced mitochondrial dynamics and inhibit mitochondrial fragmentation, followed by a robust suppression of cytochrome c release, and prevention of neuronal apoptosis by inhibition of caspase activation. Our work suggests that PBM‐P can attenuate HI‐induced brain injury by maintaining mitochondrial dynamics and inhibiting the mitochondrial apoptotic pathway.      

TRPV1 Properties in Thoracic Dorsal Root Ganglia Neurons are Modulated by Intraperitoneal Capsaicin Administration in the Late Phase of Type-1 Autoimmune Diabetes

Pharmacological therapies in type 1 diabetes for efficient control of glycemia and changes in pain alterations due to diabetic neuropathy are a continuous challenge. Transient receptor potential vanilloid type 1 (TRPV1) from dorsal root ganglia (DRG) neurons is one of the main pharmacological targets in diabetes, and its ligand capsaicin can be a promising compound for blood-glucose control. Our goal is to elucidate the effect of intraperitoneal (i.p.) capsaicin administration in type 1 diabetic mice against TRPV1 receptors from pancreatic DRG primary afferent neurons. A TCR^+/?/Ins-HA^+/? diabetic mice (dTg) was used, and patch-clamp and immunofluorescence microscopy measurements have been performed on thoracic T₉–T₁₂ DRG neurons. Capsaicin (800 μg/kg, i.p. three successive days) administration in the late-phase diabetes reduces blood-glucose levels, partly reverses the TRPV1 current density and recovery time constant, without any effect on TRPV1 expression general pattern, in dTg mice. A TRPV1 hypoalgesia profile was observed in late-phase diabetes, which was partly reversed to normoalgesic profile upon capsaicin i.p. administration. According to the soma dimensions of the thoracic DRG neurons, a detailed analysis of the TRPV1 expression upon capsaicin i.p. treatment was done, and the proportion of large A-fiber neurons expressing TRPV1 increased in dTg capsaicin-treated mice. In conclusion, the benefits of low-dose capsaicin intraperitoneal treatment in late-phase type-1 diabetes should be further exploited.     

Photobiomodulation at a wavelength of 633 nm leads to faster functional recovery than 804 nm after facial nerve injury

We analyzed the effects of photobiomodulation (PBM) of various wavelengths on regeneration of the facial nerve using in vitro and in vivo experimental models. We assessed the antioxidative effect of PBM in geniculate ganglion neurons irradiated with a diode laser at 633 nm, 780 nm and 804 nm. Wavelengths of 633 and 780 nm but not 804 nm inhibited cell death by oxidative stress. We assessed the effects of PBM on functional and morphologic recovery in rats divided into control, facial nerve damage (FND) and FND irradiated with a 633 nm or 804 nm lasers. Injured rats treated with 633-nm light had better facial palsy scores, larger axon diameter and higher expression of Schwann cells compared with the FND group. No positive results were observed in rats irradiated at 804-nm light. These findings indicate that 633-nm PBM promotes accelerated nerve regeneration and improved functional recovery in an injured facial nerve.     

Cortistatin modulates calcitonin gene-related peptide release from neuronal tissues of rat. Comparison with somatostatin

Cortistatin (CST) is an endogenous neuropeptide bearing strong structural and functional analogies with somatostatin (SST). Gene expression of CST and its putative receptor MrgX2 in dorsal root ganglia (DRG) neurons in man suggests the involvement of CST in pain transmission. In this study we have investigated the effects of CST and SST on calcitonin gene-related peptide (CGRP, the main neuropeptide mediator of pain transmission) from primary cultures of rat trigeminal neurons. Moreover, here for the first time we used organotypic cultures of rat brainstem to investigate the release of CGRP form nucleus caudalis as a model of pre-synaptic peptide release. In both experimental paradigm CGRP release was evaluated in the presence of CST or SST, with or without the addition of known secretagogues (namely high KCl concentrations, veratridine and capsaicin). We found that CST and SST do not modify basal CGRP secretion from trigeminal neurons, but both peptides were able to inhibit in a concentration-dependent manner the release of CGRP stimulated by KCl, veratridine or capsaicin. Likewise, in brainstem organotypic cultures CST and SST did not modify baseline CGRP secretion. Of the secretagogues used, capsaicin proved to be most effective compared to KCl and veratridine (8-fold vs 2-fold increase, respectively). Thereafter, CST and SST were tested on capsaicin-stimulated CGPR release only. Under these conditions, CST but not SST was able to inhibit in a significant manner pre-synaptic CGRP release from the brainstem, providing further evidence in support of a role for CST in pain transmission.     

