Nociceptive Behavior Research Articles

Behavioral characterization of G-protein-coupled receptor 160 knockout mice.

Neuropathic pain is a devastating condition where current therapeutics offer little to no pain relief. Novel nonnarcotic therapeutic targets are needed to address this growing medical problem. Our work identified the G-protein-coupled receptor 160 (GPR160) as a potential target for therapeutic intervention. However, the lack of small-molecule ligands for GPR160 hampers our understanding of its role in health and disease. To address this void, we generated a global Gpr160 knockout (KO) mouse using CRISPR-Cas9 genome editing technology to validate the contributions of GPR160 in nociceptive behaviors in mice. Gpr160 KO mice are healthy and fertile, with no observable physical abnormalities. Gpr160 KO mice fail to develop behavioral hypersensitivities in a model of neuropathic pain caused by constriction of the sciatic nerve. On the other hand, responses of Gpr160 KO mice in the hot-plate and tail-flick assays are not affected. We recently deorphanized GPR160 and identified cocaine- and amphetamine-regulated transcript peptide (CARTp) as a potential ligand. Using Gpr160 KO mice, we now report that the development of behavioral hypersensitivities after intrathecal or intraplantar injections of CARTp are dependent on GPR160. Cocaine- and amphetamine-regulated transcript peptide plays a role in various affective behaviors, such as anxiety, depression, and cognition. There are no differences in learning, memory, and anxiety between Gpr160 KO mice and their age-matched and sex-matched control floxed mice. Results from these studies support the pronociceptive roles of CARTp/GPR160 and GPR160 as a potential therapeutic target for treatment of neuropathic pain.

Prolonged testosterone 17β-cyclopentylpropionate exposition induces behavioral, ovarian, oviductal, uterine and reproductive disturbances in female mice

Anabolic-androgenic steroids (AAS) abuse is often associated with metabolic disorders and infertility. However, the current evidence on AAS-induced reproductive toxicity is mainly based on male studies. Thus, AAS repercussions on female reproductive capacity remain poorly understood, despite scarce evidence that fertility determinants may be more severely impaired in females than males exposed to these drugs. Accordingly, this study used an integrated framework to investigate the impact of different testosterone 17β-cyclopentylpropionate (TC) doses on pain sensitivity, aggressiveness, anxiety, sexual behavior, ovarian, oviductal, uterine and reproductive morphofunctional and molecular outcomes. These parameters were used to explore the reproductive capacity in female mice exposed to this synthetic testosterone ester. The animals were untreated or intraperitoneally treated with 5, 10 and 20 mg/kg TC every 48 h for 12 weeks. Our findings indicated that testosterone was upregulated while the hormones luteinizing, follicle-stimulating, estrogen and progesterone were down-regulated by TC. This AAS also exerted deleterious effects on anxiety, aggressivity, nociception, exploratory and sexual behavior in female mice. Concurrently, TC attenuated ovarian follicle maturation, interrupted the estrous cycle, induced oviductal and uterine hypotrophy. Estrous cyclicity was reestablished 60 days after AAS treatment. However, TC-treated mice still exhibited impaired reproductive capacity, a disturbance potentially related to deficiency in folliculogenesis, sex hormones production, and endometrial receptivity mediate by ER-α, PR, HOXA-10 and LIF down-regulation. Taken together, our findings indicated that in addition to female behavior, reproductive organs microstructure and function are markedly impaired by TC in a dose-dependent manner, whose time-dependent reversibility remains to be clarified.

Cobalt-doped zinc oxide based memristors with nociceptor characteristics for bio-inspired technology.

