Photocatalytic Activation of Aryl(trifluoromethyl) Diazos to Carbenes for High-Resolution Protein Labeling with Red Light.

State-of-the-art methods in photoproximity labeling center on the targeted generation and capture of short-lived reactive intermediates to provide a snapshot of local protein environments. Diazirines are the current gold standard for high-resolution proximity labeling, generating short-lived aryl(trifluoromethyl) carbenes. Here, we present a method to access aryl(trifluoromethyl) carbenes from a stable diazo source via tissue-penetrable, deep red to near-infrared light (600-800 nm). The operative mechanism of this activation involves Dexter energy transfer from photoexcited osmium(II) photocatalysts to the diazo, thus revealing an aryl(trifluoromethyl) carbene. The labeling preferences of the diazo probe with amino acids are studied, showing high reactivity toward heteroatom-H bonds. Upon the synthesis of a biotinylated diazo probe, labeling studies are conducted on native proteins as well as proteins conjugated to the Os photocatalyst. Finally, we demonstrate that the conjugation of a protein inhibitor to the photocatalyst also enables selective protein labeling in the presence of spectator proteins and achieves specific labeling of a membrane protein on the surface of mammalian cells via a two-antibody photocatalytic system.

In Vitro and In Vivo Pharmacological Characterization of a Novel TRPM8 Inhibitor Chemotype Identified by Small-Scale Preclinical Screening.

Transient receptor potential melastatin type 8 (TRPM8) is a target for the treatment of different physio-pathological processes. While TRPM8 antagonists are reported as potential drugs for pain, cancer, and inflammation, to date only a limited number of chemotypes have been investigated and thus a limited number of compounds have reached clinical trials. Hence there is high value in searching for new TRPM8 antagonistic to broaden clues to structure-activity relationships, improve pharmacological properties and explore underlying molecular mechanisms. To address this, the EDASA Scientific in-house molecular library has been screened in silico, leading to identifying twenty-one potentially antagonist compounds of TRPM8. Calcium fluorometric assays were used to validate the in-silico hypothesis and assess compound selectivity. Four compounds were identified as selective TRPM8 antagonists, of which two were dual-acting TRPM8/TRPV1 modulators. The most potent TRPM8 antagonists (BB 0322703 and BB 0322720) underwent molecular modelling studies to highlight key structural features responsible for drug-protein interaction. The two compounds were also investigated by patch-clamp assays, confirming low micromolar potencies. The most potent compound (BB 0322703, IC50 1.25 ± 0.26 µM) was then profiled in vivo in a cold allodinya model, showing pharmacological efficacy at 30 µM dose. The new chemotypes identified showed remarkable pharmacological properties paving the way to further investigations for drug discovery and pharmacological purposes.

Targeted activation in localized protein environments via deep red photoredox catalysis.

State-of-the-art photoactivation strategies in chemical biology provide spatiotemporal control and visualization of biological processes. However, using high-energy light (λ < 500 nm) for substrate or photocatalyst sensitization can lead to background activation of photoactive small-molecule probes and reduce its efficacy in complex biological environments. Here we describe the development of targeted aryl azide activation via deep red-light (λ = 660 nm) photoredox catalysis and its use in photocatalysed proximity labelling. We demonstrate that aryl azides are converted to triplet nitrenes via a redox-centric mechanism and show that its spatially localized formation requires both red light and a photocatalyst-targeting modality. This technology was applied in different colon cancer cell systems for targeted protein environment labelling of epithelial cell adhesion molecule (EpCAM). We identified a small subset of proteins with previously known and unknown association to EpCAM, including CDH3, a clinically relevant protein that shares high tumour-selective expression with EpCAM.

Contribution of CD4+ cells in the emotional alterations induced by endometriosis in mice.

