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A new, simple, and selective colorimetric method of determining formaldehyde in Hevea brasiliensis latex was developed by using a casein/ferric chloride/polyvinyl alcohol hydrogel composite (casein/FeCl3/PVA) in a modified Leach test. Under heating, formaldehyde reacted with 8% casein in the presence of 0.1% FeCl3 and 4.3% HCl (v/v) entrapped in a 30% PVA hydrogel packed in a syringe. A purple-colored product was formed with a maximum absorbance of 525 nm. The color change was evaluated at the color detection zone indicated on the the syringe. The %magenta values were easily evaluated by using a mobile phone application and employed to determine formaldehyde content. The casein/FeCl3/PVA composite gave a readable response in a formaldehyde detection range from 0.04 to 0.80% with a linear response between %magenta and formaldehyde concentration (R2 = 0.9955). The detection limit was 0.032%, and precisions were in the range 0.67-4.94%. The casein/FeCl3/PVA composite was applied to the analysis of ammonia-preserved latex samples, and recoveries of formaldehyde from samples spiked at 0.1, 0.3, and 0.5% ranged from 81.55 to 99.51% (RSDs ≤ 5.41%). The recoveries and precision of the proposed method were comparable with those of high-performance liquid chromatography (HPLC). The developed method was also selective, showing no interference from other latex preservatives, i.e., phenol, ammonia, or tetramethylthiuram disulfide.

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Stress substantially increases the risk of developing painful temporomandibular disorders (TMDs) by influencing the release of endogenous catecholamines. Propranolol, an antagonist of ß-adrenergic receptors, has shown potential in alleviating TMD-associated pain, particularly when the level of catecholamines is elevated. The aim of this study was to explore whether intra-articular propranolol administration is effective in diminishing temporomandibular joint (TMJ) pain during repeated stress situations. Additionally, we investigated the effect of repeated stress on the expression of genes encoding ß-adrenoceptors in the trigeminal ganglion. In the present study, rats were exposed to a stress protocol induced by sound, then to the administration of formalin in the TMJ (to elicit a nociceptive response), followed immediately afterward by different doses of propranolol, after which the analgesic response to propranolol was evaluated. We also assessed the levels of beta-1 and beta-2 adrenergic receptor mRNAs (Adrb1 and Adrb2, respectively) using reverse transcription-quantitative PCR (RT-qPCR). Our findings revealed that propranolol administration reduces formalin-induced TMJ nociception more effectively in stressed rats than in non-stressed rats. Furthermore, repeated stress decreases the expression of the Adrb2 gene within the trigeminal ganglion. The findings of this study are noteworthy as they suggest that individuals with a chronic stress history might find potential benefits from ß-blockers in TMD treatment.

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Descending control of nociception (DCN), a measure of efficiency of descending pain inhibition, can be assessed in animals by the combined application of test and conditioning noxious stimuli. Evidence from pre-clinical and clinical studies indicates that this mechanism of pain control may differ between sexes and might be impaired in many chronic pain states. However, little is known about sex differences in DCN efficiency in models of acute and chronic orofacial pain. Herein, we first evaluated DCN responses in male and female rats by the applying formalin into the upper lip or capsaicin into the forepaw as the conditioning stimulus, followed by mechanical stimulation (Randall-Selitto) of the hind paw as the test stimulus. The same protocol (i.e., capsaicin in the forepaw followed by mechanical stimulation of the hind paw) was evaluated in male and female rats on day 3 after intraoral incision and on day 15 and 30 after chronic constriction injury of the infraorbital nerve (CCI-ION). Additionally, we assessed the effect of the kappa opioid receptor (KOR) antagonist Norbinaltorphimine (nor-BNI) on DCN responses of female nerve-injured rats. This study shows that naïve female rats exhibit less efficient DCN compared to males. Postoperative pain did not alter DCN responses in female and male rats, but CCI-ION induced loss of DCN responses in females but not in males. Systemic pretreatment with nor-BNI prevented the loss of DCN induced by CCI-ION in female rats. The results reveal sex differences in DCN responses and female-specific impairment of DCN following chronic orofacial pain. Moreover, the findings suggest that, at least for females, blocking KOR could be a promising therapeutic approach to prevent maladaptive changes in chronic orofacial pain.