Neuromorphic computing is a new field of information technology, which is inspired by the biomimetic properties of the memristor as an electronic synapse and neuron. If there are electronic receptors that can transmit exterior impulses to the internal nervous system, then the use of memristors can be expanded to artificial nerves. In this study, a layer type memristor is used to build an artificial nociceptor in a very feasible and straightforward manner. An artificial nociceptor is demonstrated here through the fabrication and characterization of a cobalt-doped zinc oxide (CZO)/Au based memristor. In order to increase threshold switching performance, the surface effects of the CZO layer are eliminated by adding cobalt cobalt-doped zinc oxide (CZO) layer between the P++-Si and Au electrodes. Allodynia, hyperalgesia, threshold, and relaxation are the four distinct nociceptive behaviours that the device displays based on the strength, rate of relapse, and duration of the external stimuli. The electrons that are trapped in or released from the CZO layer's traps are responsible for these nociceptive behaviours. A multipurpose nociceptor performance is produced by this type of CZO-based device, which is crucial for artificial intelligence system applications such as neural integrated devices with nanometer-sized characteristics.

Impact of streptozotocin-induced diabetes on experimental masseter pain in rats.

This study aimed to assess the influence of streptozotocin (STZ)-induced diabetes on the nociceptive behavior evoked by the injection of hypertonic saline (HS) into the masseter muscle of rats. Forty male rats were equally divided into four groups: a) isotonic saline control, which received 0.9% isotonic saline (IS), (Ctrl-IS); b) hypertonic saline control, which received 5% HS (Ctrl-HS); c) STZ-induced diabetic, which received IS, (STZ-IS); d) STZ-induced diabetic, which received HS (STZ-HS). Experimental diabetes was induced by a single intraperitoneal injection of STZ at dose of 60 mg/kg dissolved in 0.1 M citrate buffer, and 100 μL of HS or IS were injected into the left masseter to measure the nociceptive behavior. Later on, muscle RNA was extracted to measure the relative expression of the following cytokines: cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF-α), and interleukins (IL)-1β, -2, -6, and -10. One-way analysis of variance (ANOVA) was applied to the data (p < 0.050). We observed a main effect of group on the nociceptive response (ANOVA: F = 11.60, p < 0.001), where the Ctrl-HS group presented the highest response (p < 0.001). However, nociceptive response was similar among the Ctrl-IS, STZ-IS, and STZ-HS group (p > 0.050). In addition, the highest relative gene expression of TNF-α and IL-6 was found in the masseter of control rats following experimental muscle pain (p < 0.050). In conclusion, the loss of somatosensory function can be observed in deep orofacial tissues of STZ-induced diabetic rats.

Mitochondrial DNA drives neuroinflammation through the cGAS-IFN signaling pathway in the spinal cord of neuropathic pain mice.

Neuroinflammation is pivotal in the development of neuropathic pain (NeP). While mitochondrial deoxyribonucleic acid (mtDNA) and cyclic GMP-AMP synthase (cGAS) are recognized for inducing inflammation in various neurological disorders, their involvement in NeP remains ambiguous. In this study, we examined: (1) the changes in mtDNA and cGAS in mice with NeP induced by chronic constriction injury (CCI) of the sciatic nerve, whether mtDNA triggers inflammation via the cGAS signaling; (2) the effects of RU.521, a cGAS antagonist, on CCI-induced nociception (allodynia and hyperalgesia) and relative inflammatory protein expression; (3) the activation of microglia and the cGAS-IFN pathway mediated by mtDNA in BV2 cell; (4) the effect of RU.521 on mtDNA-induced inflammatory response in BV2 cells. Results revealed reduced mtDNA levels in the sciatic nerve but increased levels in the spinal cord of CCI mice, along with elevated cGAS expression and inflammatory factors. RU.521 alleviated nociceptive behaviors in CCI mice, possibly by normalizing cGAS levels and suppressing inflammation. Neuron-derived mtDNA provoked cellular activation and upregulated cGAS signaling in BV2 cells. Additionally, RU.521 and DNase I effectively inhibited cGAS-induced inflammation. These findings underscore the critical role of mtDNA accumulation and mtDNA-mediated cGAS signaling in NeP development after peripheral nerve injury.

Effect of curcumin on formalin-induced muscle pain in male rats: role of local cyclooxygenase system.

