Effects of EDTA and saline as the final irrigation in regenerative endodontic procedures on the migration, proliferation, and differentiation of human stem cells from the apical papilla.

OBJECTIVES: To evaluate the effect of EDTA and saline as the final irrigation in regenerative endodontic procedures (REPS) on the attachment, proliferation, migration, and differentiation of stem cells from the apical papilla (SCAPs). MATERIALS AND METHODS: Dentin specimens from 140 human third molars were irrigated with various protocols-group 1: normal sterile saline (NSS), group 2: EDTA, group 3: EDTA then 5 mL NSS, or group 4: EDTA then 20 mL NSS. The specimens were used in cell assays. For cell proliferation, SCAPs were seeded on dentin, and the cell viability on days 1, 3, and 7 was determined using an MTT assay. At day 3, the attached cells' morphology was observed using SEM, and cell migration was investigated using a transwell migration assay. The ALP activity and odonto/osteogenic differentiation gene expression were evaluated at days 7, 14, and 21 using an ALP activity assay and RT-qPCR. RESULTS: On days 3 and 7, group 4 demonstrated more viable cells than group 1 (p < 0.01). The amount of migrated cells in groups 2, 3, and 4 was greater compared with group 1 (p < 0.05). Moreover, SCAP differentiation was similar between groups. CONCLUSIONS: Irrigating dentin with EDTA alone or with EDTA then NSS promoted SCAP migration. However, a final irrigation with 20 mL NSS after EDTA promoted SCAP proliferation without affecting their differentiation. CLINICAL RELEVANCE: When using a blood clot as a scaffold, a final flushing with 20 mL NSS after EDTA could be beneficial for clinical REP protocols.

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.

Serotonergic neuromodulation of peripheral nociceptors.

Nociception, the encoding and processing of noxious environmental stimuli by sensory neurons, functions to protect an organism from bodily damage. Activation of the terminal endings of certain sensory neurons, termed nociceptors, triggers a train of impulses to neurons in the spinal cord. Signals are integrated and processed in the dorsal spinal cord and then projected to the brain where they elicit the perception of pain. A number of neuromodulators that can affect nociceptors are released in the periphery during the inflammation that follows an initial injury. Serotonin (5-HT) is a one such proinflammatory mediator. This review discusses our current understanding of the neuromodulatory role of 5-HT, and specifically how this monoamine activates and sensitizes nociceptors. Potential therapeutic targets to treat pain are described.

Activation of estrogen receptor α enhances bradykinin signaling in peripheral sensory neurons of female rats.

Numerous studies have demonstrated that females have a higher risk of experiencing several pain disorders with either greater frequency or severity than males. Although the mechanisms that underlie this sex disparity remain unclear, several studies have shown an important role for sex steroids, such as estrogen, in the modulation of nociception. Receptors for estrogen are present in primary afferent neurons in the trigeminal and dorsal root ganglia, and brief exposure to estrogen increases responses to the inflammatory mediator bradykinin (BK). However, the mechanism for estrogen-mediated enhancement of BK signaling is not fully understood. The aim of the present study was to evaluate the relative contributions of estrogen receptor α (ERα), ERß, and G protein-coupled estrogen receptor 1 (GPER) to the enhanced signaling of the inflammatory mediator BK by 17ß-estradiol (17ß-E2) in primary sensory neurons from female rats in culture (ex vivo) and in behavioral assays of nociception in vivo. The effects of 17ß-E2 on BK responses were mimicked by ERα-selective agonists and blocked by ERα-selective antagonists and by small interfering RNA knockdown of ERα. The data indicate that ERα is required for 17ß-E2-mediated enhancement of BK signaling in peripheral sensory neurons in female rats.

Web- Based survey on Regenerative Endodontic Practices Among Members of the American Association of Endodontists.

