Decision letter: Disease-modifying effects of natural Δ9-tetrahydrocannabinol in endometriosis-associated pain

Article Figures and data Abstract eLife digest Introduction Results and discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract 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. eLife digest Endometriosis is a common disease in women caused by tissue that lines the uterus growing outside the uterine cavity on to other organs in the pelvis. This can cause a variety of symptoms including chronic pelvic pain, infertility, and pain during menstruation or sexual intercourse. These symptoms may contribute to anxiety, depression, loss of working ability and a reduced quality of life. Currently available treatments for endometriosis, including hormonal therapy and surgery, have a limited effect and can produce unwanted side effects. For example, women who undergo surgery to remove the growths may experience post-surgical pain or a recurrence. As a result, women with endometriosis often rely on self-management strategies like dietary changes or exercise. Although cannabis consumption has a large number of potential side effects and can lead to substance abuse, it has been shown to provide pain relief in some conditions. But it is unknown whether it could be useful for treating endometriosis. Now, Escudero-Lara et al. have created a mouse model that mimics some of the conditions of human endometriosis: pelvic pain, anxiety and memory impairments. The mice were treated with moderate doses of Δ9-tetrahydrocannabinol (THC), which is the main pain-relieving component of cannabis. The THC reduced pelvic pain and cognitive impairments in the mice with the endometriosis-like condition, but it had no effect on their anxious behavior. Escudero-Lara et al. also noticed that endometrial growths were also smaller in the treated mice indicating that THC may also inhibit endometriosis development. These experiments suggest that THC may be a useful treatment for patients with endometriosis. Clinical trials are already ongoing to test whether these findings translate to patients with the condition. Although THC and cannabis are readily available in some areas, Escudero-Lara et al. discourage using unregulated cannabis products due to the potential risks. Introduction Endometriosis is a chronic inflammatory disease that affects 1 in 10 women of childbearing age (Zondervan et al., 2019). It is characterized by the growth of endometrium in extrauterine locations, chronic pain in the pelvis and the lower abdomen, infertility, emotional distress and loss of working ability (Fourquet et al., 2011; Márki et al., 2017; Zondervan et al., 2019). Current clinical management provides unsatisfactory outcomes. On the one hand, hormonal therapy has unwanted effects including contraception and emotional disturbances (Ross and Kaiser, 2017; Skovlund et al., 2016), whereas surgical excision of the growths is associated with high-recurrence rates and post-surgical pain (Garry, 2004). Hence, clinical treatments are limited and women often unsatisfactorily self-manage their pain (Armour et al., 2019). In this context, marijuana legalization for medical purposes in American and European states has led to increased availability of phytocannabinoids (Abuhasira et al., 2018). While cannabis may provide pain relief in certain conditions (Campbell et al., 2001), it is unclear whether it may modify endometriosis symptoms or development. Δ9-tetrahydrocannabinol (THC) is the main psychoactive constituent of the Cannabis sativa plant, and multiple animal and clinical studies suggest its efficacy relieving chronic pain (De Vry et al., 2004; Harris et al., 2016; King et al., 2017; Ueberall et al., 2019; Williams et al., 2008), although controversial results have been obtained in human clinical trials (Stockings et al., 2018). However, THC has important side effects including cognitive deficits and anxiety (Célérier et al., 2006; Kasten et al., 2017; Puighermanal et al., 2013). This work investigates the effects of natural THC in a mouse model of endometriosis that reproduces the ectopic endometrial growths and some of the behavioral alterations of clinical endometriosis. Our data show that THC is effective inhibiting hypersensitivity in the caudal abdominal area without inducing tolerance, as well as reducing the pain unpleasantness associated with endometriosis. Notably, THC also prevents the cognitive impairment observed in mice with ectopic endometrium without modifying anxiety-like behavior at this particular dose. Interestingly, THC shows efficacy limiting the development of ectopic endometrium, revealing disease-modifying effects of this natural cannabinoid. Results and discussion Ectopic endometrium leads to pain sensitivity in the caudal abdomen, anxiety-like behavior and memory impairment Our first aim was to characterize a novel experimental procedure to evaluate at the same time nociceptive, cognitive and emotional manifestations of endometriosis pain in female mice. Mice were subjected to a surgical implantation