Investigating the mechanisms responsible for pain processing of natural and synthetic chemical compounds is necessary to optimize pain management. Curcumin (Cur), the active ingredient of turmeric, exhibits potent analgesic and anti-inflammatory properties by employing multiple mechanisms at the local peripheral, spinal and supra-spinal levels. This study was aimed to investigate the effect of oral administration of Cur on muscle pain induced by intramuscular (IM) injection of formalin. To explore the possible local mechanisms, a cyclooxygenase (COX) inhibitor, diclofenac (Dic) and a COX product, prostaglandin E2 (PGE2), were applied. The IM injection of formalin (25.00 µL, 2.50%) into the gastrocnemius muscle induced two distinct phases of hind leg flinching. A short-lasting (10 min) hind leg lifting was observed following IM injection of PGE2 (2 µg kg-1, 25.00 µL). Oral administration of Cur (25.00 and 100 mg kg-1) and IM injection of 40.00 µg kg-1 Dic attenuated formalin and PGE2 induced nociceptive behaviors. Contra-lateral IM injection of Dic did not change muscle pain induced by ipsilateral IM injection of formalin and PGE2. The second phase of formalin induced flinching as well as PGE2 evoked lifting were more suppressed when 40.00 µg kg-1 Dic and 100 mg kg-1 Cur were used together. Locomotor activity was not changed by the above-mentioned treatments. It was concluded that the reducing effect of muscle pain of Cur might be related to the local inhibition of COX.

Assessment of orofacial nociceptive behaviors of mice with the sheltering tube method: Oxaliplatin-induced mechanical and cold allodynia in orofacial regions.

Preclinical studies on pathological pain rely on the von Frey test to examine changes in mechanical thresholds and the acetone spray test to determine alterations in cold sensitivity in rodents. These tests are typically conducted on rodent hindpaws, where animals with pathological pain show reliable nocifensive responses to von Frey filaments and acetone drops applied to the hindpaws. Pathological pain in orofacial regions is also an important clinical problem and has been investigated with rodents. However, performing the von Frey and acetone spray tests in the orofacial region has been challenging, largely due to the high mobility of the head of testing animals. To solve this problem, we implemented a sheltering tube method to assess orofacial nociception in mice. In experiments, mice were sheltered in elevated tubes, where they were well accommodated because the tubes provided safe shelters for mice. Examiners could reliably apply mechanical stimuli with von Frey filament, cold stimuli with acetone spray, and light stimuli with a laser beam to the orofacial regions. We validated this method in Nav1.8-ChR2 mice treated with oxaliplatin that induced peripheral neuropathy. Using the von Frey test, orofacial response frequencies and nociceptive response scores were significantly increased in Nav1.8-ChR2 mice treated with oxaliplatin. In the acetone spray test, the duration of orofacial responses was significantly prolonged in oxaliplatin-treated mice. The response frequencies to laser light stimulation were significantly increased in Nav1.8-ChR2 mice treated with oxaliplatin. Our sheltering tube method allows us to reliably perform the von Frey, acetone spray, and optogenetic tests in orofacial regions to investigate orofacial pain.

Antinociceptive Effects of Cannabichromene (CBC) in Mice: Insights from von Frey, Tail-Flick, Formalin, and Acetone Tests.

Cannabis sativa contains minor cannabinoids that have potential therapeutic value in pain management. However, detailed experimental evidence for the antinociceptive effects of many of these minor cannabinoids remains lacking. Here, we employed artificial intelligence (AI) to perform compound-protein interaction estimates with cannabichromene (CBC) and receptors involved in nociceptive signaling. Based on our findings, we investigated the antinociceptive properties of CBC in naïve or neuropathic C57BL/6 male and female mice using von Frey (mechanical allodynia), tail-flick (noxious radiant heat), formalin (acute and persistent inflammatory pain), and acetone (cold thermal) tests. For von Frey assessments, CBC dose (0-20 mg/kg, i.p.) and time (0-6 h) responses were measured in male and female neuropathic mice. For tail-flick, formalin, and acetone assays, CBC (20 mg/kg, i.p.) was administered to naïve male and female mice 1 h prior to testing. The results show that CBC (10 and 20 mg/kg, i.p.) significantly reduced mechanical allodynia in neuropathic male and female mice 1-2 h after treatment. Additionally, CBC treatment caused significant reductions in nociceptive behaviors in the tail-flick assay and in both phase 1 and phase 2 of the formalin test. Finally, we found a significant interaction in neuropathic male mice in the acetone test. In conclusion, our results suggest that CBC targets receptors involved in nociceptive signaling and imparts antinociceptive properties that may benefit males and females afflicted with diverse forms of acute or chronic/persistent pain.