INTRODUCTION: The AAE Regenerative Endodontics Committee (REC) reports here the web-based survey data of regenerative endodontic procedures (REP) performed by the AAE members from 2008 to 2019. METHODS: The web-based survey consisted of 2 questionnaires, a revascularization and a follow-up, including clinical and radiographic data at 3, 6,12 or >12 months after treatment. Data from demographics, etiology, clinical protocols, radiographic and clinical outcome as perceived by the clinicians were recorded. From 927 entries, 184 full cases were submitted and 126 were suitable for radiographic analysis. The data were divided into cases with 6-12 months and cases with > 12 months recall time. Descriptive statistics and univariate analyses were performed. RESULTS: Predominantly patients were male (63%), average 10 years-old with anterior teeth (77.2%) due to trauma (69,6%). A wide variation in REP protocols were reported by AAE members. The most common clinical protocols used minimal instrumentation (75.5%), 2.5% or higher concentrations of sodium hypochlorite (83.7%), and antibiotic paste as intracanal medication (58.7%). The majority reported blood clot formation (56%) and most common coronal capping material was white MTA (50.5%). Increases in radiographic root length and radiographic root area were proportional to the time lapsed after treatment. Overall, case outcomes were categorized by clinicians as successful (54.3%), uncertain (23.9%) and unsuccessful (3.8%). CONCLUSIONS: This web-based survey provides a valuable perspective on case selection, clinical protocols and perceived outcomes for REPs and supports the need for future higher level evidence studies.

Intrafibrillar silicification of collagen scaffolds for sustained release of stem cell homing chemokine in hard tissue regeneration.

Traditional bone regeneration strategies relied on supplementation of biomaterials constructs with stem or progenitor cells or growth factors. By contrast, cell homing strategies employ chemokines to mobilize stem or progenitor cells from host bone marrow and tissue niches to injured sites. Although silica-based biomaterials exhibit osteogenic and angiogenic potentials, they lack cell homing capability. Stromal cell-derived factor-1 (SDF-1) plays a pivotal role in mobilization and homing of stem cells to injured tissues. In this work, we demonstrated that 3-dimensional collagen scaffolds infiltrated with intrafibrillar silica are biodegradable and highly biocompatible. They exhibit improved compressive stress-strain responses and toughness over nonsilicified collagen scaffolds. They are osteoconductive and up-regulate expressions of osteogenesis- and angiogenesis-related genes more significantly than nonsilicified collagen scaffolds. In addition, these scaffolds reversibly bind SDF-1α for sustained release of this chemokine, which exhibits in vitro cell homing characteristics. When implanted subcutaneously in an in vivo mouse model, SDF-1α-loaded silicified collagen scaffolds stimulate the formation of ectopic bone and blood capillaries within the scaffold and abrogate the need for cell seeding or supplementation of osteogenic and angiogenic growth factors. Intrafibrillar-silicified collagen scaffolds with sustained SDF-1α release represent a less costly and complex alternative to contemporary cell seeding approaches and provide new therapeutic options for in situ hard tissue regeneration.

Biomimetic silicification of demineralized hierarchical collagenous tissues.

Unlike man-made composite materials, natural biominerals containing composites usually demonstrate different levels of sophisticated hierarchical structures which are responsible for their mechanical properties and other metabolic functions. However, the complex spatial organizations of the organic-inorganic phases are far beyond what they achieved by contemporary engineering techniques. Here, we demonstrate that carbonated apatite present in collagen matrices derived from fish scale and bovine bone may be replaced by amorphous silica, using an approach that simulates what is utilized by phylogenetically ancient glass sponges. The structural hierarchy of these collagen-based biomaterials is replicated by the infiltration and condensation of fluidic polymer-stabilized silicic acid precursors within the intrafibrillar milieu of type I collagen fibrils. This facile biomimetic silicification strategy may be used for fabricating silica-based, three-dimensional functional materials with specific morphological and hierarchical requirements.

Oral Cancer Pain Includes Thermal Allodynia That May Be Attenuated by Chronic Alcohol Consumption.