of endometrial tissue in the peritoneal wall of the abdominal compartment or to a sham procedure. Mice receiving ectopic endometrial implants developed persistent mechanical hypersensitivity in the caudal abdominal area, whereas sham mice recovered their baseline sensitivity and showed significant differences in comparison to endometriosis mice since the second week of implantation (Figure 1a and Figure 1—figure supplement 1). To test whether mechanical hypersensitivity of endometriosis mice was specific to this abdominal region, nociceptive responses were also measured in the hind paw. In this distant area, mechanical sensitivity remained unaltered, indicating that pain sensitization did not generalize to other sites (Figure 1b and Figure 1—figure supplement 2). To discern whether increased nociception was accompanied by a component of negative affect, a measure of pain unpleasantness was taken on day 14 after the surgeries (Figure 1c). Endometriosis mice showed increased nocifensive behaviors to mechanical stimuli when compared with sham mice. Similarly, endometriosis mice exhibited enhanced anxiety-like behavior reflected in lower percentages of time and entries to the open arms of the elevated plus maze (Figure 1d). Total arm entries were similar in both groups (Figure 1d). In line with these findings, previous rodent models of endometriosis found increased mechanosensitivity in the lower abdomen (Arosh et al., 2015; Greaves et al., 2017) and affective-like disturbances (Filho et al., 2019; Li et al., 2018). Previous works associate nociceptive and emotional distress in chronic pain settings with cognitive decline (Bushnell et al., 2015; La Porta et al., 2015; You et al., 2018), although this cognitive impairment has not yet been revealed in rodent models of endometriosis. We found in our model a dramatic impairment of long-term memory in endometriosis mice (Figure 1e). While mnemonic effects of this pathology have not been thoroughly evaluated, a cognitive impairment may contribute to the loss of working ability consistently reported in women with endometriosis (Hansen et al., 2013; Sperschneider et al., 2019). Hence, mice with ectopic endometrium recapitulate in our model some of the symptomatology observed in the clinics, although manifestations of spontaneous pain could not be evaluated in this work. Figure 1 with 3 supplements see all Download asset Open asset Behavioral and histological alterations in female mice with ectopic endometrial implants. Endometriosis mice showed (a) persistent mechanical abdominal hypersensitivity that (b) was localized in the caudal abdominal area but not detectable in distant areas (hind paw). Mechanical sensitivity is represented by the area under the curve of frequency of response to von Frey filaments. Higher values mean higher mechanical pain. Mice receiving endometrial implants also showed (c) increased nocifensive behavior, (d) anxiety-like behavior in the elevated plus maze test and (e) cognitive impairment in the novel object recognition task. (f) From left to right: cysts were recovered from endometriosis mice, were filled with fluid (scale bar = 1 mm), contained endometrial epithelium and stroma (scale bar = 100 μm) and were innervated by beta-III tubulin-labeled fibers (scale bar = 100 μm, blue is DAPI and white is β-III tubulin). Error bars are mean ± SEM. One-way repeated measures ANOVA + Bonferroni (a and b) and Student t-test (c, d and e). *p<0.05, **p<0.01, ***p<0.001 vs sham. ##p<0.01, ###p<0.001 vs baseline. Endo, endometriosis, AUC, area under the curve. Figure 1—source data 1 Effects of ectopic endometrium. https://cdn.elifesciences.org/articles/50356/elife-50356-fig1-data1-v2.xlsx Download elife-50356-fig1-data1-v2.xlsx Mice receiving endometrial implants developed 3 to 5 endometrial cysts in the peritoneal wall of the abdominal compartment. Cysts were of 2.59 ± 0.34 mm diameter, filled with fluid, with glandular epithelium and stroma and innervated by beta-III tubulin positive fibers (Figure 1f), as shown in women (Tokushige et al., 2006; Wang et al., 2009) and other rodent models (Arosh et al., 2015; Berkley et al., 2004). Interestingly, we also found increased expression of the neuronal marker beta-III tubulin in the uteri of endometriosis mice (Figure 1—figure supplement 3), mimicking not only some of the symptoms but also the histological phenotype observed in women with endometriosis (Miller and Fraser, 2015; Tokushige et al., 2006). Δ9-tetrahydrocannabinol alleviates pain in the caudal abdomen, restores cognitive function and limits the growth of ectopic endometrium Our second objective was to assess the effects of THC exposure on the endometriosis model to select an appropriate dose for a chronic treatment. Acute doses of THC were first tested in endometriosis and sham mice at a time point in which endometriotic lesions and hypersensitivity in the caudal abdomen were fully developed. Acute THC administration produced a dose-dependent reduction of abdominal mechanical hypersensitivity (Figure 2). The acute ED50 of THC 1.916 