Capsaicin Up-Regulates Protease-Activated Receptor-4 mRNA and Protein in Primary Cultured Dorsal Root Ganglion Neurons

Previous study has shown that there is a functional link between the transient receptor potential vanilloid type 1 (TRPV1) receptor and protease-activated receptor-4 (PAR4) in modulation of inflammation and pain. Capsaicin activation of TRPV1 is involved in enhancement of the expression of TRPV1 in mRNA and protein in dorsal root ganglion (DRG) in vivo. Whether capsaicin could influence expression of PAR4 in primary sensory neurons remains unknown. In the present study, expression of PAR4 in cultured rat DRG neurons was observed using immunofluorescence, real-time PCR and Western blots to examine whether increases in PAR4 mRNA and protein levels are induced by capsaicin treatment with or without pre-treatment of forskolin, a cyclic AMP/protein kinase A (cAMP/PKA) activator or PKA inhibitor fragment 14-22 (PKI14-22), a PKA inhibitor. Capsaicin treatment of cultured DRG neurons significantly increased the expression of PAR4 in mRNA and protein levels. The percentage of PAR4-, TRPV1-immunoreactive neurons and their co-localization in cultured DRG neurons increased significantly in the presence of capsaicin as compared with that in the absence of capsaicin. Compared with capsaicin-only group, pre-incubation with forskolin strongly enhanced the capsaicin-induced increase of PAR4 in mRNA and protein levels. Consistent with the involvement of PKA in the modulation of PAR4 expression, this evoked expression both at mRNA and protein levels was significantly inhibited after PKA was inhibited by pre-incubation with PKI14-22. Taken together, these results provide evidence that TRPV1 activation significantly increases the expression of PAR4 mRNA and protein levels in primary cultures of DRG neurons after capsaicin incubation. Effects of capsaicin on PAR4 expression appear to be mediated by cAMP/PKA signal pathways in DRG neurons.     

Chronic pregabalin inhibits synaptic transmission between rat dorsal root ganglion and dorsal horn neurons in culture

In this study, we have examined the properties of synaptic transmission between dorsal root ganglion (DRG) and dorsal horn (DH) neurons, placed in co-culture. We also examined the effect of the anti-hyperalgesic gabapentinoid drug pregabalin (PGB) at this pharmacologically relevant synapse. The main method used was electrophysiological recording of excitatory post synaptic currents (EPSCs) in DH neurons. Synaptic transmission between DRG and DH neurons was stimulated by capsaicin, which activates transient receptor potential vanilloid-1 (TRPV1) receptors on small diameter DRG neurons. Capsaicin (1 μM) application increased the frequency of EPSCs recorded in DH neurons in DRG-DH co-cultures, by about 3-fold, but had no effect on other measured properties of the EPSCs. There was also no effect of capsaicin in the absence of co-cultured DRGs. Application of PGB (100 μM) for 40–48 h caused a reduction in the capsaicin-induced increase in EPSC frequency by 57%. In contrast, brief preincubation of PGB had no significant effect on the capsaicin-induced increase in EPSC frequency. In conclusion, this study shows that PGB applied for 40–48 h, but not acute application inhibits excitatory synaptic transmission at DRG-DH synapses, in response to nociceptive stimulation, most likely by a presynaptic effect on neurotransmitter release from DRG presynaptic terminals.     