Endometriosis is a disease defined by the presence of endometrial tissue in extrauterine locations. This chronic condition is frequently associated with pain and emotional disorders and has been related with altered immune function. However, the specific involvement of immune cells in pain and behavioral symptoms of endometriosis has not been yet elucidated. Here, we implement a mouse model of non-surgical endometriosis in which immunocompetent mice develop abdomino-pelvic hypersensitivity, cognitive deficits, anxiety and depressive-like behaviors. This behavioral phenotype correlates with expression of inflammatory markers in the brain, including the immune cell marker CD4. Depletion of CD4 + cells decreases the anxiety-like behavior of mice subjected to the endometriosis model, whereas abdomino-pelvic hypersensitivity, depressive-like behavior and cognitive deficits remain unaltered. The present data reveal the involvement of the immune response characterized by CD4 + white blood cells in the anxiety-like behavior induced by endometriosis in mice. This model, which recapitulates the symptoms of human endometriosis, may be a useful tool to study the immune mechanisms involved in pain and behavioral alterations associated to endometriosis.

Operant Self-medication for Assessment of Spontaneous Pain Relief and Drug Abuse Liability in Mouse Models of Chronic Pain.

The search for safe and efficient chronic pain treatments is dampened by the lack of reliable models that faithfully reproduce current pharmacological treatments for chronic spontaneous pain in humans. Preclinical models often assess the antinociceptive efficacy of non-contingent pharmacological treatments evaluated in the short-term. Here, we provide a protocol of contingent operant self-medication in mice, which allows the estimation of spontaneous pain relief and drug abuse liability in models of persistent pain. This paradigm requires preliminary habituation and animal handling, followed by training of mice in operant conditioning boxes, to allow subsequent analgesic drug self-administration. After the initial acquisition of food-maintained operant behavior, a chronic pain sensitization is induced. Posterior intravenous jugular catheterization and coupling of operant conditioning boxes to perfusion pumps allow quantification of operant responding for intravenous drug self-administration. All mice show an initial operant drug self-administration behavior associated with the previous food-maintained operant training. This initial operant responding is extinguished after administration of ineffective treatments, but continues when the compounds have analgesic efficacy or intrinsic reinforcing properties. The identification of a significant drug self-administration selectively expressed in mice exposed to the chronic pain condition is indicative of analgesic drug effects, whereas persistent self-administration in control mice is indicative of abuse liability. The present protocol provides the behavioral and surgical procedures needed to assess spontaneous pain relief and potential for abuse of pharmacological treatments, through contingent analgesic self-medication in mice. Graphic abstract: Experimental design. Animals are subjected to a 5-day food self-administration protocol with a fixed ratio of reinforcement of 1 (FR1, 1 interaction with the active nose-poke causes the release of 1 reinforcer/infusion), to acquire the operant behavior. After this training, mice are subjected to the chronic pain or sham procedure, and four days later an intravenous (i.v.) catheterization is performed, to allow self-administration with the selected compound or its vehicle. Three days after the catheterization, animals start the drug/vehicle self-administration protocol at FR1. The patency of the catheter is evaluated with the thiopental test after the last self-administration session. Adapted from Bura et al. (2018).

ThermoTRP channels in pain sexual dimorphism: new insights for drug intervention.

Chronic pain is a major burden for the society and remains more prevalent and severe in females. The presence of chronic pain is linked to persistent alterations in the peripheral and the central nervous system. One of the main types of peripheral pain transducers are the transient receptor potential channels (TRP), also known as thermoTRP channels, which intervene in the perception of hot and cold external stimuli. These channels, and especially TRPV1, TRPA1 and TRPM8, have been subjected to profound investigation because of their role as thermosensors and also because of their implication in acute and chronic pain. Surprisingly, their sensitivity to endogenous signaling has been far less studied. Cumulative evidence suggests that the function of these channels may be differently modulated in males and females, in part through sexual hormones, and this could constitute a significant contributor to the sex differences in chronic pain. Here, we review the exciting advances in thermoTRP pharmacology for males and females in two paradigmatic types of chronic pain with a strong peripheral component: chronic migraine and chemotherapy-induced peripheral neuropathy (CIPN). The possibilities of peripheral druggability offered by these channels and the differential exploitation for men and women represent a development opportunity that will lead to a significant increment of the armamentarium of analgesic medicines for personalized chronic pain treatment.

