1. Human Biology and Medicine
  2. Neuroscience
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Disease-modifying effects of natural Δ9-tetrahydrocannabinol in endometriosis-associated pain

  1. Alejandra Escudero-Lara
  2. Josep Argerich
  3. David Cabañero  Is a corresponding author
  4. Rafael Maldonado  Is a corresponding author
  1. Universitat Pompeu Fabra, Spain
  2. IMIM (Hospital del Mar Medical Research Institute), Spain
Short Report
Cite this article as: eLife 2020;9:e50356 doi: 10.7554/eLife.50356
8 figures, 3 tables and 1 additional file

Figures

Figure 1 with 3 supplements
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—figure supplement 1
Nociceptive responses to abdominal mechanical stimulation with von Frey filaments.

(a) Significantly higher frequency of responses and AUC was observed in endometriosis mice when compared to sham mice on days 14, 21 and 28 after the surgery. For each day, left panel is frequency of response to each von Frey filament and right panel is the corresponding mechanical sensitivity represented by the area under the curve of frequency of response to von Frey filaments. Higher values mean higher mechanical pain. (b) Site of application of the von Frey filaments (Test area). Error bars are mean ± SEM. For each day, one-way repeated measures ANOVA (left panels) and Student t-test (right panels). *p<0.05, **p<0.01 vs sham. Endo, endometriosis; AUC, area under the curve.

Figure 1—figure supplement 2
Nociceptive responses to abdominal and paw mechanical stimulation with von Frey filaments.

(a) Frequency of responses to von Frey filaments applied against the caudal abdominal area and corresponding AUCs on day 14 after surgery were significantly higher in endometriosis mice when compared to sham mice. (b) Frequency of responses to von Frey filaments applied against the hind paw and corresponding AUCs were similar before and after surgery in endometriosis and sham mice. For each day, left panel is frequency of response to each von Frey filament and right panel is the corresponding mechanical sensitivity represented by the area under the curve of frequency of response to von Frey filaments. Higher values mean higher mechanical pain. Error bars are mean ± SEM. One-way repeated measures ANOVA (left panels) and Student t-test (right panels). ***p<0.001 vs sham. Endo, endometriosis; AUC, area under the curve.

Figure 1—figure supplement 3
Density of beta-III tubulin-labeled fibers in uteri of endometriosis and sham mice.

(a) The percentage of immunoreactive area of the mesometrial aspect of the uterus was higher in endometriosis mice. (b) The percentage of immunoreactive area of myometrium did not differ between groups. Blue is DAPI and white is β-III tubulin. Scale bar = 100 μm. Error bars are mean ± SEM. Student t-test. **p<0.01 vs sham. Endo, endometriosis.

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 3 with 3 supplements
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
Figure 3—figure supplement 1
Effect of chronic THC treatment on nociceptive responses to abdominal mechanical stimulation with von Frey filaments.

Endometriosis mice treated with vehicle showed higher frequency of response than endometriosis mice treated with THC and sham mice treated with vehicle. THC dose: 2 mg/kg/day. Error bars are mean ± SEM. Two-way repeated measures ANOVA + Bonferroni. *p<0.05, ***p<0.001 vs sham. +p<0.05, +++p<0.001 vs vehicle. Endo, endometriosis; THC, Δ9-tetrahydrocannabinol.

Figure 3—figure supplement 2
Effect of a repeated THC treatment starting on day 8 after surgeries on nociceptive responses to abdominal mechanical stimulation with von Frey filaments.

Endometriosis mice treated with THC for 7 days (Endo – 7daysTHC) and endometriosis mice treated acutely with THC (Endo – AcuteTHC) showed a reduction in the frequency of response to von Frey filaments on day 14. THC dose: 2 mg/kg/day. Error bars are mean ± SEM. Mixed model + Bonferroni. ***p<0.001 vs sham; +++p<0.001 vs vehicle. Endo, endometriosis; THC, Δ9-tetrahydrocannabinol.

Figure 3—figure supplement 3
Effect of a repeated THC treatment starting on day eight after surgeries on nociceptive responses to hind paw mechanical stimulation with von Frey filaments.

(a) Mechanical sensitivity in the hind paw remained stable after endometriosis surgery or THC treatment. 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) Similar frequency of responses was observed in all groups of mice before and after the surgery. THC dose: 2 mg/kg/day. Error bars are mean ± SEM. Mixed model + Bonferroni. Endo, endometriosis; THC, Δ9-tetrahydrocannabinol, AUC, area under the curve.

Figure 4 with 1 supplement
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
Figure 4—figure supplement 1
Histological features of reproductive tissues after chronic THC treatment.

