Sensory innervation of masseter, temporal and lateral pterygoid muscles in common marmosets.

Myogenous temporomandibular disorders is associated with an increased responsiveness of nerves innervating the masseter (MM), temporal (TM), and lateral pterygoid muscles (LPM). This study aimed to examine sensory nerve types innervating MM, TM and LPM of adult non-human primate-common marmosets. Sensory nerves were localized in specific regions of these muscles. Pgp9.5, marker for all nerves, and NFH, a marker for A-fibers, showed that masticatory muscles were primarily innervated with A-fibers. The proportion of C- to A-fibers was highest in LPM, and lowest in MM. All C-fibers (pgp9.5+/NFH-) observed in masticatory muscles were peptidergic (CGRP+) and lacked mrgprD and CHRNA3, a silent nociceptive marker. TrpV1 was register in 17% of LPM nerves. All fibers in masticatory muscles were labeled with GFAP+, a myelin sheath marker. There were substantially more peptidergic A-fibers (CGRP+/NFH+) in TM and LPM compared to MM. MM, TM and LPM NFH+ fibers contained different percentages of trkC+ and parvalbumin+, but not trkB+ fibers. Tyrosine hydroxylase antibodies, which did not label TG, highlighted sympathetic fibers around blood vessels of the masticatory muscles. Overall, masticatory muscle types of marmosets have similarities and differences in innervation patterns.

Lingual innervation in male and female marmosets.

Several gaps in knowledge exists in our understanding of orofacial pain. Some of these include type of peripheral sensory innervation in specific tissues, differences in innervation between sexes and validation of rodent studies in higher order species. The current study addresses these gaps by validating mouse studies for sensory innervation of tongue tissue in non-human primates as well as assesses sex-specific differences. Tongue and trigeminal ganglia were collected from naïve male and female marmosets and tested for nerve fibers using specific markers by immunohistochemistry and number of fibers quantified. We also tested whether specific subgroups of nerve fibers belonged to myelinating or non-myelinating axons. We observed that similar to findings in mice, marmoset tongue was innervated with nerve filaments expressing nociceptor markers like CGRP and TRPV1 as well as non-nociceptor markers like TrkB, parvalbumin (PV) and tyrosine hydroxylase (TH). Furthermore, we found that while portion of TrkB and PV may be sensory fibers, TH-positive fibers were primarily sympathetic nerve fibers. Moreover, number of CGRP, TrkB and TH-positive nerve fibers were similar in both sexes. However, we observed a higher proportion of myelinated TRPV1 positive fibers in females than in males as well as increased number of PV + fibers in females. Taken together, the study for the first time characterizes sensory innervation in non-human primates as well as evaluates sex-differences in innervation of tongue tissue, thereby laying the foundation for future orofacial pain research with new world smaller NHPs like the common marmoset.

Transcriptional profiles of non-neuronal and immune cells in mouse trigeminal ganglia.

Non-neuronal cells constitute 90%-95% of sensory ganglia. These cells, especially glial and immune cells, play critical roles in the modulation of sensory neurons. This study aimed to identify, profile, and summarize the types of trigeminal ganglion (TG) non-neuronal cells in naïve male mice using published and our own data generated by single-cell RNA sequencing, flow cytometry, and immunohistochemistry. TG has five types of non-neuronal cells, namely, glial, fibroblasts, smooth muscle, endothelial, and immune cells. There is an agreement among publications for glial, fibroblasts, smooth muscle, and endothelial cells. Based on gene profiles, glial cells were classified as myelinated and non-myelinated Schwann cells and satellite glial cells. Mpz has dominant expression in Schwann cells, and Fabp7 is specific for SCG. Two types of Col1a2+ fibroblasts located throughout TG were distinguished. TG smooth muscle and endothelial cells in the blood vessels were detected using well-defined markers. Our study reported three types of macrophages (Mph) and four types of neutrophils (Neu) in TG. Mph were located in the neuronal bodies and nerve fibers and were sub-grouped by unique transcriptomic profiles with Ccr2, Cx3cr1, and Iba1 as markers. A comparison of databases showed that type 1 Mph is similar to choroid plexus-low (CPlo) border-associated Mph (BAMs). Type 2 Mph has the highest prediction score with CPhi BAMs, while type 3 Mph is distinct. S100a8+ Neu were located in the dura surrounding TG and were sub-grouped by clustering and expressions of Csf3r, Ly6G, Ngp, Elane, and Mpo. Integrative analysis of published datasets indicated that Neu-1, Neu-2, and Neu-3 are similar to the brain Neu-1 group, while Neu-4 has a resemblance to the monocyte-derived cells. Overall, the generated and summarized datasets on non-neuronal TG cells showed a unique composition of myeloid cell types in TG and could provide essential and fundamental information for studies on cell plasticity, interactomic networks between neurons and non-neuronal cells, and function during a variety of pain conditions in the head and neck regions.