TRPM8 contributes to sex dimorphism by promoting recovery of normal sensitivity in a mouse model of chronic migraine.

TRPA1 and TRPM8 are transient receptor potential channels expressed in trigeminal neurons that are related to pathophysiology in migraine models. Here we use a mouse model of nitroglycerine-induced chronic migraine that displays a sexually dimorphic phenotype, characterized by mechanical hypersensitivity that develops in males and females, and is persistent up to day 20 in female mice, but disappears by day 18 in male mice. TRPA1 is required for development of hypersensitivity in males and females, whereas TRPM8 contributes to the faster recovery from hypersensitivity in males. TRPM8-mediated antinociception effects required the presence of endogenous testosterone in males. Administration of exogenous testosterone to females and orchidectomized males led to recovery from hypersensitivity. Calcium imaging and electrophysiological recordings in in vitro systems confirmed testosterone activity on murine and human TRPM8, independent of androgen receptor expression. Our findings suggest a protective function of TRPM8 in shortening the time frame of hypersensitivity in a mouse model of migraine.

A ¹8F-fluorodeoxyglucose MicroPET imaging study to assess changes in brain glucose metabolism in a rat model of surgery-induced latent pain sensitization.

BACKGROUND: Neuroplastic changes involved in latent pain sensitization after surgery are poorly defined. We assessed temporal changes in glucose brain metabolism in a postoperative rat model using positron emission tomography. We also investigated brain metabolism after naloxone administration. METHODS: Rats were given remifentanil anesthetic and underwent a plantar incision, with 1 mg/kg of (-)-naloxone subcutaneously administered on postoperative days 20 and 21. Using the von Frey test, mechanical thresholds were measured pre- and postoperatively at different time points in awake animals during F-fluorodeoxyglucose (F-FDG) uptake. Brain images were also obtained the day before mechanical testing, using a positron emission tomography R4 scanner (Concorde Microsystems, Siemens, Knoxville, TN). Differences in brain activity were assessed utilizing a statistical parametric mapping. RESULTS: Surgery induced minor changes in F-FDG uptake in the cerebellum, hippocampus, and posterior cortex, which extended to the thalamus, hypothalamus, and brainstem on days 6 and 7. Changes were still present on day 21. Maximal postoperative hypersensitivity was observed on day 2. The administration of (-)-naloxone on day 21 induced significant hypersensitivity, greatly enhancing the effect on F-FDG uptake. In sham-operated rats, naloxone induced changes limited to the striatum and the cerebellum. Nonnociceptive stimulation with von Frey filaments had no effect on F-FDG uptake. CONCLUSIONS: Surgery, remifentanil, and their combination induced long-lasting and significant metabolic changes in the pain brain matrix, with a positive correlation with hypersensitivity after naloxone. Changes in brain F-FDG precipitated by naloxone suggest that surgery under remifentanil anesthetic induces the greatest neuroplastic brain adaptations in opioid-related pathways involved in nociceptive processing and long-lasting pain sensitization.

Synergism between oral paracetamol and nefopam in a murine model of postoperative pain.

