Endometriosis as a neurovascular condition: estrous variations in innervation, vascularization, and growth factor content of ectopic endometrial cysts in the rat

Abstract

Endometriosis is a poorly understood, estradiol-dependent condition associated with severe pelvic pains and defined by vascularized endometrial growths outside the uterus. Endometriosis is produced in cycling rats by autotransplanting pieces of uterus onto abdominal arteries where they develop into cysts. The surgery induces vaginal and abdominal muscle hyperalgesia, whose severity is greatest in proestrus and nearly absent in estrus. The cysts contain growth factors and cytokines and develop their own sympathetic and sensory C- and Adelta-fiber innervation. Here, we used quantitative immunostaining and protein array analyses to test the hypothesis that the innervation and growth factor/cytokine content of the cysts, but not uterine horn, contribute to proestrous-to-estrous changes in hyperalgesic severity. If so, these characteristics in the cysts, but not the uterine horn, should change with estrous stage. In cysts, the density of sympathetic (but not sensory) neurites and amounts of NGF and VEGF proteins (but not cytokines IL-1, IL-6, IL-10, or TNF-alpha) were greater in proestrus than estrus. These changes were accompanied by vascular changes. Both sympathetic and sensory fibers in both stages colabeled with TrkA, indicating that changes in NGF could act on both afferent and efferent fibers. In contrast with the cysts, no changes occurred in the uterine horn between proestrus and estrus. Together, these results suggest that coordinated proestrous-to-estrous changes in innervation and vascularization of the cysts contribute to similar changes in hyperalgesic severity. The findings also encourage consideration of endometriosis as a neurovascular condition.

Fig. 1

Photomicrographs of parts of a section immunostained with vesicular monoamine transporter (VMAT). A: Low-power view of area where photomicrographs in B and C were taken. B: at the entrance to the cyst, neurites are very dense and associated mainly with blood vessels (arrows) as they enter the wall of the cyst in the hilus region. C: further into the cyst, these neurites become less dense but also extend into the myometrium and epithelial lining. Note that this pattern was the same for neurites stained with other markers and for the uterine horn. Calibration bar: 500 μm for A; 100 μm for B and C.

Fig. 2

The regions outlined in A and B indicate the hilus region of two cysts, within which neurite labeling was counted (see Immunohistochemistry section in METHODS). The boxes in A and B (within the outline) indicate the approximate region where the higher-power photomicrographs (C–H) were taken. Photomicrographs of sections immunostained with antibodies for VMAT2 (C and D), CGRP (E and F), or transient receptor potential vanilloid-1 (TRPV1) (G and H) in the wall of cysts in proestrus (left) or estrus (right). Note that the density of neurites positively immunostained by VMAT2 (sympathetic fibers) was greater in proestrus than estrus, whereas there were no proestrous-to-estrous differences in the density of neurites positively immunostained by CGRP and TRPV1 (sensory fibers). Calibration bar: 500 μm for A and B; 50 μm for C–H.

Fig. 3

The regions outlined in A and B indicate an area in the uterine horn comparable to the hilus in the cysts (see Fig. 2, A and B), within which neurite labeling was counted (see Immunohistochemistry section in METHODS). The boxes in A and B (within the outlines) indicate the approximate regions where the higher-power photomicrographs (C–H) were taken. Photomicrographs of sections immunostained with antibodies for VMAT2 (C and D), CGRP (E and F), and TRPV1 (G and H) in the wall of the uterine horn in proestrus (left) or estrus (right). There were no proestrous-to-estrous differences in the density of neurite labeling for any of the markers. Calibration bar: 500 μm for A and B; 50 μm for C–H.

Fig. 4

Density of neurites in the hilus region of the cysts (A) and uterine horn (B) immunostained with VMAT2, CGRP, and TRPV1 ∼11 wk after endometriosis (ENDO) surgery. In the cysts, the density of sympathetic fibers labeled by VMAT2 was significantly greater in proestrus than estrus, whereas the density of neurites labeled with the other markers showed no significant proestrous-to-estrous differences. In proestrus (but not estrus), the density of TRPV1 neurites was less than the density of sympathetic neurites. In the uterine horn, there were no proestrous-to-estrous differences in the density of neurites labeled by any of the markers. In both stages, the density of TRPV1 neurites was signficantly less than the density of sympathetic and CGRP labeled neurites. Data are shown as means ± SE. *P < 0.05 compared with proestrus.

Fig. 5

Normalized spot signal intensities on films of protein array membranes of growth factors and cytokines in the cysts (A) and in the uterine horn (B) in proestrus or estrus ∼11 wk after ENDO surgery. In the cysts, the levels of growth factors VEGF and NGF, but not the cytokines, including IL-1β, IL-6, IL-10, and TNF-α, were significantly higher in proestrus than estrus. In contrast, there were no proestrus-to-estrus differences in the levels of any of these six proteins in the uterine horn. VEGF levels were greater in the cysts than in the uterine horn in proestrus but not estrus. There were no differences in cyst-uterine horn levels for the other proteins. Data are shown as means ± SE. *P < 0.05, compared with proestrus. A, inset: examples of a portion of developed films from two arrays showing double spots for the VEGF antibody, and the positive (Pos) and negative (Neg) controls. One film was from an array of a cyst in proestrus (left spots) and the other was from an array of another cyst in estrus (right spots). A key for the two arrays is shown in the table next to them.

Fig. 6

Vascularization of the cysts and uterine horn in proestrus and estrus. A and B: percentage of the total area occupied by blood vessels (BV) in the cysts and uterine horn, respectively. C and D: number of BVs within that area of the cysts and uterine horn, respectively. E and F: mean diameter of BVs in that area of the cysts and uterine horn, respectively. Note that there were significant proestrous-to-estrous changes in vascularization of the cysts but not the uterine horn. In the cysts, from proestrus-to-estrus, there was a small insignificant decrease in the diameter of BVs (E) and a large, significant increase in the number of BVs (C), resulting in an overall significant increase in the percentage of the total area occupied by BVs (A). In contrast, there were no significant estrous changes in vascularization of the uterine horn. Data are shown as means ± SE. **P < 0.01, compared with estrus.

Fig. 7

Examples showing photomicrographs of sections of cysts double-immunostained with TrkA and VMAT2, or TrkA and CGRP antibodies. Green indicates single labeling for VMAT2 (A) or CGRP (B). Red indicates single labeling for TrkA (C and D). Yellow and/or orange indicates double labeling of TrKA with VMAT2 (E) or TrkA with CGRP (F). Calibration bar, 50 μm for A–F.