Rodent Animal Models of Endometriosis-Associated Pain: Unmet Needs and Resources Available for Improving Translational Research in Endometriosis.

Chronic pain induced by endometriosis is a maladaptive pain experienced by half of women with this disease. The lack of pharmacological treatments suitable for the long-term relief of endometriosis-associated pain, without an impact on fertility, remains an urgent unmet need. Progress has been slowed by the absence of a reproducible rodent endometriosis model that fully replicates human physiopathological characteristics, including pain symptoms. Although pain assessment in rodents is a complicated task requiring qualified researchers, the choice of the behavioral test is no less important, since selecting inappropriate tests can cause erroneous data. Pain is usually measured with reflex tests in which hypersensitivity is evaluated by applying a noxious stimulus, yet this ignores the associated emotional component that could be evaluated via non-reflex tests. We conducted a systematic review of endometriosis models used in rodents and the number of them that studied pain. The type of behavioral test used was also analyzed and classified according to reflex and non-reflex tests. Finally, we determined the most used reflex tests for the study of endometriosis-induced pain and the main non-reflex behavioral tests utilized in visceral pain that can be extrapolated to the study of endometriosis and complement traditional reflex tests.

Variable transcriptional responsiveness of the P2X3 receptor gene during CFA-induced inflammatory hyperalgesia.

The purinergic receptor P2X3 (P2X3-R) plays important roles in molecular pathways of pain, and reduction of its activity or expression effectively reduces chronic inflammatory and neuropathic pain sensation. Inflammation, nerve injury, and cancer-induced pain can increase P2X3-R mRNA and/or protein levels in dorsal root ganglia (DRG). However, P2X3-R expression is unaltered or even reduced in other pain studies. The reasons for these discrepancies are unknown and might depend on the applied traumatic intervention or on intrinsic factors such as age, gender, genetic background, and/or epigenetics. In this study, we sought to get insights into the molecular mechanisms responsible for inflammatory hyperalgesia by determining P2X3-R expression in DRG neurons of juvenile male rats that received a Complete Freund's Adjuvant (CFA) bilateral paw injection. We demonstrate that all CFA-treated rats showed inflammatory hyperalgesia, however, only a fraction (14-20%) displayed increased P2X3-R mRNA levels, reproducible across both sides. Immunostaining assays did not reveal significant increases in the percentage of P2X3-positive neurons, indicating that increased P2X3-R at DRG somas is not critical for inducing inflammatory hyperalgesia in CFA-treated rats. Chromatin immunoprecipitation (ChIP) assays showed a correlated (R2 = 0.671) enrichment of the transcription factor Runx1 and the epigenetic active mark histone H3 acetylation (H3Ac) at the P2X3-R gene promoter in a fraction of the CFA-treated rats. These results suggest that animal-specific increases in P2X3-R mRNA levels are likely associated with the genetic/epigenetic context of the P2X3-R locus that controls P2X3-R gene transcription by recruiting Runx1 and epigenetic co-regulators that mediate histone acetylation.

Anxiety-related behaviors without observation of generalized pain in a mouse model of endometriosis.

Endometriosis is a chronic, hormone-dependent, inflammatory disease, characterized by the presence and growth of endometrial tissue outside the uterine cavity. It is associated with moderate to severe pelvic and abdominal pain symptoms, subfertility and a marked reduction in health-related quality of life. Furthermore, relevant co-morbidities with affective disorders like depression or anxiety have been described. These conditions have a worsening effect on pain perception in patients and might explain the negative impact on quality of life observed in those suffering from endometriosis-associated pain. Whereas several studies using rodent models of endometriosis focused on biological and histopathological similarities with the human situation, the behavioral characterization of these models was never performed. This study investigated the anxiety-related behaviors in a syngeneic model of endometriosis. Using elevated plus maze and the novel environment induced feeding suppression assays we observed the presence of anxiety-related behaviors in endometriosis-induced mice. In contrast, locomotion or generalized pain did not differ between groups. These results indicate that the presence of endometriosis lesions in the abdominal cavity could, similarly to patients, induce profound psychopathological changes/impairments in mice. These readouts might provide additional tools for preclinical identification of mechanisms relevant for development of endometriosis-related symptoms.