BACKGROUND: Oral cancer is one of the most painful cancer types, and is often refractory to existing analgesics. Oral cancer patients frequently develop a tolerance to opioids, the mainstay of current cancer pain therapy, leaving them with limited therapeutic options. Thus, there is a great need to identify molecular mechanisms driving oral cancer pain in an effort to develop new analgesics. Previous reports demonstrate that oral cancer patients experience intense mechanical pain and pain in function. To date, no studies have examined thermal pain in oral cancer patients or the role that alcohol consumption plays in oral cancer pain. This study aims to evaluate patient-reported pain levels and thermal allodynia, potential molecular mechanisms mediating thermal allodynia, and the effects of alcohol consumption on patient-perceived pain. METHODS: This study evaluated human oral squamous cell carcinoma (OSCC) cell lines for their ability to activate thermosensitive channels in vitro and validated these findings in a rat model of orofacial pain. Patient-reported pain in a south Texas OSCC cohort (n = 27) was examined using a visual analog scale (VAS). Covariant analysis examined variables such as tobacco and alcohol consumption, ethnicity, gender, and cancer stage. RESULTS: We determined that OSCC secretes factors that stimulate both the Transient Receptor Potential Ankyrin type 1 channel (TRPA1; noxious cold sensor) and the Transient Receptor Potential Vanilloid type 1 channel (TRPV1; noxious heat sensor) in vitro and that OSCC-secreted factors sensitize TRPV1 nociceptors in vivo. These findings were validated in this cohort, in which allodynia to cold and heat were reported. Notably, subjects that reported regular alcohol consumption also reported lower pain scores for every type of pain tested, with significantly reduced cold-induced pain, aching pain, and burning pain. CONCLUSION: Oral cancer patients experience multiple types of cancer pain, including thermal allodynia. Alcohol consumption correlates with reduced OSCC pain and reduced thermal allodynia, which may be mediated by TRPA1 and TRPV1. Hence, reduced pain in these patients may contribute to a delay in seeking care, and thus a delay in early detection and treatment.

Allosteric interactions between δ and κ opioid receptors in peripheral sensory neurons.

The peripheral δ opioid receptor (DOR) is an attractive target for analgesic drug development. There is evidence that DOR can form heteromers with the κ-opioid receptor (KOR). As drug targets, heteromeric receptors offer an additional level of selectivity and, because of allosteric interactions between protomers, functionality. Here we report that selective KOR antagonists differentially altered the potency and/or efficacy of DOR agonists in primary cultures of adult rat peripheral sensory neurons and in a rat behavioral model of thermal allodynia. In vitro, the KOR antagonist nor-binaltorphimine (nor-BNI) enhanced the potency of [D-Pen(2,5)]-enkephalin (DPDPE), decreased the potency of [D-Ala(2),D-Leu(5)]-enkephalin (DADLE), and decreased the potency and efficacy of 4-[(R)-[(2S,5R)-4-allyl-2,5-dimethylpiperazin-1-yl](3-methoxyphenyl)methyl]-N,N-diethylbenzamide (SNC80) to inhibit prostaglandin E(2) (PGE(2))-stimulated adenylyl cyclase activity. In vivo, nor-BNI enhanced the effect of DPDPE and decreased the effect of SNC80 to inhibit PGE(2)-stimulated thermal allodynia. In contrast to nor-BNI, the KOR antagonist 5'-guanidinonaltrindole (5'-GNTI) reduced the response of DPDPE both in cultured neurons and in vivo. Evidence for DOR-KOR heteromers in peripheral sensory neurons included coimmunoprecipitation of DOR with KOR, a DOR-KOR heteromer selective antibody augmented the antinociceptive effect of DPDPE in vivo, and the DOR-KOR heteromer agonist 6'-GNTI inhibited adenylyl cyclase activity in vitro as well as PGE(2)-stimulated thermal allodynia in vivo. Taken together, these data suggest that DOR-KOR heteromers exist in rat primary sensory neurons and that KOR antagonists can act as modulators of DOR agonist responses most likely through allosteric interactions between the protomers of the DOR-KOR heteromer.