Chronic pregabalin inhibits synaptic transmission between rat dorsal root ganglion and dorsal horn neurons in culture

In this study, we have examined the properties of synaptic transmission between dorsal root ganglion (DRG) and dorsal horn (DH) neurons, placed in co-culture. We also examined the effect of the anti-hyperalgesic gabapentinoid drug pregabalin (PGB) at this pharmacologically relevant synapse. The main method used was electrophysiological recording of excitatory post synaptic currents (EPSCs) in DH neurons. Synaptic transmission between DRG and DH neurons was stimulated by capsaicin, which activates transient receptor potential vanilloid-1 (TRPV1) receptors on small diameter DRG neurons. Capsaicin (1 μM) application increased the frequency of EPSCs recorded in DH neurons in DRG-DH co-cultures, by about 3-fold, but had no effect on other measured properties of the EPSCs. There was also no effect of capsaicin in the absence of co-cultured DRGs. Application of PGB (100 μM) for 40-48 h caused a reduction in the capsaicin-induced increase in EPSC frequency by 57%. In contrast, brief preincubation of PGB had no significant effect on the capsaicin-induced increase in EPSC frequency. In conclusion, this study shows that PGB applied for 40-48 h, but not acute application inhibits excitatory synaptic transmission at DRG-DH synapses, in response to nociceptive stimulation, most likely by a presynaptic effect on neurotransmitter release from DRG presynaptic terminals.     

Photobiomodulation therapy in knee osteoarthritis reduces oxidative stress and inflammatory cytokines in rats

Knee osteoarthritis (OA) is a chronic disease that causes pain and gradual degeneration of the articular cartilage. In this study, MIA‐induced OA knee model was used in rats to test the effects of the photobiomodulation therapy (PBM). We analyzed the inflammatory process (pain and cytokine levels), and its influence on the oxidative stress and antioxidant capacity. Knee OA was induced by monosodium iodoacetate (MIA) intra‐articular injection (1.5 mg/50 μL) and the rats were treated with eight sessions of PBM 3 days/week (904 nm, 6 or 18 J/cm²). For each animal, mechanical and cold hyperalgesia and spontaneous pain were evaluated; biological analyses were performed in blood serum, intra‐articular lavage, knee structures, spinal cord and brainstem. Cytokine assays were performed in knee, spinal cord and brainstem samples. The effects of the 18 J/cm² dose of PBM were promising in reducing pain and neutrophil activity in knee samples, together with reducing oxidative stress damage in blood serum and spinal cord samples. PBM improved the antioxidant capacity in blood serum and brainstem, and decreased the knee pro‐inflammatory cytokine levels. Our study demonstrated that PBM decreased oxidative damage, inflammation and pain. Therefore, this therapy could be an important tool in the treatment of knee OA.     

Effects of the neurotrophic factor artemin on sensory afferent development and sensitivity

Artemin is a neuronal survival and differentiation factor in the glial cell line-derived neurotrophic factor family.Its receptor GFRα3 is expressed by a subpopulation of nociceptor type sensory neurons in the dorsal root and trigeminal ganglia(DRG and TG).These neurons co-express the heat,capsaicin and proton-sensitive channel TRPV1 and the cold and chemical-sensitive channel TRPA1.To further investigate the effects of artemin on sensory neurons,we isolated transgenic mice(ARTN-OE mice) that overexpress art...     

Involvement of spinal NR2B-containing NMDA receptors in oxaliplatin-induced mechanical allodynia in rats

Background

Two main classes of peripheral sensory neurons contribute to thermal pain sensitivity: the unmyelinated C fibers and thinly myelinated Aδ fibers. These two fiber types may differentially underlie different clinical pain states and distinctions in the efficacy of analgesic treatments. Methods of differentially testing C and Aδ thermal pain are widely used in animal experimentation, but these methods are not optimal for human volunteer and patient use. Thus, this project aimed to provide psychophysical and electrophysiological evidence that whether different protocols of infrared diode laser stimulation, which allows for direct activation of nociceptive terminals deep in the skin, could differentially activate Aδ or C fiber thermonociceptors in volunteers.

Results

Short (60 ms), high intensity laser pulses (SP) evoked monomodal "pricking" pain which was not enhanced by topical capsaicin, whereas longer, lower power pulses (LP) evoked monomodal "burning" pain which was enhanced by topical capsaicin. SP also produced cortical evoked EEG potentials consistent with Aδ mediation, the amplitude of which was directly correlated with pain intensity but was not affected by topical capsaicin. LP also produced a distinct evoked potential pattern the amplitude of which was also correlated with pain intensity, which was enhanced by topical capsaicin, and the latency of which could be used to estimate the conduction velocity of the mediating nociceptive fibers.