Nerve Growth Factor/Tyrosine Kinase A Receptor Pathway Enhances Analgesia in an Experimental Mouse Model of Bone Cancer Pain by Increasing Membrane Levels of δ-Opioid Receptors.

The role of nerve growth factor (NGF)/tyrosine kinase A receptor (TrKA) signaling, which is activated in a variety of pain states, in regulating membrane-associated δ-opioid receptor (mDOR) expression is poorly understood. The hypothesis was that elevated NGF in bone cancer tumors could upregulate mDOR expression in spinal cord neurons and that mDOR agonism might alleviate bone cancer pain. Bone cancer pain (BCP) was induced by inoculating Lewis lung carcinoma cells into the femoral marrow cavity of adult C57BL/6J mice of both sexes. Nociceptive behaviors were evaluated by the von Frey and Hargreaves tests. Protein expression in the spinal dorsal horn of animals was measured by biochemical analyses, and excitatory synaptic transmission was recorded in miniature excitatory synaptic currents. The authors found that mDOR expression was increased in BCP mice (BCP vs. sham, mean ± SD: 0.18 ± 0.01 g vs. mean ± SD: 0.13 ± 0.01 g, n = 4, P < 0.001) and that administration of the DOR agonist deltorphin 2 (Del2) increased nociceptive thresholds (Del2 vs. vehicle, median [25th, 75th percentiles]: 1.00 [0.60, 1.40] g vs. median [25th, 75th percentiles]: 0.40 [0.16, 0.45] g, n = 10, P = 0.001) and reduced miniature excitatory synaptic current frequency in lamina II outer neurons (Del2 vs. baseline, mean ± SD: 2.21 ± 0.81 Hz vs. mean ± SD: 2.43 ± 0.90 Hz, n = 12, P < 0.001). Additionally, NGF expression was increased in BCP mice (BCP vs. sham, mean ± SD: 0.36 ± 0.03 vs. mean ± SD: 0.16 ± 0.02, n = 4, P < 0.001), and elevated NGF was associated with enhanced mDOR expression via TrKA signaling. Activation of mDOR produces analgesia that is dependent on the upregulation of the NGF/TrKA pathway by increasing mDOR levels under conditions of BCP in mice.

Astrocyte PERK and IRE1 Signaling Contributes to Morphine Tolerance and Hyperalgesia through Upregulation of Lipocalin-2 and NLRP3 Inflammasome in the Rodent Spinal Cord.

