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.

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.

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.

Released lipids regulate transient receptor potential channel (TRP)-dependent oral cancer pain.

BACKGROUND: Pain in the head neck area is an early symptom in oral cancer, supporting the hypothesis that cancer cells control the activities of surrounding nociceptors at the site of the tumor. Several reports implicate TRPV1 and TRPA1 in cancer pain, although there is a large gap in knowledge since the mechanisms for tumor-induced activation of these TRP receptors are unknown. Interestingly, TRP-active lipids such as linoleic acid, arachidonic acid, hydroxyoctadecadienoic acid and hydroxyeicosatetraenoic acid are significantly elevated in the saliva of oral cancer patients compared to normal patients, supporting a possible linkage between these lipids and oral cancer pain. We therefore hypothesize that oral squamous cell carcinomas release certain lipids that activate TRPV1 and/or TRPA1 on sensory neurons, contributing to the development of oral cancer pain. METHODS: Lipid extracts were made from conditioned media of three human oral squamous cell carcinoma (OSCC) cell lines as well as one normal human oral keratinocytes cell line. These were then injected intraplantarly into rat hindpaws to measure spontaneous nocifensive behavior, as well as thermal and mechanical allodynia. For interventional experiments, the animals were pretreated with AMG517 (TRPV1 antagonist) or HC030031 (TRPA1 antagonist) prior to extract injection. RESULTS: These studies demonstrate that lipids released from the three OSCC cell lines, but not the normal cell line, were capable of producing significant spontaneous nocifensive behaviors, as well as thermal and mechanical allodynia. Notably each of the cell lines produced a different magnitude of response for each of three behavioral assays. Importantly, pre-treatment with a TRPVI antagonist blocked lipid-mediated nocifensive and thermal hypersensitivity, but not mechanical hypersensitivity. In addition, pre-treatment with a TRPA1 antagonist only reversed thermal hypersensitivity without affecting lipid-induced nocifensive behavior or mechanical allodynia. CONCLUSIONS: These data reveal a novel mechanism for cancer pain and provide strong direction for future studies evaluating the cellular mechanism regulating the TRP-active lipids by OSCC tumors.

Oxidized linoleic acid metabolite-cytochrome P450 system (OLAM-CYP) is active in biopsy samples from patients with inflammatory dental pain.

Endogenous TRPV1 agonists such as oxidized linoleic acid metabolites (OLAMs) and the enzymes releasing them [eg, cytochrome P450 (CYP)] are up-regulated after inflammation in the rat. However, it is not known whether such agonists are elevated in human inflammatory pain conditions. Because TRPV1 is expressed in human dental pulp nociceptors, we hypothesized that OLAM-CYP machinery is active in this tissue type and is increased under painful inflammatory conditions such as irreversible pulpitis (IP). The aim of this study was to compare CYP expression and linoleic acid (LA) metabolism in normal vs inflamed human dental pulp. Our data showed that exogenous LA metabolism was significantly increased in IP tissues compared to normal tissues and that pretreatment with a CYP inhibitor, ketoconazole, significantly inhibited LA metabolism. Additionally, extracts obtained from LA-treated inflamed tissues evoked significant inward currents in trigeminal ganglia neurons and were blocked by pretreatment with the TRPV1 antagonist IRTX. Moreover, extracts obtained from ketoconazole-pretreated inflamed tissues significantly reduced inward currents in trigeminal ganglia neurons. These data suggest that LA metabolites produced in human inflamed tissues act as TRPV1 agonists and that the metabolite production can be targeted by CYP inhibition. In addition, immunohistochemical analysis of 2 CYP isoforms, CYP2J and CYP3A1, were shown to be predominately expressed in immune cells infiltrating the inflamed dental pulp, emphasizing the paracrine role of CYP enzymes in OLAM regulation. Collectively, our data indicate that the machinery responsible for OLAM production is up-regulated during inflammation and can be targeted to develop potential analgesics for inflammatory-induced dental pain.

Omega-3 fatty acid inhibition of prostate cancer progression to hormone independence is associated with suppression of mTOR signaling and androgen receptor expression.

Currently, progression of prostate cancer to androgen independence remains the primary obstacle to improved survival. In order to improve overall survival, novel treatment strategies that are based upon specific molecular mechanisms that prolong the androgen-dependent state and that are useful for androgen-independent disease need to be identified. Both epidemiological as well as preclinical data suggest that omega-3 fatty acids are effective primary tumor prevention agents; however, their efficacy at preventing and treating refractory prostate cancer has not been as thoroughly investigated. We used an in vitro model of androgen ablation to determine the effect of treatment with omega-3 fatty acids on the progression to an androgen-independent state. The omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were able to prevent progression of LNCaP cells while the omega-6 fatty acid arachidonic acid (AA) actually promoted cell growth under conditions of hormone depletion. These results correlated with a decrease in the expression of the androgen receptor as well as suppression of the Akt/mTOR signaling pathway. Connecting the mechanisms by which omega-3 fatty acids affect phenotypic outcome is important for effective exploitation of these nutrient agents as a therapeutic approach. Understanding these processes is critical for the development of effective dietary intervention strategies that improve overall survival.

In vitro sarcoma cells release a lipophilic substance that activates the pain transduction system via TRPV1.

BACKGROUND: Despite success in treating many forms of cancer, pain associated with malignancy remains a serious clinical issue with a poorly understood etiology. This study determined if certain sarcoma cell lines produced a soluble factor that activates the TRPV1 ion channel expressed on nociceptive sensory neurons, thereby activating a major pain transduction system. MATERIALS AND METHODS: Trigeminal ganglia were harvested from rats and cultured. A rhabdomyosarcoma (CRL1598) and osteosarcoma (CRL 1543) cell line were grown to 75% confluency. Conditioned media (CM) was collected after 24 h of exposure and subjected to reverse phase chromatography. Neuronal activation in the presence of CM was measured using iCGRP RIA and calcium imaging after treatment with vehicle or I-RTX, a potent TRPV1 antagonist. Data were analyzed by ANOVA/Bonferroni or t test. RESULTS: The rhabdomyosarcoma CM produced a 4-fold increase in iCGRP release compared with control media (P < 0.001). The osteosarcoma cell line CM produced a 7-fold increase in iCGRP release compared with control media (P < 0.001). This evoked iCGRP release was via TRPV1 activation since the effect was blocked by the antagonist I-RTX. The application of rhabdomyosarcoma CM produced about a 4-fold increase in [Ca(2+)]I levels (P < 0.001), and this effect was blocked by pretreatment with the TRPV1 antagonist, I-RTX. CONCLUSIONS: We have shown that certain sarcoma cell lines produce a soluble, lipophilic factor that activates the peripheral nociceptor transduction system via TRPV1 activation, thereby contributing to cancer pain. Further investigations are needed to develop tumor-specific analgesics that do not produce unwanted or harmful side-effects.