The cytochrome P450 inhibitor, ketoconazole, inhibits oxidized linoleic acid metabolite-mediated peripheral inflammatory pain.

BACKGROUND: Oxidized linoleic acid metabolites (OLAMs) are a class of endogenous agonists to the transient receptor potential V1 (TRPV1) receptor. Although TRPV1 mediates inflammatory heat hyperalgesia, it is not known if the OLAMs contribute to the peripheral activation of this receptor during tissue inflammation. In the present study, we evaluated whether the OLAM system is activated during inflammation and whether cytochrome P450 enzymes mediate OLAM contributions to heat hyperalgesia using the complete Freund's adjuvant (CFA) model of inflammation. RESULTS: Our results demonstrate that the intraplantar (ipl) injection of anti-OLAM antibodies significantly reversed CFA-induced heat hyperalgesia. Moreover, application of lipid extracts from inflamed rat skin to cultured sensory neurons triggered a significant release of iCGRP that is blocked by co-treatment with I-RTX, a TRPV1 antagonist. To determine the role of CYP enzymes in mediating OLAM effects, we used a broad spectrum CYP inhibitor, ketoconazole. Pretreatment with ketoconazole inhibited the release of TRPV1 agonists in lipid extracts from inflamed skin and significantly reversed CFA-induced heat hyperalgesia by a peripheral mechanism of action. Moreover, the ipl injection of linoleic acid to rats 24 hr after CFA evoked spontaneous nocifensive behaviors that were significantly reduced by capsazepine, by knockout of the TRPV1 gene, or by pretreatment with either anti-OLAM antibodies or ketoconazole. CONCLUSIONS: Taken together, our data suggests that OLAMs contribute to inflammatory nociception in the periphery and that cytochrome P450 enzymes play a crucial role in mediating OLAM contributions to inflammatory heat hyperalgesia.

Activation of TRPV1 in the spinal cord by oxidized linoleic acid metabolites contributes to inflammatory hyperalgesia.

Transient receptor potential vanilloid 1 (TRPV1) plays a major role in hyperalgesia and allodynia and is expressed both in the peripheral and central nervous systems (CNS). However, few studies have evaluated mechanisms by which CNS TRPV1 mediates hyperalgesia and allodynia after injury. We hypothesized that activation of spinal cord systems releases endogenous TRPV1 agonists that evoke the development of mechanical allodynia by this receptor. Using in vitro superfusion, the depolarization of spinal cord triggered the release of oxidized linoleic acid metabolites, such as 9-hydroxyoctadecadienoic acid (9-HODE) that potently activated spinal TRPV1, leading to the development of mechanical allodynia. Subsequent calcium imaging and electrophysiology studies demonstrated that synthetic oxidized linoleic acid metabolites, including 9-HODE, 13-HODE, and 9 and 13-oxoODE, comprise a family of endogenous TRPV1 agonists. In vivo studies demonstrated that intrathecal application of these oxidized linoleic acid metabolites rapidly evokes mechanical allodynia. Finally, intrathecal neutralization of 9- and 13-HODE by antibodies blocks CFA-evoked mechanical allodynia. These data collectively reveal a mechanism by which an endogenous family of lipids activates TRPV1 in the spinal cord, leading to the development of inflammatory hyperalgesia. These findings may integrate many pain disorders and provide an approach for developing analgesic drugs.

Biologically based treatment of immature permanent teeth with pulpal necrosis: a case series.