Understanding painful versus non-painful dental pain in female and male patients: A transcriptomic analysis of human biopsies.

Dental pain from apical periodontitis is an infection induced-orofacial pain condition that presents with diversity in pain phenotypes among patients. While 60% of patients with a full-blown disease present with the hallmark symptom of mechanical allodynia, nearly 40% of patients experience no pain. Furthermore, a sexual dichotomy exists, with females exhibiting lower mechanical thresholds under basal and diseased states. Finally, the prevalence of post-treatment pain refractory to commonly used analgesics ranges from 7-19% (∼2 million patients), which warrants a thorough investigation of the cellular changes occurring in different patient cohorts. We, therefore, conducted a transcriptomic assessment of periapical biopsies (peripheral diseased tissue) from patients with persistent apical periodontitis. Surgical biopsies from symptomatic male (SM), asymptomatic male (AM), symptomatic female (SF), and asymptomatic female (AF) patients were collected and processed for bulk RNA sequencing. Using strict selection criteria, our study found several unique differentially regulated genes (DEGs) between symptomatic and asymptomatic patients, as well as novel candidate genes between sexes within the same pain group. Specifically, we found the role of cells of the innate and adaptive immune system in mediating nociception in symptomatic patients and the role of genes involved in tissue homeostasis in potentially inhibiting nociception in asymptomatic patients. Furthermore, sex-related differences appear to be tightly regulated by macrophage activity, its secretome, and/or migration. Collectively, we present, for the first time, a comprehensive assessment of peripherally diseased human tissue after a microbial insult and shed important insights into the regulation of the trigeminal system in female and male patients.

Sex-dependent differences in the genomic profile of lingual sensory neurons in naïve and tongue-tumor bearing mice.

Mechanisms of sex-dependent orofacial pain are widely understudied. A significant gap in knowledge exists about comprehensive regulation of tissue-specific trigeminal sensory neurons in diseased state of both sexes. Using RNA sequencing of FACS sorted retro-labeled sensory neurons innervating tongue tissue, we determined changes in transcriptomic profiles in males and female mice under naïve as well as tongue-tumor bearing conditions Our data revealed the following interesting findings: (1) FACS sorting obtained higher number of neurons from female trigeminal ganglia (TG) compared to males; (2) Naïve female neurons innervating the tongue expressed immune cell markers such as Csf1R, C1qa and others, that weren't expressed in males. This was validated by Immunohistochemistry. (3) Accordingly, immune cell markers such as Csf1 exclusively sensitized TRPV1 responses in female TG neurons. (4) Male neurons were more tightly regulated than female neurons upon tumor growth and very few differentially expressed genes (DEGs) overlapped between the sexes, (5) Male DEGs contained higher number of transcription factors whereas female DEGs contained higher number of enzymes, cytokines and chemokines. Collectively, this is the first study to characterize the effect of sex as well as of tongue-tumor on global gene expression, pathways and molecular function of tongue-innervating sensory neurons.

Transcriptional Profiles of Non-neuronal and Immune Cells in Mouse Trigeminal Ganglia.

