Endocannabinoid involvement in endometriosis

Abstract

Endometriosis is a disease common in women that is defined by abnormal extrauteral growths of uterine endometrial tissue and associated with severe pain. Partly because how the abnormal growths become associated with pain is poorly understood, the pain is difficult to alleviate without resorting to hormones or surgery, which often produce intolerable side effects or fail to help. Recent studies in a rat model and women showed that sensory and sympathetic nerve fibers sprout branches to innervate the abnormal growths. This situation, together with knowledge that the endocannabinoid system is involved in uterine function and dysfunction and that exogenous cannabinoids were once used to alleviate endometriosis-associated pain, suggests that the endocannabinoid system is involved in both endometriosis and its associated pain. Herein, using a rat model, we found that CB1 cannabinoid receptors are expressed on both the somata and fibers of both the sensory and sympathetic neurons that innervate endometriosis's abnormal growths. We further found that CB1 receptor agonists decrease, whereas CB1 receptor antagonists increase, endometriosis-associated hyperalgesia. Together these findings suggest that the endocannabinoid system contributes to mechanisms underlying both the peripheral innervation of the abnormal growths and the pain associated with endometriosis, thereby providing a novel approach for the development of badly-needed new treatments.

Figure 1

CB1 co-localization on sensory and sympathetic axons in ectopic endometrial cysts, and on cyst-innervating sensory and post-ganglionic sympathetic ganglion cells in thoracic dorsal root ganglion (DRG) and coeliac ganglion (CG), respectively. Boxed areas in b show approximate locations of photomicrographs in the other panels. Panel a shows cyst-innervating neurons in a T10 DRG retrogradely-labeled with Dil dye (blue), some of which co-label with both CB1 (red) and sensory-calcitonin gene related peptide (CGRP-sens; green) antibodies. Tripe-labeled neurons appear white. Panel c shows cyst-innervating neurons in a CG, some of which co-label with both CB1 (red) and sympathetic-tyrosine hydroxylase (TH-symp; green) antibodies. Tripe-labeled neurons appear white. Panel d shows CB1-labeled (red) either CGRP-sens or vesicular monoamine transporter 2-sympathetic (VMAT2-symp)-labeled fibers (both green) in the cyst. Double-labeled fibers appear yellow. Arrows in all merged images point to examples of either tripe-labeled ganglion cells (panels a and c) or double-labeled axons (panel d). See Methods for details of image acquisition and preparation used to generate this figure. Calibration bars, 100 µm.

Figure 2

Density of CB1-positive fibers is greater in the cysts and uterine horn. The regions outlined in the photomicrographs (a, b) show the approximate area in which counts were made in each tissue (i.e., the hilus of the cyst; the region in the uterine horn at the entrance of the uterine artery). Note that the size of this area did not differ between the two tissues (P = 0.89, unpaired Student’s t-test). For the graph in c, *, P = 0.024; unpaired Student’s t-test. Data shown as means and s.e.m. Calibration bar = 0.5 mm for both photomicrographs.

Figure 3

Effects of WIN2 (CB1/CB2 agonist), AM 251 (CB1 antagonist), AM 630 (CB2 antagonist) on the visceromotor reflex threshold (VMRth) to vaginal distention. The diagram in a shows the testing set-up. The graph in b compares the effect of 2 doses of WIN2 on the VMRth in shamENDO and ENDO rats. Comparing only the back bars shows that the pre-drug VMRth was significantly greater in shamENDO than in ENDO rats; P = 0.00025, unpaired Student’s t-test, which confirms earlier studies that ENDO produces referred muscle hyperalgesia [38]. In the shamENDO group, WIN2 had no significant affects; ANOVA (P = 0.353). In contrast, in the ENDO group, both doses of WIN2 increased the VMRth significantly. **P < 0.01 compared with pre-drug; ANOVA followed by post-hoc paired Student’s t-tests. Thus, WIN2 alleviates referred muscle hyperalgesia in the ENDO group. The graph in c illustrates, in ENDO rats, the effects of WIN2 when combined with antagonists AM 251 (CB1-antagonist; CB1 ant) and AM630 (CB2 antagonist; CB2-ant) and with the antagonists delivered alone. **P < 0.008 compared with pre-drug; ANOVA followed by post-hoc Student’s t-tests, with alpha set at 0.008 (Bonferroni correction). The CB1, but not the CB2 antagonist prevented the effect of WIN2, which indicates that WIN2 acts via the CB1 receptor. The CB1, not the CB2, antagonist alone significantly reduced the VMRth (increases nociception), indicating that endocannabinoids normally suppress endometriosis-induced hyperalgesia. Data shown as means and ± s.e.m.

Figure 4

Lack of tolerance to repeated administration of WIN2 over 8 days. The graph compares the effect on the VMRth of a single treatment of WIN2 (3 mg/kg) with multiple treatments in ENDO rats. In the multiple group, WIN2 was given three times at four-day intervals. No values between single-and multiply-dosed groups differed significantly; i.e., pre drug (P = 0.775), post-drug (P = 0.437), or the change in VMRth from pre to post drug (P = 0.711, not shown on graph); Students t-tests. Data are shown as means ± s.e.m.