This case series reports the outcomes of 8 patients (ages 9-4 years) who presented with 9 immature permanent teeth with pulpal necrosis and apical periodontitis. During treatment, 5 of the teeth were found to have at least some residual vital tissue remaining in the root canal systems. After NaOCI irrigation and medication with ciprofloxacin, metronidazole, and minocycline, these teeth were sealed with mineral trioxide aggregate and restored. The other group of 4 teeth had no evidence of any residual vital pulp tissue. This second group of teeth was treated with NaOCl irrigation and medicated with ciprofloxacin, metronidazole, and minocycline followed by a revascularization procedure adopted from the trauma literature (bleeding evoked to form an intracanal blood clot). In both groups of patients, there was evidence of satisfactory postoperative clinical outcomes (1-5 years); the patients were asymptomatic, no sinus tracts were evident, apical periodontitis was resolved, and there was radiographic evidence of continuing thickness of dentinal walls, apical closure, or increased root length.

Effect of Sodium Hypochlorite in Dental Unit Waterline on Aerosolized Bacteria Generated from Endodontic Procedures.

INTRODUCTION: Aerosol generation in a dental setting is a critical concern, and approaches that aim at decreasing bacterial load in aerosols are of high priority for dental professionals. The objectives of this study were to evaluate the relative effect of various endodontic procedures on the generation and dissemination of aerosols and the effect of 0.1% sodium hypochlorite (NaOCl) in dental unit waterlines (DUWLs) on the bacterial load in the generated aerosols in a clinical setting. METHODS: The study was completed in 2 phases. The classic passive sampling technique using brain-heart infusion agar plates was used. Agar plates were strategically placed throughout the operatory at predefined locations. In phase 1, to evaluate the effect of different endodontic procedures on the generation and dissemination of aerosols, we collected a total of 38 samples. After baseline collection, test samples were collected during vital pulp therapy (VPT) full pulpotomy (n = 10), nonsurgical root canal therapy (NSRCT, n = 10), surgical root canal therapy (SRCT, n = 10), and incision and drainage (n = 8) procedures. Bacterial growth was expressed as colony-forming units at 48 hours after sample collection. Data were analyzed using 1-way analysis of variance with the Tukey multiple comparison post hoc test. In phase 2, to evaluate the effect of 0.1% NaOCl in the DUWL on the bacterial load in the generated aerosols, a total of 30 samples were collected. All procedures including VPT (n = 10), NSRCT (n = 10), and SRCT (n = 10) were performed with 0.1% NaOCl in the DUWL. Bacterial growth was expressed as colony-forming units at 48 hours after sample collection. Data were analyzed using 2-way analysis of variance with the Tukey multiple comparison post hoc test. RESULTS: All endodontic procedures generated aerosols at all tested locations, except incision and drainage. Aerosols were disseminated as far as 3 m from the patient's head with no significant difference between various locations (P > .05). VPT procedures generated the maximum number of aerosols compared with NSRCT and SRCT. Adding 0.1% NaOCl to DUWLs significantly reduced the bacterial load in the generated aerosols in all treatment groups compared with groups treated with untreated waterlines (P < .05). No significant difference was noted in the bacterial load between all groups with treated waterlines (P > .05). CONCLUSIONS: All tested endodontic procedures led to the generation and dissemination of contaminated aerosols, and the addition of 0.1% NaOCl as a biocide to the DUWL led to a statistically significant reduction in the bacterial load.

Microbiome Changes during Regenerative Endodontic Treatment Using Different Methods of Disinfection.