Non-neuronal cells constitute 90-95% of sensory ganglia. These cells play critical roles in modulation of nociceptive signal transmissions by sensory neurons. Accordingly, the aim of this review-study was to identify, profile and summarize TG non-neuronal cell types in naïve male mice using published and our own data generated by single-cell RNA sequencing (scRNA-seq), flow cytometry (FC) and immunohistochemistry (IHC). TG contains 5 types of non-neuronal cells: glial, fibroblasts, smooth muscle, endothelial and immune cells. There is agreement among publications for glial, fibroblasts, smooth muscle and endothelial cells. Based on gene profiles, glial cells were classified as Schwann cells and satellite glial cells (SGC). Mpz had dominant expression in Schwann cells, and Fabp7 is specific for SCG. Two types of Col1a2 + fibroblasts located throughout TG were distinguished using gene profiles. TG smooth muscle and endothelial cells representing blood vessels were detected with well recognized markers. Our study split reported single TG immune cell group into 3 types of macrophages and 4 types of neutrophils. Macrophages were located among neuronal bodies and nerve fibers, and were sub-grouped by unique transcriptomic profiles and using Ccr2 , Cx3cr1 and Iba1 as markers. S100a8 + neutrophils were located in dura surrounding TG and were sub-grouped by clustering and expressions of Csf3r , Ly6G, Ngp, Elane and Mpo . Overall, generated and summarized here dataset on non-neuronal TG cells could provide essential and fundamental information for studies on cell plasticity, interactomic network between neurons and non-neuronal cells and function during variety of pain conditions in the head and neck region.

Sensory innervation of masseter, temporal and lateral pterygoid muscles in common marmosets.

Myogenous temporomandibular disorders (TMDM) is associated with an increased responsiveness of nerves innervating the masseter (MM), temporal (TM), medial pterygoid (MPM) and lateral pterygoid muscles (LPM). This study aimed to examine sensory nerve types innervating MM, TM and LPM of adult non-human primate - common marmosets. Sensory nerves are localized in specific regions of these muscles. Pgp9.5, marker for all nerves, and NFH, a marker for A-fibers, showed that masticatory muscles were predominantly innervated with A-fibers. The proportion of C- to A-fibers was highest in LPM, and minimal (6-8%) in MM. All C-fibers (pgp9.5+/NFH-) observed in masticatory muscles were peptidergic (CGRP+) and lacked mrgprD, trpV1 and CHRNA3, a silent nociceptive marker. All fibers in masticatory muscles were labeled with GFAP+, a myelin sheath marker. There were substantially more peptidergic A-fibers (CGRP+/NFH+) in TM and LPM compared to MM. Almost all A-fibers in MM expressed trkC, with some of them having trkB and parvalbumin. In contrast, a lesser number of TM and LPM nerves expressed trkC, and lacked trkB. Tyrosine hydroxylase antibodies, which did not label TG, highlighted sympathetic fibers around blood vessels of the masticatory muscles. Overall, masticatory muscle types of marmosets have distinct and different innervation patterns.

Sex-dependent Differences in the Genomic Profile of Lingual Sensory Neurons in Naïve and Tongue-Tumor Bearing Mice.

Mechanisms of sex-dependent orofacial pain are widely understudied. A significant gap in knowledge exists about comprehensive regulation of tissue-specific trigeminal sensory neurons in diseased state of both sexes. Using RNA sequencing of FACS sorted retro-labeled sensory neurons innervating tongue tissue, we determined changes in transcriptomic profiles in males and female mice under naïve as well as tongue-tumor bearing conditions Our data revealed the following interesting findings: 1) Tongue tissue of female mice was innervated with higher number of trigeminal neurons compared to males; 2) Naïve female neurons innervating the tongue exclusively expressed immune cell markers such as Csf1R, C1qa and others, that weren't expressed in males. This was validated by Immunohistochemistry. 4) Accordingly, immune cell markers such as Csf1 exclusively sensitized TRPV1 responses in female TG neurons. 3) Male neurons were more tightly regulated than female neurons upon tumor growth and very few differentially expressed genes (DEGs) overlapped between the sexes, 5) Male DEGs contained higher number of transcription factors whereas female DEGs contained higher number of enzymes, cytokines and chemokines. Collectively, this is the first study to characterize the effect of sex as well as of tongue-tumor on global gene expression, pathways and molecular function of tongue-innervating sensory neurons.