BACKGROUND: The use of paracetamol or nefopam for postoperative pain control is limited by the need of high doses associated with unwanted effects. Previous works suggest positive interactions between both compounds that may be exploited to obtain potentiation of antinociception. METHODS: Mechanical and heat antinociception induced by oral doses of paracetamol, nefopam or their combination was studied by isobolographic analysis in a murine model of postsurgical pain. The effective doses that produced 50% antinociception (ED50 ) were calculated from the log dose-response curves for each compound. Subsequently, the effects of ED8.7 s, ED12.5 s, ED17.5 s and ED35 s of nefopam and paracetamol combined were assessed. RESULTS: Oral paracetamol induced dose-dependent relief of postoperative sensitivity and showed higher efficacy reducing mechanical hypersensitivity (ED50 177.3 ± 15.4 mg/kg) than heat hyperalgesia (ED50 278.6 ± 43 mg/kg). Oral nefopam induced dose-dependent antinociception with similar efficacy for mechanical and heat hypersensitivity (ED50 s 5.42 ± 0.81 vs. 5.83 ± 0.72). Combinations of increasing isoeffective doses revealed that combined ED17.5 s (85.76 mg/kg paracetamol and 1.9 mg/kg nefopam) and ED35 s (132.67 mg/kg and 3.73 mg/kg) showed synergistic effects leading to 75% and 90% mechanical antinociception, respectively. These mixtures were defined by interaction indexes of 0.43 and 0.41 and ratios 45:1 and 35:1 paracetamol:nefopam, respectively. The same combinations showed additive effects for the inhibition of incisional thermal hyperalgesia. CONCLUSIONS AND LIMITATIONS: This work describes a synergistic antinociceptive interaction between low doses of nefopam and paracetamol for the treatment of postoperative hypersensitivity to peripheral stimuli. The promising results obtained on reflexive nociceptive responses of young male mice subjected to plantar surgery highlight the interest of further research evaluating the effects of this mixture on the affective-motivational component of pain and in females and additional age groups. Confirmation of pain-relieving efficacy and safety of this oral combination clinically available in European and Asian countries could provide a useful tool for postsurgical pain management. SIGNIFICANCE: Early postoperative pain is currently undertreated and has been recognized as a relevant source of chronic postsurgical pain. Oral efficient treatments could facilitate fast-track surgeries and patient recovery at home. Here, we identify in a mouse model of postoperative pain a potent synergistic oral combination consisting of low paracetamol and nefopam doses that provides relief of postsurgical hypersensitivity to mechanical and thermal stimuli. Oral multimodal paracetamol-nefopam mixtures represent a potential clinically available pharmacological strategy for the relief of incisional sensitivity and the promotion of patient recovery.

Kappa opioid receptor modulation of endometriosis pain in mice.

The kappa opioid receptor is a constituent of the endogenous opioid analgesia system widely expressed in somatosensory nervous pathways and also in endometrial tissues. This work investigates the possible involvement of kappa opioid receptor on the nociceptive, behavioral and histopathological manifestations of endometriosis in a murine model. Female mice receiving endometrial implants develop a persistent mechanical hypersensitivity in the pelvic area that is stronger during the estrus phase of the estrous cycle. The kappa opioid receptor agonist U50,488H produces a dose-dependent relief of this mechanical hypersensitivity, regardless of the cycle phase. Repeated exposure to a low dose of U50,488H (1 mg/kg/day s.c. for one month) provides sustained relief of mechanical hypersensitivity, without tolerance development or sedative side effects. Interestingly, this treatment also inhibits a decreased rearing behavior associated with spontaneous pain or discomfort in endometriosis mice. This KOR-mediated pain relief does not prevent the anxiety-like behavior or the cognitive impairment exhibited by endometriosis mice, and the growth of endometriotic cysts is also unaltered. These data provide evidence of strong pain-relieving properties of kappa opioid receptor stimulation in female mice with endometriosis pain. The persistence of affective and cognitive manifestations suggests that these comorbidities are independent of pelvic pain and simultaneous treatment of these comorbidities may be necessary for successful management of endometriosis.

The CB2 cannabinoid receptor as a therapeutic target in the central nervous system.

INTRODUCTION: Targeting CB2 cannabinoid receptor (CB2r) represents a promising approach for the treatment of central nervous system disorders. These receptors were identified in peripheral tissues, but also in neurons in the central nervous system. New findings have highlighted the interest to target these central receptors to obtain therapeutic effects devoid of the classical cannabinoid side-effects. AREAS COVERED: In this review, we searched PubMed (January 1991-May 2021), ClinicalTrials.gov and Cochrane Library databases for articles, reviews and clinical trials. We first introduce the relevance of CB2r as a key component of the endocannabinoid system. We discuss CB2r interest as a possible novel target in the treatment of pain. This receptor has raised interest as a potential target for neurodegenerative disorders treatment, as we then discussed. Finally, we underline studies revealing a novel potential CB2r interest in mental disorders treatment. EXPERT OPINION: In spite of the interest of targeting CB2r for pain, clinical trials evaluating CB2r agonist analgesic efficacy have currently failed. The preferential involvement of CB2r in preventing the development of chronic pain could influence the failure of clinical trials designed for the treatment of already established pain syndromes. Specific trials should be designed to target the prevention of chronic pain development.