INTRODUCTION: The purpose of this study was to characterize qualitatively and quantitatively the changes in the endodontic microbiome, in teeth with necrotic pulp, open apexes, and apical periodontitis, with 3 antimicrobial protocols, undertaken in a multicenter clinical trial. METHODS: Microbiological samples were collected from 116 regenerative endodontic teeth, and 97 qualified for inclusion. The teeth were randomly divided into 3 treatment groups: apexification (APEX), regeneration (REGEN), and revascularization (REVASC), all in 2 appointments. The group variables in the first appointment irrigants, and second appointment irrigants and medicaments were as follows: APEX: 5.25%-6% NaOCl, 5.25%-6% NaOCl + 17% EDTA and calcium hydroxide; REGEN: 1.25% NaOCl, 17% EDTA, and 0.1 mg/mL triple antibiotic paste (TAP); and REVASC 5.25% NaOCl, saline, and 1 g/mL TAP, respectively. Sampling was done upon access (S0), after irrigation in the first appointment (S1), and after using medication and irrigation in the second appointment (S2). RESULTS: Quantitative polymerase chain reaction analysis of the 16S ribosomal RNA gene showed significant reduction in bacterial load from S0 to S2 in all groups; however, the APEX and REVASC groups had significantly less residual DNA than the REGEN group (P = .0045). The relative abundance of Bacteroidetes, Fusobacteria, Spirochaetes, and Synergistetes were reduced with the treatment rendered. However, relative abundance of Firmicutes and Actinobacteria was not changed, and that of Proteobacteria increased. LEfSe analysis showed that reduction in bacterial taxa was more in REVASC than APEX, which in turn was more than in REGEN. CONCLUSION: Enhanced antimicrobial protocols lead to better reduction in quantitative and qualitative parameters of the endodontic microflora.

Desensitization of transient receptor potential ankyrin 1 (TRPA1) by the TRP vanilloid 1-selective cannabinoid arachidonoyl-2 chloroethanolamine.

Recent studies on cannabinoid-induced analgesia implicate certain transient receptor potential (TRP) channels as a therapeutic target along with metabotropic cannabinoid receptors. Although TRP ankyrin 1 (TRPA1)-selective cannabinoids, such as (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo-[1,2,3-d,e]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone (WIN55,212), are effective at desensitizing TRPA1 and TRP vanilloid 1 (TRPV1), there is a gap in knowledge in understanding the opposite situation, namely whether TRPV1-selective cannabinoids desensitize TRPA1. We selected the TRPV1-specific synthetic cannabinoid, arachidonoyl-2 chloroethanolamine (ACEA), to study peripheral antihyperalgesic properties because ACEA is known to activate TRPV1. Hence, we used in vitro as well as in vivo assays to evaluate the following: 1) the effects of ACEA on the TRPA1-selective agonist, mustard oil (MO), for calcitonin gene-related peptide (CGRP) release from rat hindpaw skin in vitro; 2) the effects of a peripherally selective dose of ACEA on MO-induced nocifensive behavior in vivo; and 3) the effects of five ACEA-insensitive TRPV1 mutations on ACEA-inhibition of MO-evoked calcium accumulation using a Chinese hamster ovary cell expression system. Our results demonstrate that 1) ACEA significantly attenuated (∼40%) MO-evoked CGRP release from rat hindpaw skin, and this effect was not antagonized by the TRPV1 antagonist, capsazepine; 2) ACEA significantly inhibited (∼40%) MO-induced nocifensive behavior in wild-type mice but not in TRPV1 knockout mice; and 3) all TRPV1 mutations insensitive to ACEA lacked the ability to inhibit MO-evoked calcium accumulation in Chinese hamster ovary cells transfected with TRPV1 and TRPA1. Taken together, the results indicate that a TRPV1-selective cannabinoid, ACEA, inhibits MO-evoked responses via a TRPV1-dependent mechanism. This study strengthens the hypothesis that cannabinoids mediate their peripheral analgesic properties, at least in part, via the TRP channels.

Endogenous prolactin generated during peripheral inflammation contributes to thermal hyperalgesia.