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Pain is a common symptom associated with disorders involving the orofacial structures. Most acute orofacial painful conditions are easily recognized, but the pharmacological treatment may be limited by the adverse events of current available drugs and/or patients' characteristics. In addition, chronic orofacial pain conditions represent clinical challenges both, in terms of diagnostic and treatment. There is growing evidence that specialized pro-resolution lipid mediators (SPMs) present potent analgesic effects, in addition to their well characterized role in the resolution of inflammation. Maresins (MaR-1 and MaR-2) were the last described members of this family, and MaR-2 analgesic action has not yet been reported. Herein the effect of MaR-2 in different orofacial pain models was investigated. MaR-2 (1 or 10 ng) was always delivered via medullary subarachnoid injection, which corresponds to the intrathecal treatment. A single injection of MaR-2 caused a significant reduction of phases I and II of the orofacial formalin test in rats. Repeated injections of MaR-2 prevented the development of facial heat and mechanical hyperalgesia in a model of post-operative pain in rats. In a model of trigeminal neuropathic pain (CCI-ION), repeated MaR-2 injections reversed facial heat and mechanical hyperalgesia in rats and mice. CCI-ION increased c-Fos positive neurons and CGRP+ activated (nuclear pNFkB) neurons in the trigeminal ganglion (TG), which were restored to sham levels by MaR-2 repeated treatment. In conclusion, MaR-2 showed potent and long-lasting analgesic effects in inflammatory and neuropathic pain of orofacial origin and the inhibition of CGRP-positive neurons in the TG may account for MaR-2 action.

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Orofacial pain has significant psychological and physiological effects. Citral (3,7-dimethyl-2,6-octadienal) is the main component of Cymbopogon citratus (DC) Stapf, an herb with analgesic properties. Although citral has been considered a potent analgesic, its putative effects on orofacial pain are still unknown. OBJECTIVE: The objective of this study is to test the hypothesis that citral modulates orofacial pain using two experimental models: formalin-induced hyperalgesia in the vibrissae area and during persistent temporomandibular hypernociception using Complete Freund's Adjuvant - CFA test. METHODS: For the formalin test, citral (100 and 300 mg/kg, oral gavage) or its vehicle (Tween 80, 1 %) were given 1 h before the formalin injection subcutaneously (sc) into the vibrissae area. For the CFA model, we analyzed the prophylactic (100 mg/kg of citral by oral gavage, 1 h before CFA injection) and the chronic therapeutic (citral treatment 1-hour post-CFA injection and daily post-CFA injection) effect of citral or its vehicle in animals treated with CFA for 8 days. RESULTS: Citral caused a decrease in formalin-induced local inflammation and the time spent performing nociceptive behavior in a dose-dependent fashion. Similarly, prophylactic and therapeutic citral treatment decreased the CFA-induced persistent mechanical hypernociception in the temporomandibular area. CONCLUSION: Our data strengthen the notion that citral plays a powerful antinociceptive role by decreasing orofacial hypernociception in formalin and CFA models.

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OBJECTIVE: Evidence supports the local application of non-steroidal antiinflammatory drugs such as dexketoprofen trometamol (DXT) for pain management, but little is known about the potential antinociceptive effect of chlorhexidine gluconate (CHX) and its possible synergistic effect when combined with DXT. The aim of this study was to evaluate the local effect of a DXT-CHX combination using isobolographic analysis in a formalin pain model in rats. MATERIALS AND METHODS: Briefly, 60 female Wistar rats were used for the formalin test. Individual dose effect curves were obtained using linear regression. For each drug, the percentage of antinociception and median effective dose (ED50; 50% of antinociception) were calculated, and drug combinations were prepared using the ED50s for DXT (phase 2) and CHX (phase 1). The ED50 of the DXT-CHX combination was determined, and an isobolographic analysis was performed for both phases. RESULTS: The ED50 of local DXT was 5.3867 mg/mL in phase 2 and for CHX was 3.9233 mg/mL in phase 1. When the combination was evaluated, phase 1 showed an interaction index (II) of less than 1, indicating synergism but without statistical significance. For phase 2, the II was 0.3112, with a reduction of 68.88% in the amounts of both drugs to obtain the ED50; this interaction was statistically significant (P < .05). CONCLUSION: DXT and CHX had a local antinociceptive effect and exhibited synergistic behavior when combined in phase 2 of the formalin model.

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The use of immunohistochemical techniques to study the patterns of protein phosphorylation has revolutionized the study of signaling pathways. This technique allows detecting the phosphorylated state of signaling proteins in formalin-fixed and paraffin-embedded tissue sections by using phosphospecific antibodies. This chapter describes in detail the immunohistocshemical protocols from which the study of phosphoproteins in tissue sections can be approached.