Delta Opioid Receptor in Astrocytes Contributes to Neuropathic Cold Pain and Analgesic Tolerance in Female Mice.

Background: The delta opioid receptor (DOR) contributes to pain control, and a major challenge is the identification of DOR populations that control pain, analgesia, and tolerance. Astrocytes are known as important cells in the pathophysiology of chronic pain, and many studies report an increased prevalence of pain in women. However, the implication of astrocytic DOR in neuropathic pain and analgesia, as well as the influence of sex in this receptor activity, remains unknown. Experimental Approach: We developed a novel conditional knockout (cKO) mouse line wherein DOR is deleted in astrocytes (named GFAP-DOR-KO), and investigated neuropathic mechanical allodynia as well as analgesia and analgesic tolerance in mutant male and female mice. Neuropathic cold allodynia was also characterized in mice of both sexes lacking DOR either in astrocytes or constitutively. Results: Neuropathic mechanical allodynia was similar in GFAP-DOR-KO and floxed DOR control mice, and the DOR agonist SNC80 produced analgesia in mutant mice of both sexes. Interestingly, analgesic tolerance developed in cKO males and was abolished in cKO females. Cold neuropathic allodynia was reduced in mice with decreased DOR in astrocytes. By contrast, cold allodynia was exacerbated in full DOR KO females. Conclusions: These findings show that astrocytic DOR has a prominent role in promoting cold allodynia and analgesic tolerance in females, while overall DOR activity was protective. Altogether this suggests that endogenous- and exogenous-mediated DOR activity in astrocytes worsens neuropathic allodynia while DOR activity in other cells attenuates this form of pain. In conclusion, our results show a sex-specific implication of astrocytic DOR in neuropathic pain and analgesic tolerance. These findings open new avenues for developing tailored DOR-mediated analgesic strategies.

Role of the endocannabinoid system in a mouse model of Fragile X undergoing neuropathic pain.

BACKGROUND: Neuropathic pain is a complex condition characterized by sensory, cognitive and affective symptoms that magnify the perception of pain. The underlying pathogenic mechanisms are largely unknown and there is an urgent need for the development of novel medications. The endocannabinoid system modulates pain perception and drugs targeting the cannabinoid receptor type 2 (CB2) devoid of psychoactive side effects could emerge as novel analgesics. An interesting model to evaluate the mechanisms underlying resistance to pain is the fragile X mental retardation protein knockout mouse (Fmr1KO), a model of fragile X syndrome that exhibits nociceptive deficits and fails to develop neuropathic pain. METHODS: A partial sciatic nerve ligation was performed to wild-type (WT) and Fmr1KO mice having (HzCB2 and Fmr1KO-HzCB2, respectively) or not (WT and Fmr1KO mice) a partial deletion of CB2 to investigate the participation of the endocannabinoid system on the pain-resistant phenotype of Fmr1KO mice. RESULTS: Nerve injury induced canonical hypersensitivity in WT and HzCB2 mice, whereas this increased pain sensitivity was absent in Fmr1KO mice. Interestingly, Fmr1KO mice partially lacking CB2 lost this protection against neuropathic pain. Similarly, pain-induced depressive-like behaviour was observed in WT, HzCB2 and Fmr1KO-HzCB2 mice, but not in Fmr1KO littermates. Nerve injury evoked different alterations in WT and Fmr1KO mice at spinal and supra-spinal levels that correlated with these nociceptive and emotional alterations. CONCLUSIONS: This work shows that CB2 is necessary for the protection against neuropathic pain observed in Fmr1KO mice, raising the interest in targeting this receptor for the treatment of neuropathic pain. SIGNIFICANCE: Neuropathic pain is a complex chronic pain condition and current treatments are limited by the lack of efficacy and the incidence of important side effects. Our findings show that the pain-resistant phenotype of Fmr1KO mice against nociceptive and emotional manifestations triggered by persistent nerve damage requires the participation of the cannabinoid receptor CB2, raising the interest in targeting this receptor for neuropathic pain treatment. Additional multidisciplinary studies more closely related to human pain experience should be conducted to explore the potential use of cannabinoids as adequate analgesic tools.