Endoplasmic reticulum stress plays a crucial role in the pathogenesis of neuroinflammation and chronic pain. This study hypothesized that PRKR-like endoplasmic reticulum kinase (PERK) and inositol-requiring enzyme type 1 (IRE1) regulate lipocalin-2 (LCN2) and Nod-like receptor family pyrin domain containing 3 (NLRP3) expression in astrocytes, thereby contributing to morphine tolerance and hyperalgesia. The study was performed in Sprague-Dawley rats and C57/Bl6 mice of both sexes. The expression of LCN2 and NLRP3 was assessed by Western blotting. The tail-flick, von Frey, and Hargreaves tests were used to evaluate nociceptive behaviors. Chromatin immunoprecipitation was conducted to analyze the binding of activating transcription factor 4 (ATF4) to the promoters of LCN2 and TXNIP. Whole-cell patch-clamp recordings were used to evaluate neuronal excitability. Pharmacologic inhibition of PERK and IRE1 attenuated the development of morphine tolerance and hyperalgesia in male (tail latency on day 7, 8.0 ± 1.13 s in the morphine + GSK2656157 [10 μg] group vs. 5.8 ± 0.65 s in the morphine group; P = 0.04; n = 6 rats/group) and female (tail latency on day 7, 6.0 ± 0.84 s in the morphine + GSK2656157 [10 μg] group vs. 3.1 ± 1.09 s in the morphine group; P = 0.0005; n = 6 rats/group) rats. Activation of PERK and IRE1 upregulated expression of LCN2 and NLRP3 in vivo and in vitro. Chromatin immunoprecipitation analysis showed that ATF4 directly bound to the promoters of the LCN2 and TXNIP. Lipocalin-2 induced neuronal hyperexcitability in the spinal cord and dorsal root ganglia via melanocortin-4 receptor. Astrocyte endoplasmic reticulum stress sensors PERK and IRE1 facilitated morphine tolerance and hyperalgesia through upregulation of LCN2 and NLRP3 in the spinal cord.

Adolescent morphine exposure changes the endogenous vlPAG opioid response to inflammatory pain in rats.

Adolescence is one of the most critical periods for brain development, and exposure to morphine during this period can have long-life effects on pain-related behaviors. The opioid system in the periaqueductal gray (PAG) is highly vulnerable to drug exposure. However, the impact of adolescent morphine exposure (AME) on the endogenous opioid system in the PAG is currently unknown. This study aims to investigate the long-lasting effects of AME on the endogenous opioid system and its involvement in altering nociceptive behaviors. Adolescent rats were given escalating doses of morphine (2.5-25mg/kg, subcutaneous) or an equal volume of saline twice daily for 10 consecutive days (PND 31-40). After a 30-day washout period, adult rats underwent formalin tests following microinjection of morphine, naloxone, or saline into the ventrolateral PAG (vlPAG) region. The results indicated that morphine microinjection into the vlPAG of the adolescent morphine-treated group significantly reduced the nociceptive score. However, the analgesic response to morphine in this group was significantly lower compared to the saline-treated group during adolescence. Additionally, the nociceptive score significantly increased following naloxone but not saline microinjection into the vlPAG of the saline-treated group during adolescence, rather than the morphine-treated one. These findings indicate that AME has long-lasting effects on the endogenous opioid system in the vlPAG, which can consequently alter behaviors related to inflammatory pain in adulthood.

Oral treatment with Berberine reduces peripheral nociception: Possible interaction with different nociceptive pathways activated by different allogeneic substances

Ethnopharmacological relevanceBerberine was identified in extracts of Berberis ruscifolia Lam., a plant used in traditional medicine as an analgesic. Its presence may be involved in the reported pharmacological activity of this species. However, there is still a lack of scientific research concerning its analgesic activity in the peripheral nervous system. Aim of the studyTo investigate Berb-induced antinociception in the formalin test and to evaluate several pathways related to its pharmacological antinociceptive effects in chemical models of nociception in mice. Materials and methodsThe antinociceptive activity of Berb was assessed by inducing the paw licking in mice with different allodynic agents. In the formalin test, the antiedematous and antithermal effect of Berb was evaluated simultaneously in the same experiment. Other nociceptive behavior produced by endogenous [prostaglandin E2 (PGE2), histamine (His), glutamate (Glu) or bradykinin (BK)] or exogenous [capsaicin (Caps) and cinnamaldehyde (Cin)] chemical stimuli, and activators as protein kinase A (PKA) and C (PKC), were also evaluated.The in vivo doses for p.o. were 3 and 30 mg/kg. ResultsBerb, at 30 mg/kg p.o., showed a significant inhibition of the nociceptive action in formalin in both phases being stronger at the inflammatory phase (59 ± 9%) and more active than Asp (positive control) considering the doses evaluated. Moreover, Berb inhibited the edema (34 ± 10%), but not the temperature in the formalin test. Regarding the different nociceptive signaling pathways evaluated, the most relevant data were that the administration of p.o. of Berb, at 30 mg/kg, caused significant inhibition of nociception induced by endogenous [His (72 ± 11%), PGE2 (78 ± 4%), and BK (51 ± 7%)], exogenous [Cap (68 ± 4%) and Cinn (57 ± 5%)] compounds, and activators of the PKA [(FSK (86 ± 3%)] and PKC [(PMA(86 ± 6%)] signaling pathway. Berb did not inhibit the nociceptive effect produced by Glu. ConclusionThe present study demonstrated, for the first time, the potential of Berb in several nociceptive tests, with the compound present in B. ruscifolia contributing to the analgesic effect reported for this species.