Identification of Altered Evoked and Non-Evoked Responses in a Heterologous Mouse Model of Endometriosis-Associated Pain.

The aim of this study was to develop and refine a heterologous mouse model of endometriosis-associated pain in which non-evoked responses, more relevant to the patient experience, were evaluated. Immunodeficient female mice (N = 24) were each implanted with four endometriotic human lesions (N = 12) or control tissue fat (N = 12) on the abdominal wall using tissue glue. Evoked pain responses were measured biweekly using von Frey filaments. Non-evoked responses were recorded weekly for 8 weeks using a home cage analysis (HCA). Endpoints were distance traveled, social proximity, time spent in the center vs. outer areas of the cage, drinking, and climbing. Significant differences between groups for von Frey response, climbing, and drinking were detected on days 14, 21, and 35 post implanting surgery, respectively, and sustained for the duration of the experiment. In conclusion, a heterologous mouse model of endometriosis-associated evoked a non-evoked pain was developed to improve the relevance of preclinical models to patient experience as a platform for drug testing.

Preclinical models of endometriosis and interstitial cystitis/bladder pain syndrome: an Innovative Medicines Initiative-PainCare initiative to improve their value for translational research in pelvic pain.

ABSTRACT: Endometriosis (ENDO) and interstitial cystitis/bladder pain syndrome (IC/BPS) are chronic pain conditions for which better treatments are urgently needed. Development of new therapies with proven clinical benefit has been slow. We have conducted a review of existing preclinical in vivo models for ENDO and IC/BPS in rodents, discussed to what extent they replicate the phenotype and pain experience of patients, as well as their relevance for translational research. In 1009 publications detailing ENDO models, 41% used autologous, 26% syngeneic, 18% xenograft, and 11% allogeneic tissue in transplantation models. Intraperitoneal injection of endometrial tissue was the subcategory with the highest construct validity score for translational research. From 1055 IC/BPS publications, most interventions were bladder centric (85%), followed by complex mechanisms (8%) and stress-induced models (7%). Within these categories, the most frequently used models were instillation of irritants (92%), autoimmune (43%), and water avoidance stress (39%), respectively. Notably, although pelvic pain is a hallmark of both conditions and a key endpoint for development of novel therapies, only a small proportion of the studies (models of ENDO: 0.5%-12% and models of IC/BPS: 20%-44%) examined endpoints associated with pain. Moreover, only 2% and 3% of publications using models of ENDO and IC/BPS investigated nonevoked pain endpoints. This analysis highlights the wide variety of models used, limiting reproducibility and translation of results. We recommend refining models so that they better reflect clinical reality, sharing protocols, and using standardized endpoints to improve reproducibility. We are addressing this in our project Innovative Medicines Initiative-PainCare/Translational Research in Pelvic Pain.

Anti-nociceptive action of peripheral mu-opioid receptors by G-beta-gamma protein-mediated inhibition of TRPM3 channels.

Opioids, agonists of µ-opioid receptors (µORs), are the strongest pain killers clinically available. Their action includes a strong central component, which also causes important adverse effects. However, µORs are also found on the peripheral endings of nociceptors and their activation there produces meaningful analgesia. The cellular mechanisms downstream of peripheral µORs are not well understood. Here, we show in neurons of murine dorsal root ganglia that pro-nociceptive TRPM3 channels, present in the peripheral parts of nociceptors, are strongly inhibited by µOR activation, much more than other TRP channels in the same compartment, like TRPV1 and TRPA1. Inhibition of TRPM3 channels occurs via a short signaling cascade involving Gßγ proteins, which form a complex with TRPM3. Accordingly, activation of peripheral µORs in vivo strongly attenuates TRPM3-dependent pain. Our data establish TRPM3 inhibition as important consequence of peripheral µOR activation indicating that pharmacologically antagonizing TRPM3 may be a useful analgesic strategy.