Increased spinal dynorphin levels and phospho-extracellular signal-regulated kinases 1 and 2 and c-Fos immunoreactivity after surgery under remifentanil anesthesia in mice.

In humans, remifentanil anesthesia enhances nociceptive sensitization in the postoperative period. We hypothesized that activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the expression of c-Fos, prodynorphin (mRNA), and dynorphin in the spinal cord could participate in the molecular mechanisms underlying postoperative opioid-induced sensitization. In a mouse model of incisional pain, we evaluated thermal (Hargreaves test) and mechanical (von Frey) hyperalgesia during the first 21 postoperative days. Moreover, prodynorphin (mRNA, real-time polymerase chain reaction), dynorphin (enzymatic immunoassay), c-Fos expression, and ERK1/2 phosphorylation (both by immunohistochemistry) in the lumbar spinal cord were assessed. Surgery performed under remifentanil anesthesia induced a maximal decrease in nociceptive thresholds between 4 h and 2 days postoperatively (p < 0.001) that lasted 10 to 14 days compared with noninjured animals. In the same experimental conditions, a significant increase in prodynorphin mRNA expression (at 2 and 4 days) followed by a sustained increase of dynorphin (days 2 to 10) in the spinal cord was observed. We also identified an early expression of c-Fos immunoreactivity in the superficial laminae of the dorsal horn of the spinal cord (peak at 4 h; p < 0.001), together with a partial activation of ERK1/2 (4 h; p < 0.001). These findings suggest that activated ERK1/2 could induce c-Fos expression and trigger the transcription of prodynorphin in the spinal cord. This in turn would result in long-lasting increased levels of dynorphin that, in our model, could participate in the persistence of pain but not in the manifestation of first pain.

The endocannabinoid system in modulating fear, anxiety, and stress
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The endocannabinoid system is widely expressed in the limbic system, prefrontal cortical areas, and brain structures regulating neuroendocrine stress responses, which explains the key role of this system in the control of emotions. In this review, we update recent advances on the function of the endocannabinoid system in determining the value of fear-evoking stimuli and promoting appropriate behavioral responses for stress resilience. We also review the alterations in the activity of the endocannabinoid system during fear, stress, and anxiety, and the pathophysiological role of each component of this system in the control of these protective emotional responses that also trigger pathological emotional disorders. In spite of all the evidence, we have not yet taken advantage of the therapeutic implications of this important role of the endocannabinoid system, and possible future strategies to improve the treatment of these emotional disorders are discussed.
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El sistema endocannabinoide tiene una amplia expresión en el sistema límbico, las áreas corticales prefrontales y las estructuras cerebrales que regulan las respuestas al estrés neuroendocrino, lo que explica el papel clave de este sistema en el control de las emociones. En esta revisión, se actualizan los avances recientes sobre la función del sistema endocannabinoide para determinar el valor de los estímulos que provocan miedo y promueven respuestas conductuales apropiadas para la resiliencia frente al estrés. También se revisan las alteraciones en la actividad del sistema endocannabinoide durante el miedo, el estrés y la ansiedad, y el papel fisiopatológico de cada componente de este sistema en el control de estas respuestas emocionales protectoras que también desencadenan trastornos emocionales patológicos. A pesar de todas las evidencias, todavía no se han aprovechado las traducciones terapéuticas de este importante papel del sistema endocannabinoide. También se discuten las posibles estrategias futuras para mejorar el tratamiento de estos trastornos emocionales.