mg/kg (≈2 mg/kg) was chosen for the repeated administration. Figure 2 Download asset Open asset Effect of acute THC administration on the nociceptive responses to mechanical stimulation. (a) Acute THC produced a dose-dependent reduction of mechanical hypersensitivity in the caudal abdominal area. Mechanical sensitivity is represented by the area under the curve of frequency of response to von Frey filaments. Higher values mean higher mechanical pain. (b) Administration of 2, 2.5 and 5 mg/kg of THC decreased the frequency of response to von Frey filaments in endometriosis mice. Error bars are mean ± SEM. One-way repeated measures ANOVA + Bonferroni. *p<0.05, **p<0.01 vs sham; +p<0.05, ++p<0.01, +++p<0.001 vs vehicle. Endo, endometriosis; THC, Δ9-tetrahydrocannabinol, AUC, area under the curve. Figure 2—source data 1 Acute THC effects. https://cdn.elifesciences.org/articles/50356/elife-50356-fig2-data1-v2.xlsx Download elife-50356-fig2-data1-v2.xlsx Repeated exposure to THC 2 mg/kg, once daily for 28 days, provided a sustained alleviation of mechanical hypersensitivity during the whole treatment period (Figure 3a and Figure 3—figure supplement 1). Repeated THC starting on day 1 could have exerted a preventive effect at endometriosis stages in which pain sensitivity may have not been fully developed. To discern whether the absence in loss of efficacy was due to an inhibition of endometriosis development or to an actual lack of tolerance, we assessed the persistence of THC efficacy once pain was already present. THC given for the first time on day 14 was as effective as THC given on the same day after a daily treatment starting on day 8 (7 days long, Figure 3b and Figure 3—figure supplement 2). Therefore, THC did not lose its efficacy when repeated administration started once painful symptomatology was established. The absence of tolerance to THC-induced antinociception is in contrast with the tolerance described at higher THC doses in other pain models (Greene et al., 2018; LaFleur et al., 2018; Wakley et al., 2014). As expected, no effects of endometriosis or THC treatments were found in mechanical sensitivity of distant areas (Hind paw, Figure 3—figure supplement 3). Endometriosis mice treated with vehicle showed an increase in nocifensive behaviors compared with sham mice (Figure 3c). Interestingly, the 7 day treatment with THC inhibited this component of negative affect, while the effects of an acute administration of THC were highly variable. This variable response could be associated to aversive effects associated with a first exposure to THC, an event described in humans (MacCallum and Russo, 2018) and mice (Kubilius et al., 2018). Figure 3 with 3 supplements see all Download asset Open asset Effects of THC on the behavioral changes observed in mice with ectopic endometrium. (a) Repeated THC (28 days) alleviated mechanical hypersensitivity in the caudal abdominal area of endometriosis mice in the von Frey test. (b) THC administered on day 14 after a 6 day treatment (Endo – 7daysTHC) was as effective as an acute dose given on day 14 (Endo – AcuteTHC). Mechanical sensitivity is represented by the area under the curve of frequency of response to von Frey filaments. Higher values mean higher mechanical pain. (c) Nocifensive behaviors were abolished in endometriosis mice after a 7 day treatment with THC (Endo – 7daysTHC). (d) Endometriosis-associated anxiety-like behavior was unaltered after THC in the elevated plus maze test. (e) THC impaired object recognition memory in sham mice and prevented memory deficits of endometriosis mice in the novel object recognition test. THC dose: 2 mg/kg/day. Error bars are mean ± SEM. Two-way repeated measures ANOVA + Bonferroni (a), Mixed model + Bonferroni (b), Kruskal-Wallis + Mann Whitney U (c) and Two-way ANOVA + Bonferroni (d and e). ###p<0.001 vs baseline. *p<0.05, **p<0.01, ***p<0.001 vs sham. ++p<0.01, +++p<0.001 vs vehicle,. Endo, endometriosis; THC, Δ9-tetrahydrocannabinol; AUC, area under the curve. Figure 3—source data 1 Effects of repeated THC on behavioral alterations. https://cdn.elifesciences.org/articles/50356/elife-50356-fig3-data1-v2.xlsx Download elife-50356-fig3-data1-v2.xlsx Additional experiments were conducted to assess the effects of THC on the anxiety-like behavior induced by endometriosis pain (Figure 3d). As in previous experiments, endometriosis mice showed a lower percentage of time in the open arms of the elevated plus maze (Figure 3d), revealing increased anxiety-like behavior. However, the percentage of entries to open arms was similar in endometriosis and sham mice. Therefore, the anxiogenic-like effect of ectopic endometrium in these experimental conditions was mild and the present model was not optimal to reveal the emotional component of this painful situation. No significant effects of repeated THC 2 mg/kg were observed on the percentages of time and entries, although THC-treated mice showed a subtle increase in anxiety-like behavior (Figure 3d, percentage of time in open arms). Previous studies described anxiogenic-like