An automaton for preclinical pain testing

In this issue of Cell Reports Methods, Dedek etal. present RAMalgo-an AI-powered, automated platform for quantifying nociceptive behaviors in mice. With integrated video tracking and mechanical, thermal, and optogenetic stimulation, RAMalgo has the potential to increase standardization and throughput of pain behavior measurement in rodents.

Spinal HDAC6 mediates nociceptive behaviors induced by chronic constriction injury via neuronal activation and neuroinflammation.

Neuropathic pain (NP) is often accompanied by psychiatric comorbidities and currently lacks effective treatment. Prior research has shown that HDAC6 plays a crucial role in pain sensitization, but the specific mechanisms remain unclear. HDAC6 inhibitors have been found to alleviate mechanical allodynia caused by inflammation and peripheral nerve damage. In this study, we investigated the cellular mechanisms of HDAC6 in the development and maintenance of neuropathic pain. Our findings indicate that HDAC6 expression in the spinal cord (SC) is upregulated in a time-dependent manner following chronic constriction injury (CCI). HDAC6 is primarily expressed in neurons and microglia in the spinal cord. CCI-induced HDAC6 production was abolished by intrathecal injection of a microglia inhibitor. ACY-1215, a specific HDAC6 inhibitor, significantly reduced CCI-induced mechanical allodynia, but not thermal hyperalgesia. ACY-1215 also inhibited neuron activation and suppressed CCI-induced pyroptosis and neuroinflammatory responses. In summary, our results suggest that HDAC6 contributes to the development and maintenance of NP through neuronal activation and neuroinflammation. HDAC6 may be a promising target for treating NP.

Orexin-1 Receptor Antagonist SB-334867 Enhances Formalin-Induced Nociceptive Behaviors in Adult Male Rats.

Orexin (hypocretin) is one of the hypothalamic neuropeptides that plays a critical role in some behaviors including feeding, sleep, arousal, reward processing, and drug addiction. Neurons that produce orexin are scattered mediolaterally within the Dorsomedial Hypothalamus (DMH) and the lateral hypothalamus. In the current research, we assessed the impact of prolonged application of the antagonist of Orexin Receptor 1 (OXR1) on nociceptive behaviors in adult male rats. Sixteen Wistar rats received subcutaneous (s.c.) injections of the OXR1 antagonist, SB-334867 (20 mg/kg, i.p.), or its vehicle repetitively from Postnatal Day 1 (PND1)-PND30. On the 30th day following the final application of the OXR1 antagonist formalin-provoked pain was evaluated by injecting formalin. Administration of the OXR1 antagonist in the long-term augmented the formalin-provoked nociceptive behaviors in interphase and phase II of the formalin-induced pain. Current results showed that the continued inhibiting OXR1 might be implicated in formalin-induced nociceptive behaviors. Therefore, the present study highlighted the effect of orexin on analgesia.