Conclusions

Psychophysical and electrophysiological data were consistent with the ability of short high intensity infrared laser pulses to selectively produce Aδ mediated pain and of longer pulses to selectively produce C fiber mediated thermal pain. Thus, the use of these or similar protocols may be useful in developing and testing novel therapeutics based on the differential molecular mechanisms underlying activation of the two fiber types (e.g., TRPV1, TRPV2, etc). In addition, these protocol may be useful in determining the fiber mediation of different clinical pain types which may, in turn be useful in treatment choice.     

Photobiomodulation induces antinociception,recovers structural aspects and regulates mitochondrial homeostasis in peripheral nerve of diabetic mice

Diabetic peripheral neuropathy (DPN) is a nervous disorder caused by diabetes mellitus, affecting about 50% of patients in clinical medicine. Chronic pain is one of the major and most unpleasant symptoms developed by those patients, and conventional available treatments for the neuropathy, including the associated pain, are still unsatisfactory and benefit only a small number of patients. Photobiomodulation (PBM) has been gaining clinical acceptance once it is able to promote early nerve regeneration resulting in significant improvement in peripheral nerves disabilities. In this work, the effects of PBM (660 nm, 30 mW, 1.6 J/cm², 0.28 cm², 15 s in a continuous frequency) on treating DPN‐induced pain and nerve damage were evaluated in an experimental model of diabetic‐neuropathy induced by streptozotocin in mice. PBM‐induced antinociception in neuropathic‐pain mice was dependent on central opioids release. After 21 consecutive applications, PBM increased nerve growth factor levels and induced structural recovery increasing mitochondrial content and regulating Parkin in the sciatic nerve of DPN‐mice. Taking together, these data provide new insights into the mechanisms involved in the effects of PBM‐therapy emphasizing its therapeutic potential in the treatment of DPN.      

Reduced pain behaviors and extracellular signal-related protein kinase activation in primary sensory neurons by peripheral tissue injury in mice lacking platelet-activating factor receptor

Peripheral tissue injury causes the release of various mediators from damaged and inflammatory cells, which in turn activates and sensitizes primary sensory neurons and thereby produces persistent pain. The present study investigated the role of platelet-activating factor (PAF), a phospholipid mediator, in pain signaling using mice lacking PAF receptor (pafr-/- mice). Here we show that pafr-/- mice displayed almost normal responses to thermal and mechanical stimuli but exhibit attenuated persistent pain behaviors resulting from tissue injury by locally injecting formalin at the periphery as well as capsaicin pain and visceral inflammatory pain without any alteration in cytoarchitectural or neurochemical properties in dorsal root ganglion (DRG) neurons and a defect in motor function. However, pafr-/- mice showed no alterations in spinal pain behaviors caused by intrathecally administering agonists for N-methyl-d-aspartate (NMDA) and neurokinin(1) receptors. A PAFR agonist evoked an intracellular Ca(2+) response predominantly in capsaicin-sensitive DRG neurons, an effect was not observed in pafr-/- mice. By contrast, the PAFR agonist did not affect C- or Adelta-evoked excitatory post-synaptic currents in substantia gelatinosa neurons in the dorsal horn. Interestingly, mice lacking PAFR showed reduced phosphorylation of extracellular signal-related protein kinase (ERK), an important kinase for the sensitization of primary sensory neurons, in their DRG neurons after formalin injection. Furthermore, U0126, a specific inhibitor of the ERK pathway suppressed the persistent pain by formalin. Thus, PAFR may play an important role in both persistent pain and the sensitization of primary sensory neurons after tissue injury.     

