Innervation of ectopic endometrium in a rat model of endometriosis

Abstract

Endometriosis (ENDO) is a disorder in which vascularized growths of endometrial tissue occur outside the uterus. Its symptoms include reduced fertility and severe pelvic pain. Mechanisms that maintain the ectopic growths and evoke symptoms are poorly understood. One factor not yet considered is that the ectopic growths develop their own innervation. Here, we tested the hypothesis that the growths develop both an autonomic and a sensory innervation. We used a rat model of surgically induced ENDO whose growths mimic those in women. Furthermore, similar to women with ENDO, such rats exhibit reduced fertility and increased pelvic nociception. The ENDO was induced by autotransplanting, on mesenteric cascade arteries, small pieces of uterus that formed vascularized cysts. The cysts and healthy uterus were harvested from proestrous rats and immunostained using the pan-neuronal marker PGP9.5 and specific markers for calcitonin gene-related peptide (CGRP) (sensory C and A delta fibers), substance P (SP) (sensory C and A delta fibers) and vesicular monoamine transporter (sympathetic fibers). Cysts (like the uterus) were robustly innervated, with many PGP9.5-stained neurites accompanying blood vessels and extending into nearby luminal epithelial layers. CGRP-, SP-, and vesicular monoamine transporter-immunostained neurites also were observed, with CGRP and SP neurites extending the furthest into the cyst lining. These results demonstrate that ectopic endometrial growths develop an autonomic and sensory innervation. This innervation could contribute not only to symptoms associated with ENDO but also to maintenance of the ectopic growths.

Fig. 1.

Surgical model of ENDO (A) and sections through a cyst immunostained for the pan-neuronal marker PGP9.5 (B–E). (A) Diagram shows the surgical procedure: a partial hysterectomy is carried out (X), and 2 × 2-mm squares of uterine horn are sewn around alternate mesenteric cascade arteries (black dots). (B) Low-magnification digital image of a section through the cyst and lumen (L); note the bundle of nerves entering the hilus (arrow). (C) Higher-magnification view of the hilus area in B. Note the nerves entering the cyst wall (arrow). (D) Nerves (arrows) coursing through the cyst wall to the endometrium and approaching the epithelium (E) lining the cyst lumen (L). (E) Higher-magnification view of another part of the cyst wall in B. Note the individual neurites (arrows) in the endometrial layer and approaching the epithelium (E). (Bars: B and C, 250 μm; D and E, 25 μm.)

Fig. 2.

Specific markers for afferent and efferent fibers in control uterus (A) and cyst (B–D) from the same rat. Sections in A–C were immunostained for CGRP (afferent fibers), those in D for VMAT (sympathetic fibers). (A) Uterine wall showing neurites (arrow) in the myometrium (M); E, epithelium; L, lumen. (B) Cyst wall showing neurites (arrow) in the myometrium (M) (compare with A). (C) Higher magnification of the cyst wall showing neurites (arrows) in the endometrium subjacent to the cyst epithelium (E) and appearing to enter the epithelium. (D) Cyst wall showing sympathetic neurites (arrows) in myometrium (M), near arteries and the endometrial layer. (Bar: 25 μm.)

Fig. 3.

Confocal microscope images of sections immunostained for specific markers for afferent fibers in a cyst. (A and B) Immunostained for SP. (C) Immunostained for CGRP. (A) Cyst wall showing neurites (arrow) in the endometrial stroma subjacent to the epithelium (E) lining the lumen (L). SP-immunoreactive neurites also closely surround the blood vessels (V) coursing through the wall and muscle layer (M) of the cyst. (B) Cyst wall showing individual SP-immunoreactive varicose neurites (arrow) extending into the epithelium (extent of the epithelium indicated by the double-headed arrow) lining the cyst lumen (L). (C) Image similar to B of the cyst wall showing CGRP-immunoreactive neurites (arrows) in the epithelial layer (indicated by the double-headed arrow) lining the cyst lumen (L). (Bar: 25 μm.)