Myeloid Cell Association with Spinal Cord Injury-Induced Neuropathic Pain and Depressive-like Behaviors in LysM-eGFP Mice

Spinal cord injury (SCI) affects ∼500,000 people worldwide annually, with the majority developing chronic neuropathic pain. Following SCI, approximately 60% of these individuals are diagnosed with comorbid mood disorders, while only ∼21% of the general population will experience a mood disorder in their lifetime. We hypothesize that nociceptive and depressive-like dysregulation occurs after SCI and is associated with aberrant macrophage infiltration in segmental pain centers. We completed moderate unilateral C5 spinal cord contusion on LysM-eGFP reporter mice to visualize infiltrating macrophages. At 6-weeks post-SCI, mice exhibit nociceptive and depressive-like dysfunction compared to naïve and sham groups. There were no differences between the sexes, indicating that sex is not a contributing factor driving nociceptive or depressive-like behaviors after SCI. Utilizing hierarchical cluster analysis, we classified mice based on endpoint nociceptive and depressive-like behavior scores. Approximately 59.3% of the SCI mice clustered based on increased paw withdrawal threshold to mechanical stimuli and immobility time in the forced swim test. SCI mice displayed increased myeloid cell presence in the lesion epicenter, ipsilateral C7-8 dorsal horn, and C7-8 DRGs as evidenced by eGFP, CD68, and Iba1 immunostaining when compared to naïve and sham mice. This was further confirmed by SCI-induced alterations in the expression of genes indicative of myeloid cell activation states and their associated secretome in the dorsal horn and dorsal root ganglia. In conclusion, moderate unilateral cervical SCI caused the development of pain-related and depressive-like behaviors in a subset of mice and these behavioral changes are consistent with immune system activation in the segmental pain pathway. PerspectiveThese experiments characterized pain-related and depressive-like behaviors and correlated these changes with the immune response post-SCI. While humanizing the rodent is impossible, the results from this study inform clinical literature to closely examine sex differences reported in humans to better understand the underlying shared etiologies of pain and depressive-like behaviors following central nervous system trauma.

Lipid mediator resolvin D2 inhibits ATP currents in rat primary sensory neurons.

Resolvin D2 (RvD2), an endogenous lipid mediator derived from docosahexaenoic acid, has been demonstrated to have analgesic effects. However, little is known about the mechanism underlying RvD2 in pain relief. Herein, we demonstrate that RvD2 targeted the P2X3 receptor as an analgesic. The electrophysiological activity of P2X3 receptors was suppressed by RvD2 in rat dorsal root ganglia (DRG) neurons. RvD2 pre-application dose-dependently decreased α,β-methylene-ATP (α,β-meATP)-induced inward currents. RvD2 remarkably decreased the maximum response to α,β-meATP, without influencing the affinity of P2X3 receptors. RvD2 also voltage-independently suppressed ATP currents. An antagonist of the G protein receptor 18 (GPR18), O-1918, prevented the RvD2-induced suppression of ATP currents. Additionally, intracellular dialysis of the Gαi/o-protein antagonist pertussis toxin (PTX), the PKA antagonist H89, or the cAMP analog 8-Br-cAMP also blocked the RvD2-induced suppression. Furthermore, α,β-meATP-triggered depolarization of membrane potential along with the action potential bursts in DRG neurons were inhibited by RvD2. Lastly, RvD2 attenuated spontaneous nociceptive behaviors as well as mechanical allodynia produced by α,β-meATP in rats via the activation of the peripheral GPR18. These findings indicated that RvD2 inhibited P2X3 receptors in rat primary sensory neurons through GPR18, PTX-sensitive Gαi/o-proteins, and intracellular cAMP/PKA signaling, revealing a novel mechanism that underlies its analgesic effects by targeting P2X3 receptors.

Cuminum cyminum Ameliorates Urotoxic Effects of Cyclophosphamide by Modulating Antioxidant, Inflammatory Cytokines, and Urinary Bladder Overactivity: In vivo and in Silico Investigations.

