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.

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.

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.

Direct effect of endodontic sealers on trigeminal neuronal activity.

INTRODUCTION: Endodontic sealers are selected on the basis of their antimicrobial properties and ability to provide a tight seal. Sealer extrusions, whether intentional or unintentional, are common during obturation procedures. Such events have been correlated with increased postoperative discomfort and persistent pain states. However, the mechanisms underlying this phenomenon are largely unknown. Thus, we sought to evaluate the effect of commonly used endodontic sealers on peripheral nociceptors. We hypothesized that endodontic sealers can directly activate trigeminal nociceptors in a concentration-dependent manner, resulting in release of calcitonin gene-related peptide (CGRP), a potent modulator of neurogenic inflammation. METHODS: Rat trigeminal sensory neurons were exposed in vitro to vehicle, zinc oxide-eugenol (ZOE)-based sealer, AH Plus, EndoSequence BC sealer, or RealSeal SE. Neuronal activation was measured by quantification of neuropeptide (CGRP) release. In addition, cultured neurons were also subjected to the set form of all 4 sealers. The concentration of CGRP released was quantified by using a radioimmunoassay. Data were analyzed by using one-way analysis of variance with Newman-Keuls multiple comparison post hoc test. RESULTS: Both ZOE-based sealer and AH Plus in their fresh form evoked greater CGRP release than the control groups. Conversely, EndoSequence BC and RealSeal sealers both reduced basal GCRP release at all concentrations tested. Evaluation of the set sealers revealed that only ZOE-based sealer evoked significant CGRP release compared with its control group. CONCLUSIONS: Overall, our results suggest that sealers can directly activate trigeminal nociceptors, leading to a robust release of CGRP, and may therefore lead to pain and neurogenic inflammation. This direct activation along with the immunologic response may underlie the symptoms and flare-up occurrences often seen with sealer extrusions.

Prolactin regulates TRPV1, TRPA1, and TRPM8 in sensory neurons in a sex-dependent manner: Contribution of prolactin receptor to inflammatory pain.

Prolactin (PRL) is a hormone produced in the anterior pituitary but also synthesized extrapituitary where it can influence diverse cellular processes, including inflammatory responses. Females experience greater pain in certain inflammatory conditions, but the contribution of the PRL system to sex-dependent inflammatory pain is unknown. We found that PRL regulates transient receptor potential (TRP) channels in a sex-dependent manner in sensory neurons. At >20 ng/ml, PRL sensitizes TRPV1 in female, but not male, neurons. This effect is mediated by PRL receptor (PRL-R). Likewise, TRPA1 and TRPM8 were sensitized by 100 ng/ml PRL only in female neurons. We showed that complete Freund adjuvant (CFA) upregulated PRL levels in the inflamed paw of both male and female rats, but levels were higher in females. In contrast, CFA did not change mRNA levels of long and short PRL-R in the dorsal root ganglion or spinal cord. Analysis of PRL and PRL-R knockout (KO) mice demonstrated that basal responses to cold stimuli were only altered in females, and with no significant effects on heat and mechanical responses in both sexes. CFA-induced heat and cold hyperalgesia were not changed in PRL and PRL-R KO compared with wild-type (WT) males, whereas significant reduction of heat and cold post-CFA hyperalgesia was detected in PRL and PRL-R KO females. Attenuation of CFA-induced mechanical allodynia was observed in both PRL and PRL-R KO females and males. Thermal hyperalgesia in PRL KO females was restored by administration of PRL into hindpaws. Overall, we demonstrate a sex-dependent regulation of peripheral inflammatory hyperalgesia by the PRL system.

Major blunt trauma evokes selective upregulation of oxidative enzymes in circulating leukocytes.

Tissue injury, such as burns or inflammation, can lead to the generation of oxidized lipids capable of regulating hemodynamic, pulmonary, immune, and neuronal responses. However, it is not known whether traumatic injury leads to a selective upregulation of transcripts encoding oxidative enzymes capable of generating these mediators. Here, we analyzed microarrays taken from circulating leukocytes of 187 trauma subjects compared with 97 control volunteers for changes in the expression of 105 oxidative enzymes and related receptors. The results indicate that major blunt trauma triggers a selective change in gene expression, with some transcripts undergoing highly significant upregulation (e.g., CYP2C19), while others display significantly reduced expression (e.g., CYP2U1). This pattern in gene expression was maintained for up to 28 days after injury. In addition, the level of expression of CYP2A7, CYP2B7P1, CYP2C19, CYP2E1, CYP4A11, CYP4F3, CYP8B1, CYP19A1, CYP20A1, CYP51A1, HMOX2, NCF1, NCF2, and NOX1 and the receptors PTGER2 and ESR2 were correlated with clinical trauma indices such as APACHE II, Max Denver Scale, and the Injury Severity Score. Demonstration of a selective alteration in expression of transcripts encoding oxidative enzymes reveals a complex molecular response to major blunt trauma in circulating leukocytes. Furthermore, the association between changes in gene expression and clinical trauma scores suggests an important role in integrating pathophysiologic responses to blunt force trauma.

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.