effects of slightly higher doses (3 mg/kg) in naïve male mice (Viñals et al., 2015), and anxiolytic-like effects when using lower doses (0.3 mg/kg, Puighermanal et al., 2013; Viñals et al., 2015). Thus, possible effects of THC alleviating pain-related anxiety-like behavior in endometriosis mice could be hindered by intrinsic anxiogenic effects of this THC dose. Therefore, doses with less pain-relieving efficacy could potentially be effective promoting anxiolytic-like effects considering the intrinsic effects of THC on emotional-like behavior. Alternatively, the absence of clear effects of THC on anxiety-like behavior may be associated to the evaluation time point, which was 6 hr after administration to study the impact of pain relief on anxiety-like behavior, rather than to assess direct drug effects. Total arm entries were similar among groups (Figure 3d). Memory performance was also assessed the third week after starting the THC treatment. As expected, mice exposed to the chronic nociceptive manifestations of endometriosis showed a pronounced cognitive impairment, as well as sham mice exposed to THC, in accordance with previous reports in naïve males (Kasten et al., 2017; Puighermanal et al., 2013). Surprisingly, endometriosis mice repeatedly treated with natural THC showed intact discrimination indices (Figure 3e) suggesting protective effects of THC in this chronic inflammatory condition. In agreement, recent studies have shown cognitive improvements after THC exposure in old male and female mice (Bilkei-Gorzo et al., 2017; Sarne et al., 2018). Exogenous and endogenous cannabinoids have shown modulatory effects on the female reproductive system (Walker et al., 2019). Thus, we analyzed the effects of THC on the ectopic and eutopic endometrium and on ovarian follicle maturation. Interestingly, endometriosis mice receiving THC 2 mg/kg for 32 days showed an evident inhibition of the development of endometrial cysts (cyst diameter and area of endometrial tissue, Figure 4a) without significant effects on cyst innervation (Figure 4—figure supplement 1a). In agreement, a previous study showed antiproliferative effects of WIN 55212–2, a synthetic cannabinoid agonist, on endometrial cell cultures and in ectopic endometrium implanted in immunodepressed mice (Leconte et al., 2010). The assessment of the uterine diameter and the area of eutopic endometrium (Figure 4—figure supplement 1b) showed no effects of the THC treatment, suggesting that the antiproliferative activity of THC on endometrial cells is restricted to ectopic sites. However, possible effects of THC on established endometriosis lesions were not evaluated. Repeated THC increased the expression of neuronal markers in the uteri of sham mice, similar to the increase provoked by the ectopic endometrium (Figure 4b). Interestingly, THC prevented this increase in endometriosis mice (Figure 4b) indicating again that THC exposure may have different consequences under chronic inflammatory conditions. In agreement, recent studies showed differential effects of THC on the nervous system of rodents with and without chronic inflammation (Bilkei-Gorzo et al., 2017; Sarne et al., 2018). To investigate a possible estrogenic influence on these histological findings, we analyzed 17 β-estradiol plasma levels. As expected, 17 β-estradiol plasma levels depended on the phase of the estrous cycle: mice in proestrus had the highest concentration followed by mice in diestrus, and mice in estrus showed the lowest levels (Figure 4c, left graph). We found that 17 β-estradiol was similar in all experimental groups (Figure 4c, right graph), although the levels of this estrogen were positively correlated with cyst diameter (Figure 4d, left), proving the estrogenic influence on ectopic endometrial lesions. 17 β-estradiol levels were not correlated with endometrial area of the cysts (Figure 4d, middle), or uterine innervation (Figure 4d, right), suggesting independent THC effects on these histological changes. Figure 4 with 1 supplement see all Download asset Open asset Effects of THC on the histological changes observed in mice with ectopic endometrium. (a) Ectopic endometrial growths of mice treated with THC were smaller (left graph) and had less endometrial tissue (right graph) than those of mice receiving vehicle. Scale bar = 1 mm. (b) THC increased innervation in sham mice but prevented uterine hyperinnervation in endometriosis mice. Blue is DAPI and white is β-III tubulin. Scale bar = 100 μm. (c) As expected, 17-β estradiol levels were higher in mice in proestrus (left). Estrogen levels were similar in all experimental conditions (right). (d) There was a positive correlation between cyst diameter and plasma levels of 17-β estradiol (left, r = 0.450). Absence of correlation of estrogen levels with cyst endometrial area (middle, r = 0.263) and uterine innervation (right, r = 0.039). THC dose: 2 mg/kg/day. Error bars are mean ± SEM. Student t-test (a, left graph), Mann Whitney U (a, right graph), two-way ANOVA + Bonferroni (b), mixed model + Bonferroni (c, left); Two-way ANOVA (c, right) and Pearson correlation (d). *p<0.05, **p<0.01 vs sham. +p<0.05, ++p<0.01 vs vehicle. ^p<0.05, ^^p<0.01 vs proestrus. Endo, endometriosis; THC, Δ9-tetrahydrocannabinol. Figure 4—source data 1 Effects of repeated THC on histopathological features. https://cdn.elifesciences.org/articles/50356/elife-50356-fig4-data1-v2.xlsx Download elife-50356-fig4-data1-v2.xlsx We also assessed possible effects of THC on ovarian functioning, since previous works have suggested inhibitory effects of THC on folliculogenesis and ovulation (Adashi et al., 1983; El-Talatini et al., 2009). Numbers of preantral follicles, antral follicles and corpora lutea were similar in all groups in our experimental conditions (Figure 4—figure supplement 1c). These data suggest that endometriosis and THC were void of overt effects on ovarian follicle maturation and luteinization, however, other effects of endometriosis or THC on fertility cannot be excluded in our model. Similarly, the presence of prominent symptoms of endometriosis such as dysmenorrhea or dyspareunia could not be evaluated. Conclusions Here we show for the first time that chronic administration of a moderate dose of the phytocannabinoid THC relieves mechanical hypersensitivity of caudal abdominal area, pain unpleasantness and cognitive impairment associated with the presence of ectopic endometrial cysts. These behavioral manifestations correlate with a decrease in the size of ectopic endometrium in THC-exposed mice. However, the pain-relieving effects of this particular dose of THC were not accompanied by a modification of anxiety-like behavior associated with endometriosis and effects on spontaneous pain were not evaluated in this work. Interestingly, THC produced opposite cognitive effects in sham and endometriosis mice. THC also induced an increase in markers of uterine innervation in sham animals, but prevented such changes in endometriosis mice, suggesting again different effects of THC under chronic inflammatory conditions. Importantly, THC also inhibited the growth of ectopic endometrium without apparent consequences on the eutopic endometrium and ovarian tissues. Altogether, the present data obtained in a preclinical model of endometriosis underline the interest in conducting clinical research to assess the effects of moderate doses of THC on endometriosis patients. Based on our results, we (clinicaltrials.gov, #NCT03875261) and others (gynica.com) have planned the initiation of clinical trials to provide evidence on the translatability of these results to women with endometriosis. These novel clinical trials will evaluate this new possible endometriosis treatment under pathological human conditions. However, cannabis has a large number of potential side effects, as well as a high potential for abuse liability (Curran et al., 2016), that have to be considered by physicians and patients. Therefore, the use of cannabis in unregulated scenarios should be discouraged taking into account these serious side effects. Materials and methods Key resources table Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional informationStrain, strain background (Mus musculus, female)C57Bl/6JCharles Rivers, Lyon, FranceC57Bl/6JFemaleChemical compound, drugTHC (Tetrahydrocannabinol)THC-Pharm-GmbHNatural THCChemical compound, drugCremophor ELSigma-AldrichC5135; Kolliphor ELChemical compound, drug0.9%, NaCl physiological salineLaboratorios ErnVituliaChemical compound, compound, in for for compound, in with tubulin compound, compound, or mice Rivers, Lyon, were in all the Mice were 8 old at the of the experiments and were in of 4 to 5 mice with to and The conditions were at ± and ± in on between 8 and 8 were to conditions and for 1 week the of the animal were conducted in accordance with and for and of and by and Mice were to treatment groups and all experiments were for and surgical conditions. THC was from as natural THC with This of natural THC has been in multiple research studies et al., 2018; et al., 2011; et al., 2013; et al., et al., et al., et al., 2011; Puighermanal et al., 2013). To the of the THC – was for cannabinoid and and for These revealed no detectable of other cannabinoids or Data 2, 3 and THC was in a vehicle of and and was administered in a of 5 a The phase of the estrous was assessed by histological of cells by et al., the day of the surgeries and the day of mice were and of were 5 into the The fluid was with blue and observed at under a of endometriosis lesions were surgically-induced as described et al., with some uterine from mice at were and into (2 2 mice were with in for for and a of 1 was to the abdominal compartment. Endometriosis mice had uterine to the whereas mice of abdominal and abdominal and were using a The nociceptive, and cognitive manifestations associated with the presence of ectopic endometrium were in a first the of baseline mechanical sensitivity endometriosis or sham surgery was and nociceptive responses were assessed again and 28 days after behavior and cognitive performance were evaluated on days