Oral squamous cell carcinoma-released brain-derived neurotrophic factor contributes to oral cancer pain by peripheral tropomyosin receptor kinase B activation.

ABSTRACT: Oral cancer pain is debilitating and understanding mechanisms for it is critical to develop novel treatment strategies treatment strategies. Brain-derived neurotrophic factor (BDNF) signaling is elevated in oral tumor biopsies and is involved with tumor progression. Whether BDNF signaling in oral tumors contributes to cancer-induced pain is not known. The current study evaluates a novel peripheral role of BDNF-tropomyosin receptor kinase B (TrkB) signaling in oral cancer pain. Using human oral squamous cell carcinoma (OSCC) cells and an orthotopic mouse tongue cancer pain model, we found that BDNF levels were upregulated in superfusates and lysates of tumor tongues and that BDNF was expressed by OSCC cells themselves. Moreover, neutralization of BDNF or inhibition of TrkB activity by ANA12, within the tumor-bearing tongue reversed tumor-induced pain-like behaviors in a sex-dependent manner. Oral squamous cell carcinoma conditioned media also produced pain-like behaviors in naïve male mice that was reversed by local injection of ANA12. On a physiological level, using single-fiber tongue-nerve electrophysiology, we found that acutely blocking TrkB receptors reversed tumor-induced mechanical sensitivity of A-slow high threshold mechanoreceptors. Furthermore, single-cell reverse transcription polymerase chain reaction data of retrogradely labeled lingual neurons demonstrated expression of full-form TrkB and truncated TrkB in distinct neuronal subtypes. Last but not the least, intra-TG siRNA for TrkB also reversed tumor-induced orofacial pain behaviors. Our data suggest that TrkB activities on lingual sensory afferents are partly controlled by local release of OSCC-derived BDNF, thereby contributing to oral cancer pain. This is a novel finding and the first demonstration of a peripheral role for BDNF signaling in oral cancer pain.

Pituitary hormones are specifically expressed in trigeminal sensory neurons and contribute to pain responses in the trigeminal system.

Trigeminal (TG), dorsal root (DRG), and nodose/jugular (NG/JG) ganglia each possess specialized and distinct functions. We used RNA sequencing of two-cycle sorted Pirt-positive neurons to identify genes exclusively expressing in L3-L5 DRG, T10-L1 DRG, NG/JG, and TG mouse ganglion neurons. Transcription factor Phox2b and Efcab6 are specifically expressed in NG/JG while Hoxa7 is exclusively present in both T10-L1 and L3-L5 DRG neurons. Cyp2f2, Krt18, and Ptgds, along with pituitary hormone prolactin (Prl), growth hormone (Gh), and proopiomelanocortin (Pomc) encoding genes are almost exclusively in TG neurons. Immunohistochemistry confirmed selective expression of these hormones in TG neurons and dural nerves; and showed GH expression in subsets of TRPV1+ and CGRP+ TG neurons. We next examined GH roles in hypersensitivity in the spinal versus trigeminal systems. Exogenous GH produced mechanical hypersensitivity when injected intrathecally, but not intraplantarly. GH-induced thermal hypersensitivity was not detected in the spinal system. GH dose-dependently generated orofacial and headache-like periorbital mechanical hypersensitivity after administration into masseter muscle and dura, respectively. Periorbital mechanical hypersensitivity was reversed by a GH receptor antagonist, pegvisomant. Overall, pituitary hormone genes are selective for TG versus other ganglia somatotypes; and GH has distinctive functional significance in the trigeminal versus spinal systems.

Automated analyses for single-fiber electrophysiological recordings using a newly developed Microsoft Excel application and graphical user interface.