Mouse DRG Cell Line with Properties of Nociceptors

In vitro cell lines from DRG neurons aid drug discovery because they can be used for early stage, high-throughput screens for drugs targeting pain pathways, with minimal dependence on animals. We have established a conditionally immortal DRG cell line from the Immortomouse. Using immunocytochemistry, RT-PCR and calcium microfluorimetry, we demonstrate that the cell line MED17.11 expresses markers of cells committed to the sensory neuron lineage. Within a few hours under differentiating conditions, MED17.11 cells extend processes and following seven days of differentiation, express markers of more mature DRG neurons, such as NaV1.7 and Piezo2. However, at least at this time-point, the nociceptive marker NaV1.8 is not expressed, but the cells respond to compounds known to excite nociceptors, including the TRPV1 agonist capsaicin, the purinergic receptor agonist ATP and the voltage gated sodium channel agonist, veratridine. Robust calcium transients are observed in the presence of the inflammatory mediators bradykinin, histamine and norepinephrine. MED17.11 cells have the potential to replace or reduce the use of primary DRG culture in sensory, pain and developmental research by providing a simple model to study acute nociception, neurite outgrowth and the developmental specification of DRG neurons.     

Na+ channel Scn1b gene regulates dorsal root ganglion nociceptor excitability in vivo

Nociceptive dorsal root ganglion (DRG) neurons express tetrodotoxin-sensitive (TTX-S) and -resistant (TTX-R) Na(+) current (I(Na)) mediated by voltage-gated Na(+) channels (VGSCs). In nociceptive DRG neurons, VGSC β2 subunits, encoded by Scn2b, selectively regulate TTX-S α subunit mRNA and protein expression, ultimately resulting in changes in pain sensitivity. We hypothesized that VGSCs in nociceptive DRG neurons may also be regulated by β1 subunits, encoded by Scn1b. Scn1b null mice are models of Dravet Syndrome, a severe pediatric encephalopathy. Many physiological effects of Scn1b deletion on CNS neurons have been described. In contrast, little is known about the role of Scn1b in peripheral neurons in vivo. Here we demonstrate that Scn1b null DRG neurons exhibit a depolarizing shift in the voltage dependence of TTX-S I(Na) inactivation, reduced persistent TTX-R I(Na), a prolonged rate of recovery of TTX-R I(Na) from inactivation, and reduced cell surface expression of Na(v)1.9 compared with their WT littermates. Investigation of action potential firing shows that Scn1b null DRG neurons are hyperexcitable compared with WT. Consistent with this, transient outward K(+) current (I(to)) is significantly reduced in null DRG neurons. We conclude that Scn1b regulates the electrical excitability of nociceptive DRG neurons in vivo by modulating both I(Na) and I(K).     

Expression and function of proton-sensing G-protein-coupled receptors in inflammatory pain

Background

Chronic inflammatory pain, when not effectively treated, is a costly health problem and has a harmful effect on all aspects of health-related quality of life. Despite the availability of pharmacologic treatments, chronic inflammatory pain remains inadequately treated. Understanding the nociceptive signaling pathways of such pain is therefore important in developing long-acting treatments with limited side effects. High local proton concentrations (tissue acidosis) causing direct excitation or modulation of nociceptive sensory neurons by proton-sensing receptors are responsible for pain in some inflammatory pain conditions. We previously found that all four proton-sensing G-protein-coupled receptors (GPCRs) are expressed in pain-relevant loci (dorsal root ganglia, DRG), which suggests their possible involvement in nociception, but their functions in pain remain unclear.

Results

In this study, we first demonstrated differential change in expression of proton-sensing GPCRs in peripheral inflammation induced by the inflammatory agents capsaicin, carrageenan, and complete Freund's adjuvant (CFA). In particular, the expression of TDAG8, one proton-sensing GPCR, was increased 24 hours after CFA injection because of increased number of DRG neurons expressing TDAG8. The number of DRG neurons expressing both TDAG8 and transient receptor potential vanilloid 1 (TRPV1) was increased as well. Further studies revealed that TDAG8 activation sensitized the TRPV1 response to capsaicin, suggesting that TDAG8 could be involved in CFA-induced chronic inflammatory pain through regulation of TRPV1 function.

Conclusion

Each subtype of the OGR1 family was expressed differently, which may reflect differences between models in duration and magnitude of hyperalgesia. Given that TDAG8 and TRPV1 expression increased after CFA-induced inflammation and that TDAG8 activation can lead to TRPV1 sensitization, it suggests that high concentrations of protons after inflammation may not only directly activate proton-sensing ion channels (such as TRPV1) to cause pain but also act on proton-sensing GPCRs to regulate the development of hyperalgesia.