(a) Cyst innervation was unaffected by THC. Blue is DAPI and white is β-III tubulin. Scale bar = 100 μm. (b) Uterine diameter and area of endometrial tissue were similar among the groups. Scale bar = 1 mm. (c) Number of preantral follicles, antral follicles and corpora lutea were unchanged after endometriosis or THC treatment. Error bars are mean ± SEM. Student t-test (a) and two-way ANOVA (b and c). Endo, endometriosis; THC, Δ9-tetrahydrocannabinol.

Author response image 1
Effects of THC and stage of the estrous cycle on cyst diameter.

(a) Cyst diameter in the different group treatments and estrous cycle stages. A significant effect of the treatment was found. (b) Significance of the effects according to a Two-way ANOVA. Error bars are mean ± SEM. *p<0.05 vs vehicle. Endo, endometriosis.

Author response image 2
Effects of THC and stage of the estrous cycle on cyst endometrial area.

(a) Cyst endometrial area in the different group treatments and estrous cycle stages. (b) Shapiro-Wilk test revealed that data on endometrial area did not follow a normal distribution. Subsequent Kruskal-Wallis test showed no significant difference among these groups. Error bars are mean ± SEM. Endo, endometriosis.

Author response image 3
Effects of THC and stage of the estrous cycle on uterine innervation.

(a) Uterine innervation (represented as% of beta-III tubulin immunoreactive area) in the different experimental groups and estrous cycle stages. (b) Significance of the effects according to a Three-way ANOVA. A significant effect of the interaction between surgery and treatment was found as shown in Figure 4B. However, no effect of the estrous cycle was detected. Error bars are mean ± SEM. Endo, endometriosis.

Author response image 4
Total time exploring objects in the novel object recognition test.

The total time exploring the objects was not affected by (a,b) endometriosis or (b) THC treatment. Error bars are mean ± SEM. Student t-test (a) and two-way ANOVA (b). Endo, endometriosis.

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Strain, strain background (Mus musculus, female)C57Bl/6JCharles Rivers, Lyon, FranceC57Bl/6JFemale
Chemical compound, drugTHC
(Tetrahydrocannabinol)
THC-Pharm-GmbHNatural THC
Chemical compound, drugCremophor ELSigma-AldrichC5135; Kolliphor EL
Chemical compound, drug0.9%, NaCl physiological salineLaboratorios ErnVitulia
Chemical compound, drugEthanolScharlabET00051000
Chemical compound, drugVaporised Isoflurane in oxygenVirbacVetflurane4% V/V for induction; 2.5% V/V for maintenance
Chemical compound, drugOptimal cutting temperature compoundSakura finetek25608–930Item code 4583
Biological sample (Equus asinus)Normal donkey serumSigma-AldrichD9663-10ML3% in PBS with 0.3% Triton X-100
Biological sample (Capra aegagrus hircus)Normal goat serumVector labS-1000
AntibodyRabbit polyclonal anti-beta-III tubulin antibodyAbcamab18207(1:2000)
AntibodyDonkey polyuclonal anti-rabbit Alexa Fluor A488 antibodyThermo Fisher ScientificA21206(1:1000)
AntibodyGoat polyclonal anti-rabbit Alexa Fluor A555 antibodyAbcamab150078(1:1000)
Chemical compound, drugDAPI Fluoromount-G mounting mediaSouthernBiotech0100–20
Chemical compound, drugCalcium EDTA (Sodium calcium edetate)Sigma-AldrichSodium calcium edetate
Commercial assay or kitEnzyme-linked immunosorbent assay; ELISACalbiotechES180S-100
Software, algorithmNIH Image J softwareWayne Rasband
Software, algorithmGraphPad Prism 8GraphPad Software, Inc
Software, algorithmIBM SPSS 23 softwareIBM Corporation
Software, algorithmSmart 3.0 videotracking softwarePanlab
Author response table 1
Significance levels of the filament number in each evaluation day (Two-way repeated measures ANOVA).
FSig.
Day -119.9550.000
Day 74.6550.001
Day 145.4180.000
Day 216.6700.000
Day 282.2160.059
Author response table 2
Number of animals in each phase of the cycle.
SurgeryTreatmentPhase of the estrous cycleN
ShamVehicleProestrus0
Estrus5
Metestrus0
Diestrus5
THCProestrus1
Estrus3
Metestrus0
Diestrus4
EndoVehicleProestrus1
Estrus2
Metestrus0
Diestrus7
THCProestrus1
Estrus3
Metestrus0
Diestrus6

Data availability

All data supporting the findings of this study are available within the manuscript and its source data files. Source data files have been provided for Figures 1, 2, 3 and 4 and their figure supplements.

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