Abondamment retrouvé dans le système limbique, le cortex préfrontal et les structures cérébrales régulant les réponses neuroendocriniennes au stress, le système endocannabinoïde joue un rôle clé dans le contrôle des émotions. Sa fonction dans la détermination de l'intensité des stimuli déclenchant la peur et dans l'amélioration des réponses comportementales adaptées à la résistance au stress est étudiée ici à l'aune des données récentes actualisées. Nous reconsidérons également les modifications de l'activité du système endocannabinoïde face à la peur, au stress et à l'anxiété, ainsi que le rôle physiopathologique de chaque composante de ce système dans le contrôle de ces réponses émotionnelles protectrices, elles-mêmes génératrices de troubles émotionnels pathologiques. Malgré toutes les données disponibles, le rôle crucial du système endocannabinoïde sur le plan thérapeutique n'a pas encore été exploité. Nous analysons les futures stratégies possibles pour améliorer le traitement de ces troubles émotionnels.

Surgical Induction of Endometriosis in Female Mice.

Endometriosis is a common gynecological disease characterized by the presence of endometrial tissue outside the uterine cavity. It is frequently associated with pain, infertility and a reduced quality of life, and it lacks adequate treatment. Several rodent models of endometriosis have been developed through heterologous and homologous transplantation of endometrial tissue into the abdominal compartment. Here we describe a surgical procedure to generate a syngeneic model of endometriosis in immunocompetent mice with intact uterine and ovarian tissues. In this model, four uterine fragments from a donor mouse at diestrus are sutured to the abdominal wall of a recipient mouse. One month after surgeries, endometrial implants develop into cysts with glandular epithelium and stroma, mimicking the endometriotic lesions observed in women with endometriosis. Therefore, this mouse model provides a valuable tool to study the pathophysiology of endometriosis and the efficacy of potential treatments.

Disease-modifying effects of natural Δ9-tetrahydrocannabinol in endometriosis-associated pain.

Endometriosis is a chronic painful disease highly prevalent in women that is defined by growth of endometrial tissue outside the uterine cavity and lacks adequate treatment. Medical use of cannabis derivatives is a current hot topic and it is unknown whether phytocannabinoids may modify endometriosis symptoms and development. Here we evaluate the effects of repeated exposure to Δ9-tetrahydrocannabinol (THC) in a mouse model of surgically-induced endometriosis. In this model, female mice develop mechanical hypersensitivity in the caudal abdomen, mild anxiety-like behavior and substantial memory deficits associated with the presence of extrauterine endometrial cysts. Interestingly, daily treatments with THC (2 mg/kg) alleviate mechanical hypersensitivity and pain unpleasantness, modify uterine innervation and restore cognitive function without altering the anxiogenic phenotype. Strikingly, THC also inhibits the development of endometrial cysts. These data highlight the interest of scheduled clinical trials designed to investigate possible benefits of THC for women with endometriosis.

Mu and delta opioid receptors play opposite nociceptive and behavioural roles on nerve-injured mice.

BACKGROUND AND PURPOSE: Mu and delta opioid receptors(MOP, DOP) contribution to the manifestations of pathological pain is not understood. We used genetic approaches to investigate the opioid mechanisms modulating neuropathic pain and its comorbid manifestations. EXPERIMENTAL APPROACH: We generated conditional knockout mice with MOP or DOP deletion in sensoryNav1.8-positive neurons (Nav1.8), in GABAergic forebrain neurons (DLX5/6) orconstitutively (CMV). Mutant mice and wild-type littermates were subjected topartial sciatic nerve ligation (PSNL) or sham surgery and their nociception wascompared. Anxiety-, depressivelike behaviour and cognitive performance were also measured. Opioid receptor mRNA expression, microgliosis and astrocytosis were assessed in the dorsalroot ganglia (DRG) and/or the spinal cord (SC). KEY RESULTS: Constitutive CMV-MOP knockouts after PSNL displayed reduced mechanical allodynia and enhanced heat hyperalgesia. This phenotype was accompanied by increased DOP expression in DRG and SC, and reduced microgliosis and astrocytosis in deep dorsal horn laminae. Conditional MOP knockouts and control mice developed similar hypersensitivity after PSNL, except for anenhanced heat hyperalgesia by DLX5/6-MOP male mice. Neuropathic pain-induced anxiety was aggravated in CMV-MOP and DLX5/6-MOP knockouts. Nerve-injured CMV-DOP mice showed increased mechanical allodynia, whereas Nav1.8-DOP and DLX5/8-DOP mice had partial nociceptive enhancement. CMV-DOP and DLX5/6-DOP mutants showed increased depressive-like behaviour after PSNL. CONCLUSIONS AND IMPLICATIONS: MOP activity after nerve injury increased anxiety-like responses involving forebrain GABAergic neurons and enhanced mechanical pain sensitivity along with repression of DOP expression and spinal cord gliosis. In contrast, DOP shows a protective function limiting nociceptive and affective manifestations of neuropathic pain.