Interstitial Cystitis (IC) is a chronic inflammatory disease that lacks effective treatment. The present study aimed to investigate the potential of aqueous ethanol extract of Cuminum cyminum (AEECC) on oxidative stress, inflammation and overactivity of urinary bladder induced by cyclophosphamide (CYP). Female Sprague-Dawley rats received intraperitoneal administration of cyclophosphamide (150 mg/kg, i. p. 1st , 4th , and 7th days). To investigate the urothelial damage, the bladder weight, nociception behavior, and Evans blue dye extravasation method was used. The antioxidants CAT, GPX and NO were measured. ELISA determined the IL-6 and TNF-α levels. The spasmolytic effect of AEECC was investigated on isolated bladder strips and its mechanisms were determined. The enhanced nociception behavior, bladder weight, vascular permeability, edema, hemorrhage, nitric oxide, IL-6 and TNF-α levels by CYP administration were significantly reduced by AEECC (250 and 500 mg/kg). A significant increase in serum antioxidant system such as CAT and GPx was also observed in AEECC-treated rats. The AEECC (3 mg/ml) significantly reduced urinary bladder tone in the strips pre-contracted with carbachol in both control and CYP-treated rats. This relaxation was demolished by atropine, nifedipine, glibenclamide, and indomethacin but not with propranolol. The plant extract showed the presence of antioxidant and anti-inflammatory phytochemicals. These results suggest that Cuminum cyminum offers uroprotective activity and can ameliorate CYP-induced bladder toxicity by modulating antioxidant parameters, pro-inflammatory cytokine levels and bladder smooth muscle overactivity. The in silico binding interactions of antioxidant 2I3Y and anti-inflammatory protein 1TNF with various ligands from Cuminum cyminum seeds revealed potential bioactive compounds with promising antioxidant and anti-inflammatory properties, providing valuable insights for drug development and nutraceutical research.

Epigenetic regulation of beta-endorphin synthesis in hypothalamic arcuate nucleus neurons modulates neuropathic pain in a rodent pain model

Although beta-endorphinergic neurons in the hypothalamic arcuate nucleus (ARC) synthesize beta-endorphin (β-EP) to alleviate nociceptive behaviors, the underlying regulatory mechanisms remain unknown. Here, we elucidated an epigenetic pathway driven by microRNA regulation of β-EP synthesis in ARC neurons to control neuropathic pain. In pain-injured rats miR-203a-3p was the most highly upregulated miRNA in the ARC. A similar increase was identified in the cerebrospinal fluid of trigeminal neuralgia patients. Mechanistically, we found histone deacetylase 9 was downregulated following nerve injury, which decreased deacetylation of histone H3 lysine-18, facilitating the binding of NR4A2 transcription factor to the miR-203a-3p gene promoter, thereby upregulating miR-203a-3p expression. Further, increased miR-203a-3p was found to maintain neuropathic pain by targeting proprotein convertase 1, an endopeptidase necessary for the cleavage of proopiomelanocortin, the precursor of β-EP. The identified mechanism may provide an avenue for the development of new therapeutic targets for neuropathic pain treatment.

Cleomin Exerts Acute Antinociceptive Effects in Mice via GABAB and Muscarinic Receptors.

Cleomin, a 1,3-oxazolidine-2-thione, was recently isolated from Neocalyptrocalyx longifolium, a species traditionally used for treating painful conditions. Reports about the pharmacological activities of cleomin are lacking. Here, the antinociceptive effects of cleomin were investigated using mice models of pain, namely the formalin, the cold plate, and the tail flick tests. Motor integrity was assessed in the rota-rod test. Antagonism assays and in silico docking analyses were performed to investigate the putative mechanisms of action. Cleomin (12.5-25 mg/kg), at doses that did not induce motor impairment, induced dose-dependent antinociception in both early and late phases of the formalin test and reduced nociceptive behaviors in both the cold plate and tail flick tests. Pretreatments with phaclofen and atropine attenuated the antinociceptive effects of cleomin, implicating the involvement of GABAB and muscarinic receptors. In silico docking studies suggested satisfactory coupling between cleomin and GABAB and M2 receptors, hence corroborating their role in cleomin's activity. Pretreatments with naloxone, yohimbine, bicuculline, and methysergide did not affect the antinociception of cleomin. In silico pharmacokinetics prediction showed a good drug ability profile of cleomin. In conclusion, cleomin promoted antinociception mediated by GABAB and muscarinic receptors. These findings support further investigation of the analgesic potential of cleomin.