BACKGROUND: Electrophysiological recordings of isolated sensory afferents are commonly used in the field of pain research to investigate peripheral mechanisms of nociception in various pain models. The method involves skillful and tedious recordings of teased fibers from nerve preparations as well as time-consuming post-recording analyses. To increase efficiency and productivity of data analyses of recorded action potentials, we developed and validated a novel, easy-to-use Microsoft Excel-based application using Visual Basic Programming. NEW METHOD: A code for the novel program, shigraspike1.0, was written to create a module to include customizable subroutines for analyses for electrical and mechanical responses. Using previously recorded action potentials with tongue-lingual nerve preparations, the program was validated for appropriate execution, ease-of-use, accuracy of the output data and time taken for analyses. RESULTS: We observed appropriate execution of shigraspike1.0 on Windows and iOS desktop platforms that included computation of response latency of the spike of interest using electrical stimulus as well as estimation of the number of impulses at each force with a step-and-hold mechanical ramp of 10-200mN. Output data obtained by shigrapsike1.0 for both stimulus types were accurate and statistically insignificant from manual analyses. COMPARISON WITH EXISTING METHOD: The novel application shigraspike1.0, allows for rapid analyses for single-fiber recordings and takes less than half the time to analyze electrical and mechanical responses compared to manual analyses. CONCLUSIONS: The newly developed shigraspike1.0 application can be a very productive tool to be routinely used for efficient analyses of single-fiber electrophysiology in pain research.

Elevated dietary ω-6 polyunsaturated fatty acids induce reversible peripheral nerve dysfunction that exacerbates comorbid pain conditions.

Chronic pain is the leading cause of disability worldwide1 and is commonly associated with comorbid disorders2. However, the role of diet in chronic pain is poorly understood. Of particular interest is the Western-style diet, enriched with ω-6 polyunsaturated fatty acids (PUFAs) that accumulate in membrane phospholipids and oxidise into pronociceptive oxylipins3,4. Here we report that mice administered an ω-6 PUFA-enriched diet develop persistent nociceptive hypersensitivities, spontaneously active and hyper-responsive glabrous afferent fibres and histologic markers of peripheral nerve damage reminiscent of a peripheral neuropathy. Linoleic and arachidonic acids accumulate in lumbar dorsal root ganglia, with increased liberation via elevated phospholipase (PLA)2 activity. Pharmacological and molecular inhibition of PLA2G7 or diet reversal with high levels of ω-3 PUFAs attenuate nociceptive behaviours, neurophysiologic abnormalities and afferent histopathology induced by high ω-6 intake. Additionally, ω-6 PUFA accumulation exacerbates allodynia observed in preclinical inflammatory and neuropathic pain models and is strongly correlated with multiple pain indices of clinical diabetic neuropathy. Collectively, these data reveal dietary enrichment with ω-6 PUFAs as a new aetiology of peripheral neuropathy and risk factor for chronic pain and implicate multiple therapeutic considerations for clinical pain management.

Reliable approaches to extract high-integrity RNA from skin and other pertinent tissues used in pain research.

INTRODUCTION: Comprehensive mRNA sequencing is a powerful tool for conducting unbiased, quantitative differential gene expression analysis. However, the reliability of these data is contingent on the extraction of high-quality RNA from samples. Preserving RNA integrity during extraction can be problematic, especially in tissues such as skin with dense, connective matrices and elevated ribonuclease expression. This is a major barrier to understanding the influences of altered gene expression in many preclinical pain models and clinical pain disorders where skin is the site of tissue injury. OBJECTIVE: This study developed and evaluated extraction protocols for skin and other tissues to maximize recovery of high-integrity RNA needed for quantitative mRNA sequencing. METHODS: Rodent and human tissue samples underwent one of the several different protocols that combined either RNA-stabilizing solution or snap-freezing with bead milling or cryosectioning. Indices of RNA integrity and purity were assessed for all samples. RESULTS: Extraction of high-integrity RNA is highly dependent on the methods used. Bead-milling skin collected in RNA-stabilizing solution resulted in extensive RNA degradation. Snap-freezing in liquid nitrogen was required for skin and highly preferable for other tissues. Skin also required cryosectioning to achieve effective penetration of RNA-stabilizing solution to preserve RNA integrity, whereas bead milling could be used instead with other tissues. Each method was reproducible across multiple experimenters. Electrophoretic anomalies that skewed RNA integrity value assignment required manual correction and often resulted in score reduction. CONCLUSION: To achieve the potential of quantitative differential gene expression analysis requires verification of tissue-dependent extraction methods that yield high-integrity RNA.