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Resolvin D5 (RvD5) is a specialized pro-resolving lipid mediator with potent anti-inflammatory and analgesic properties. Orofacial pain conditions, especially those that are chronic, present clinical challenges in terms of pharmacological management. Thus, new therapeutic options are clearly warranted. Herein, we investigated the antinociceptive effect of RvD5 in the chronic constriction injury of the infraorbital nerve (CCI-ION) model and in the orofacial formalin test in female and male Wistar rats. Our results indicated that repeated subarachnoid medullary injections of RvD5 at 10 ng resulted in a significant reduction of heat and mechanical hyperalgesia induced by the CCI-ION in male and female rats, but males were more sensitive to RvD5 effects. In addition, after CCI-ION, interleukin-6 (IL-6) level was increased in the trigeminal nucleus caudalis of male, but not female rats, which was reduced by RvD5 repeated treatment. No changes in the levels of IL-1ß were found. Minocycline blocked the effect of RvD5 in male rats but failed to affect RvD5 antinociceptive effect in females. Moreover, a single medullary injection of RvD5 caused a significant reduction of formalin-induced facial grooming, in phases I and II of the test, but only in male rats. This study demonstrated for the first time the analgesic effect of RvD5 in trigeminal pain models, and corroborated previous evidence of sex dichotomy, with a greater effect in males. This article presents a translational potential of RvD5 for targeted therapies aiming at the control of acute and chronic trigeminal pain, but further studies are needed to elucidate its sex-related mechanisms. PERSPECTIVE: This study demonstrated that RvD5 may provide the benefits for trigeminal neuropathic pain treatment in male and female rats, but its effect on inflammatory orofacial pain seems to be restricted only to males. Also, it provided the evidence for sex dichotomy in the mechanisms related to the antinociceptive effect of RvD5.

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BACKGROUND: Hindpaw injection of formalin in rodents is used to assess acute persistent pain. The response to formalin is biphasic. The initial response (first minutes) is thought to be linked to inflammatory, peripheral mechanisms, while the latter (around 30 min after the injection), is linked to central mechanisms. This model is useful to analyze the effect of drugs at one or both phases, and the involvement of ion channels in the response. Acid-sensing ion channels (ASICs) regulate synaptic activities and play important roles in pain conditions. Recently, psalmotoxin-1 (Pctx-1), a toxin that inhibits ASIC1a-constituted channels, and antisense ASIC1a-RNA, intrathecal administered in mice were shown to affect both phases of the test. METHODS: The mouse formalin test was performed on C57/BL6 7- to 9-week-old mice. Behavioral tests were conducted and tissue was extracted to detect proteins (ASIC1 and pERK) and ASIC1-mRNA and mir485-5p levels. RESULTS: The injection of formalin was accompanied by an increase in ASIC1 levels. This was detected at the contralateral anterior cingulate cortex (ACC) compared to the ipsilateral side, and both sides of the ACC of vehicle-injected animals. At the spinal cord and dorsal root ganglia, ASIC1 levels followed a gradient stronger at lumbar (L) 3 and decreased towards L5. Gender differences were detected at the ACC; with female mice showing higher ASIC1a levels at the ACC. No significant changes in ASIC1-mRNA levels were detected. Evidence suggests ASIC1 upregulation depends on regulatory microRNAs. CONCLUSION: This work highlights the important role of ASIC1 in pain and the potential role of pharmacological therapies aimed at this channel.

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Objective: The molecular pathways underlying hypoxemia-induced alterations in neurodevelopment of infants with congenital heart disease have not been delineated. We used transcriptome analysis to investigate differential gene expression induced by hypoxemia in an ovine artificial-womb model. Methods: Mid-gestation fetal sheep (median [interquartile range] 109 [107-112] days' gestation) were cannulated via the umbilical vessels, attached to a pumpless, low-resistance oxygenator circuit, and incubated in a sterile, fluid environment for 22 [21-23] days. Fetuses were maintained with an oxygen delivery of 20-25 mL/kg/min (normoxemia, n = 3) or 14-16 mL/kg/min (hypoxemia, n = 4). Transcriptional profiling by RNA sequencing was carried out on left frontal brains and hypoxemia-regulated genes were identified by differential gene expression analysis. Results: A total of 228 genes whose expression was up or down regulated by ≥1.5-fold (false discovery rate ≤0.05) were identified. The majority of these genes were induced in hypoxemic animals compared to normoxemic controls, and functional enrichment analysis identified respiratory electron transport as a pathway strongly upregulated in the brain during chronic hypoxemia. Further examination of hypoxemia-induced genes showed robust induction of all 7 subunits of the mitochondrial NADH:ubiquinone oxidoreductase (complex I). Other hypoxemia-induced genes included cytochrome B, a component of complex III, and ATP6, ATP8, both of which are components of complex V. Conclusions: Chronic fetal hypoxemia leads to upregulation of multiple mitochondrial respiratory complex genes critical for energy production and reactive oxygen species generation, including complex I. These data provide valuable insight into potential pathways involved in chronic hypoxemia-induced neuropathology and offers potential therapeutic targets for fetal neuroprotection in fetuses with congenital heart defects.