Protective role of neuronal and lymphoid cannabinoid CB2 receptors in neuropathic pain.

Cannabinoid CB2 receptor (CB2) agonists are potential analgesics void of psychotropic effects. Peripheral immune cells, neurons and glia express CB2; however, the involvement of CB2 from these cells in neuropathic pain remains unresolved. We explored spontaneous neuropathic pain through on-demand self-administration of the selective CB2 agonist JWH133 in wild-type and knockout mice lacking CB2 in neurons, monocytes or constitutively. Operant self-administration reflected drug-taking to alleviate spontaneous pain, nociceptive and affective manifestations. While constitutive deletion of CB2 disrupted JWH133-taking behavior, this behavior was not modified in monocyte-specific CB2 knockouts and was increased in mice defective in neuronal CB2 knockouts suggestive of increased spontaneous pain. Interestingly, CB2-positive lymphocytes infiltrated the injured nerve and possible CB2transfer from immune cells to neurons was found. Lymphocyte CB2depletion also exacerbated JWH133 self-administration and inhibited antinociception. This work identifies a simultaneous activity of neuronal and lymphoid CB2that protects against spontaneous and evoked neuropathic pain.

Pronociceptive effects of remifentanil in a mouse model of postsurgical pain: effect of a second surgery.

BACKGROUND: Remifentanil anesthesia enhances postoperative pain in animals and humans. The authors evaluated the impact of the dose (microg x kg(-1) x min(-1)) and duration of remifentanil infusion, and the effects of a second surgery on postoperative pain sensitization. METHODS: Mice received different doses of remifentanil over 30 or 60 min. The authors assessed thermal (Hargreaves) and mechanical hyperalgesia (von Frey) at 2, 4, 7, and 10 days. In other experiments, mice had a plantar incision during sevoflurane with or without remifentanil anesthesia that was repeated 27 days later, when nociceptive thresholds returned to baseline. Linear mixed models were used for statistical analysis. RESULTS: Remifentanil induced dose-dependent pronociceptive effects with calculated ED(50)s of 1.7 (95% confidence interval, 1.3-2.1) and 1.26 (1.0-1.6) microg x kg(-1) x min(-1) for thermal and mechanical hyperalgesia, respectively, which lasted longer with higher doses (P < 0.001). The duration of infusion did not alter the pronociceptive effects of remifentanil when administered at a constant dose of infusion. When given during surgery, high (2.66 microg x kg(-1) x min(-1)) or low (0.66 microg x kg(-1) x min(-1)) remifentanil increased the extent (P < 0.05) and duration (P < 0.01) of thermal and mechanical hyperalgesia. The latter was further enhanced after a second surgery performed in the same experimental conditions (P < 0.05). Surgery or remifentanil infusion, each one individually, induced significant mechanical hyperalgesia, which was greater when repeated (P < 0.05). CONCLUSIONS: In this model of incisional pain, remifentanil induces pronociceptive effects, which are dose dependent but unaltered by the duration of administration. A second surgery performed on the same site and experimental conditions induces greater postoperative hyperalgesia that is enhanced when remifentanil is used as an anesthetic.