Depiction of Oral Tumor-Induced Trigeminal Afferent Responses Using Single-Fiber Electrophysiology.

Considerable gap in knowledge exists about the mechanisms by which oral tumors regulate peripheral sensory fibers to produce pain and altered sensations. To address this gap, we used a murine model of oral squamous cell carcinoma (OSCC) of the tongue to investigate changes in response properties of trigeminal afferent neurons. Using this model, we developed an ex vivo method for single neuron recordings of the lingual nerve from isolated tongue tissue. Our data demonstrated that the tongue tumor produced increased spontaneous firing of lingual fibers compared to control as well as produced mechanical hypersensitivity and reduced von Frey thresholds of C- and A-slow-high-threshold mechanoreceptors (HTMR) fibers but had no effect on C-LTMR, A-slow-LTMR and A-fast lingual fibers. Mechanically-insensitive fibers were also detected in lingual afferents of the control group, that were significantly decreased in tumor-bearing preparations. Collectively, using single fiber electrophysiology of lingual sensory fibers, we show that human OSCC tumors sensitize peripheral trigeminal nerve terminals, providing a unique opportunity to study mechanisms of oral cancer pain.

Characterization of sensory neuronal subtypes innervating mouse tongue.

The tongue is uniquely exposed to water-soluble environmental chemicals that may lead to injury or tumorigenesis. However, comparatively little research has focused on the molecular and functional organization of trigeminal ganglia (TG) afferent neurons innervating the tongue. The current study identified and characterized lingual sensory neurons based on a neuronal subtype classification previously characterized in the dorsal root ganglion (DRG) neurons. We employed immunohistochemistry on transgenic reporter mouse lines as well as single-cell PCR of known markers of neuronal subtypes to characterize neuronal subtypes innervating the tongue. Markers expressed in retrogradely labeled TG neurons were evaluated for the proportion of neurons expressing each marker, intensity of expression, and overlapping genes. We found that tongue-innervating sensory neurons primarily expressed CGRP, TRPV1, TrkC, 5HT3A and Parvalbumin. These markers correspond to peptidergic and a subgroup of non-peptidergic C-nociceptors, peptidergic A nociceptors, proprioceptors and myelinated low-threshold mechanoreceptors (LTMRs). Interestingly, as reported previously, we also found several differences between TG and DRG neurons indicating the need for single-cell sequencing of neuronal types based on tissue type within all TG as well as DRG neurons.

Effect of Bacterial Biofilm on the Osteogenic Differentiation of Stem Cells of Apical Papilla.

INTRODUCTION: Although clinical success in regenerative endodontics is substantially high, histological success is limited to finding bone/cementum-like tissue instead of dentin within the canal space. The aims of this study were to investigate (1) the effect of bacterial biofilm on osteogenic gene expression in stem cells of the apical papilla (SCAP) and (2) the effect of bacterial antigens on the functional differentiation of SCAP into a mineralizing phenotype. METHODS: Using an ex vivo organotypic root canal model and an American Association of Endontists-recommended regenerative endodontic procedures, we evaluated SCAP differentiation in the presence and absence of an Enterococcus faecalis biofilm. Gene expression analysis for dentinogenic and osteoblastic markers was performed with real-time polymerase chain reaction. The effect of E. faecalis antigens on SCAP differentiation into mineralizing cells in vitro was evaluated with 2 functional assays: Alizarin Red and alkaline phosphatase activity assays. RESULTS: After regenerative endodontic procedures, residual bacteria continued to sustain within the root canal system. SCAP in the presence of E. faecalis biofilm significantly downregulated dentinogenic genes such as dentin sialophosphoprotein and upregulated osteoblastic genes such as bone sialoprotein, osteocalcin, distal-less homeobox 5, and runt-related transcription factor 2. E. faecalis antigens significantly inhibited SCAP differentiation into a mineralizing phenotype when alizarin red staining and alkaline phosphatase assays were used in vitro. CONCLUSIONS: Current disinfection protocols were ineffective in eliminating bacteria from root tips and the levels of the residual bacterial biofilm, and its byproducts, were able to significantly alter osteogenic-differentiation of SCAP.

