Implication of the enteric glia in the IBS-like colonic inflammation associated with endometriosis

Abstract

Background: Endometriosis is a complex gynecological disorder characterized by the ectopic growth of endometrial tissue. Symptoms of endometriosis are known to impair the quality of life of patients, and among these are found dysmenorrhea, chronic pelvic pain, and gastrointestinal (GI) issues. GI issues such as painful bowel movements, bloating and constipation or diarrhea, are one of the common reasons for misdiagnosis with irritable bowel syndrome (IBS). Enteric glial cells (EGC) are known to play a role in pain associated with IBS, and reactive gliosis has been reported in patients with IBS, but the role of EGC in endometriosis has yet to be elucidated. We hypothesized that endometriosis will induce reactive gliosis, with increased expression of the glial fibrillary acidic protein (GFAP) and S100B, in the myenteric plexus of colonic sections in an animal model of endometriosis.

Methods: In the present study animal experiments were employed to explore the impact of endometriosis on the gastrointestinal tract. Using a surgically induced endometriosis rat model, we collected ileal and colonic segments for analysis. We used H&E to assess microscopic damage in colon and ileum, immunofluorescence to measure GFAP and S100B expression in the colon, and toluidine blue staining to measure mast cell infiltration in colon and ileum. Immunofluorescence images were captured using confocal microscope and analyzed using ImageJ software.

Results: All endometriosis animals developed vesicles. These animals had a significant increase in the colonic macroscopic damage compared to Sham and Naïve controls. Colonic and ileal sections didn't show statistical differences in microscopic damage between groups, yet endometriosis ileum had significantly increased mast cell infiltration compared to Naïve. GFAP immunostaining showed significantly increased integrated density in endometriosis when compared to Sham or Naïve, while no statistical difference was found in S100B integrated density between groups.

Conclusions: We conclude that endometriosis can alter GI homeostasis by inducing colon inflammation, reactive gliosis, and ileal mast cell infiltration. Taken together this suggests endometriosis can mimic IBS histopathology beyond the symptomatology, reinforcing this disease's complexity and the need to treat it beyond the gynecological setting.

Keywords: Colon; Endometriosis; Enteric glia; Inflammation.

Fig. 1

Pain associated with endometriosis. A) Paw withdrawal threshold of Sham Control decreased significantly by Day 12 (after surgery) but returned to normal by Day 19. Endometriosis animals paw withdrawal decreased significantly by Day 19. B) No difference in hot plate latency time was found across groups (n = 8 animals/per group ± s.d. *p < 0.05)

Fig. 2

Colonic damage associated with endometriosis. A) Colonic damage was significantly increased in endometriosis animals compared to Naïve and Sham Control. Analyzed parameters show endometriosis has increased B) adhesions, C) hyperemia and D) full thickness (n = 8 animals/group ± s.d.**p < 0.01 ***p < 0.001 ****p < 0.0001)

Fig. 3

Endometriosis increases ileal mast cell infiltration. A) No significant differences were found in colonic mast cell count among groups. B) Endometriosis animals showed a statistically significant increase in ileal mast cell infiltration when compared to Naïve (n = 6–8 animals/group ± s.d. *p < 0.05)

Fig. 4

Endometriosis induces reactive gliosis. (A) Endometriosis animals showed an increase of GFAP expression when compared to Sham Control and Naïve animals. (B) No significant differences were found among groups for S100B expression. (C) Representative pictures of GFAP, S100B, DAPI and Merged for colonic tissues (4–5 animals/group ± s.d. ***p < 0.001 ****p < 0.0001)

Fig. 5

Endometriosis induces no changes in ⍺-7 receptor expression. A) Endometriosis animals showed no statistically significant difference in ⍺-7 receptor integrated density or B) percentage of area compared to Naïve and Sham Control. C) Representative pictures of Naïve, Sham Control, and Endometriosis colonic tissues (n = 6–8 animals/per group ± s.d.)

Fig. 6

Translational assessment. Visual comparison between findings in the clinical scenario and our animal model. Increased numbers of mast cells in the ileum and the higher expression of GFAP with no change in S100B in our endometriosis animal model echo findings in IBS patients suggesting similarities in underlying mechanisms for gastrointestinal disturbances. ↑ means increased, - means no difference