Prolactin (PRL) is a hormone and a neuromodulator. It sensitizes TRPV1 (transient receptor potential cation channel subfamily V member 1) responses in sensory neurons, but it is not clear whether peripheral inflammation results in the release of endogenous PRL, or whether endogenous PRL is capable of acting as an inflammatory mediator in a sex-dependent manner. To address these questions, we examined inflammation-induced release of endogenous PRL, and its regulation of thermal hyperalgesia in female and male rats. PRL is expressed in several types of peripheral neuronal and non-neuronal cells, including TRPV1-positive nerve fibers, preadipocytes and activated macrophages/monocytes localized in the vicinity of nerves. Evaluation of PRL levels in hindpaws and plasma indicated that complete Freund's adjuvant (CFA) stimulates release of peripheral, but not systemic, PRL within 6-48 h in both ovariectomized females with estradiol replacement (OVX-E) and intact male rats. The time course of release varies in OVX-E and intact male rats. We next employed the prolactin receptor (PRL-R) antagonist Δ1-9-G129R-hPRL to assess the role of locally produced PRL in nociception. Applied at a ratio of 1 : 1 (PRL:Δ1-9-G129R-hPRL; 40 nm each), this antagonist was able to nearly (≈ 80%) reverse PRL-induced sensitization of capsaicin responses in rat sensory neurons. CFA-induced inflammatory thermal hyperalgesia in OVX-E rat hindpaws was significantly reduced in a dose-dependent manner by the PRL-R antagonist at 6 h but not at 24 h. In contrast, PRL contributed to inflammatory thermal hyperalgesia in intact male rats at 24, but not at 6 h. These findings indicate that inflammation leads to accumulation of endogenous PRL in female and male rats. Furthermore, PRL acts as an inflammatory mediator at different time points for female and intact male rats.

Regulation of κ-opioid receptor signaling in peripheral sensory neurons in vitro and in vivo.

There is considerable interest in understanding the regulation of peripheral opioid receptors to avoid central nervous system side effects associated with systemically administered opioid analgesics. Here, we investigated the regulation of the κ-opioid receptor (KOR) on rat primary sensory neurons in vitro and in a rat model of thermal allodynia. Under basal conditions, application of the KOR agonist trans-(1S,2S)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide hydrochloride hydrate (U50488) did not inhibit adenylyl cyclase (AC) activity nor release of calcitonin gene-related peptide (CGRP) in vitro and did not inhibit thermal allodynia in vivo. However, after 15-min pretreatment with bradykinin (BK), U50488 became capable of inhibiting AC activity, CGRP release, and thermal allodynia. Inhibition of AC by 5-hydroxytryptamine 1 or neuropeptide Y(1) receptor agonists and stimulation of extracellular signal-regulated kinase activity by U50488 did not require BK pretreatment. The effect of U50488 in BK-primed tissue was blocked by the KOR antagonist nor-binaltorphimine both in vitro and in vivo. The effect of BK in vitro was blocked by either indomethacin or bisindolylmaleimide, suggesting that an arachidonic acid (AA) metabolite and protein kinase C (PKC) activation mediate BK-induced regulation of the KOR system. Furthermore, the effect of U50488 in BK-treated tissue was blocked by a soluble integrin-blocking peptide (GRGDSP), but not the inactive reverse sequence peptide (GDGRSP), suggesting that, in addition to AA and PKC, RGD-binding integrins participate in the regulation of KOR signaling in response to U50488. Understanding the mechanisms by which peripheral KOR agonist efficacy is regulated may lead to improved pharmacotherapy for the treatment of pain with reduced adverse effects.

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.

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.

Role of ionotropic cannabinoid receptors in peripheral antinociception and antihyperalgesia.

Despite the wealth of information on cannabinoid-induced peripheral antihyperalgesic and antinociceptive effects in many pain models, the molecular mechanism(s) for these actions remains unknown. Although metabotropic cannabinoid receptors have important roles in many pharmacological actions of cannabinoids, recent studies have led to the recognition of a family of at least five ionotropic cannabinoid receptors (ICRs). The known ICRs are members of the family of transient receptor potential (TRP) channels and include TRPV1, TRPV2, TRPV4, TRPM8 and TRPA1. Cannabinoid activation of ICRs can result in desensitization of the TRPA1 and TRPV1 channel activities, inhibition of nociceptors and antihyperalgesia and antinociception in certain pain models. Thus, cannabinoids activate both metabotropic and ionotropic mechanisms to produce peripheral analgesic effects. Here, we provide an overview of the pharmacology of TRP channels as ICRs.