Central activation of TRPV1 and TRPA1 by novel endogenous agonists contributes to mechanical allodynia and thermal hyperalgesia after burn injury.

The primary complaint of burn victims is an intense, often devastating spontaneous pain, with persistence of mechanical and thermal allodynia. The transient receptor potential channels, TRPV1 and TRPA1, are expressed by a subset of nociceptive sensory neurons and contribute to inflammatory hypersensitivity. Although their function in the periphery is well known, a role for these TRP channels in central pain mechanisms is less well defined. Lipid agonists of TRPV1 are released from peripheral tissues via enzymatic oxidation after burn injury; however, it is not known if burn injury triggers the release of oxidized lipids in the spinal cord. Accordingly, we evaluated whether burn injury evoked the central release of oxidized lipids . Analysis of lipid extracts of spinal cord tissue with HPLC-MS revealed a significant increase in levels of the epoxide and diol metabolites of linoleic acid: 9,10-DiHOME, 12,13-DiHOME, 9(10)-EpOME, and 12(13)-EpOME, that was reduced after intrathecal (i.t.) injection of the oxidative enzyme inhibitor ketoconazole. Moreover, we found that these four lipid metabolites were capable of specifically activating both TRPV1 and TRPA1. Intrathecal injection of specific antagonists to TRPV1 (AMG-517) or TRPA1 (HC-030031) significantly reduced post-burn mechanical and thermal allodynia. Finally, i.t. injection of ketoconazole significantly reversed post-burn mechanical and thermal allodynia. Our data indicate that spinal cord TRPV1 and TRPA1 contributes to pain after burn and identifies a novel class of oxidized lipids elevated in the spinal cord after burn injury. Since the management of burn pain is problematic, these findings point to a novel approach for treating post-burn pain.

Persistent Nociception Triggered by Nerve Growth Factor (NGF) Is Mediated by TRPV1 and Oxidative Mechanisms.

Nerve growth factor (NGF) is elevated in certain chronic pain conditions and is a sufficient stimulus to cause lasting pain in humans, but the actual mechanisms underlying the persistent effects of NGF remain incompletely understood. We developed a rat model of NGF-induced persistent thermal hyperalgesia and mechanical allodynia to determine the role of transient receptor potential vanilloid 1 (TRPV1) and oxidative mechanisms in the persistent effects of NGF. Persistent thermal hypersensitivity and mechanical allodynia require de novo protein translation and are mediated by TRPV1 and oxidative mechanisms. By comparing effects after systemic (subcutaneous), spinal (intrathecal) or hindpaw (intraplantar) injections of test compounds, we determined that TRPV1 and oxidation mediate persistent thermal hypersensitivity via peripheral and spinal sites of action and mechanical allodynia via only a spinal site of action. Therefore, NGF-evoked thermal and mechanical allodynia are mediated by spatially distinct mechanisms. NGF treatment evoked sustained increases in peripheral and central TRPV1 activity, as demonstrated by increased capsaicin-evoked nocifensive responses, increased calcitonin gene-related peptide release from hindpaw skin biopsies, and increased capsaicin-evoked inward current and membrane expression of TRPV1 protein in dorsal root ganglia neurons. Finally, we showed that NGF treatment increased concentrations of linoleic and arachidonic-acid-derived oxidized TRPV1 agonists in spinal cord and skin biopsies. Furthermore, increases in oxidized TRPV1-active lipids were reduced by peripheral and spinal injections of compounds that completely blocked persistent nociception. Collectively, these data indicate that NGF evokes a persistent nociceptive state mediated by increased TRPV1 activity and oxidative mechanisms, including increased production of oxidized lipid TRPV1 agonists.