Loss of sympathetic nerve fibers in the muscle wall in intestinal endometriosis

Introduction: Few studies investigated the effect of bowel endometriosis on intestinal physiology. Nerve fibers and their neurotransmitters are known to contribute to intestinal inflammation. Substance P (SP) is the main neurotrans- mitter of sensory nerve fibers and it is a proinflammatory neuropeptide. Tyrosine hydroxylase (TH) is sympathetic neurotransmitter and has antiinflammatory activity at high concentrations. This study aims to investigate the intestinal innervation around colorectal endometriotic lesion. Materials and methods: This study included women who had bowel endometriosis and underwent laparoscopic colorectal resection because of severe pain and gastrointestinal symptoms. Bowel specimens were collected immediately after surgery. From each patient a bowel specimen including serosa, muscularis propria and mucosa was obtained at two sites: one sample was collected near the endometriotic lesion and the other sample was obtained at the limit of the resected specimen in histologically confirmed healthy tissue. Specimens were immediately fixed in formalin and treated with sucrose. Afterwards samples were frozen in Tissue-Tec and stored at 2808C until analysis. Slices of 8 mm were used for immunofluorescence. Following air drying and rehydratation, the stain of the sympathetic nerve endings was performed with a rabbit polyclonal antibody against TH (Chemicon, dilution 1:1000) and the sensory nerve fibers were stained with a goat polyclonal antibody against SP (Santa Cruz, dilution 1:50). The slices were incubated with the primary anti- body for 3 h. After washing, samples were incubated with a secondary antibody for 90 min. For the TH staining, an Alexa Fluor 546-conjugated monoclonal goat anti-rabbit antibody was used. For the SP staining, Alexa Fluor 546-conjugated monoclonal rabbit anti-goat was used. After washing, nuclei staining was performed by 0.1% 4,6-diamidino-2-pheny- lindole (DAPI) in PBS for 4 min. Finally the slices were embedded with fluorescent mounting medium and stored until microscopy at 48C. The numbers of positive nerve fibers per square millimeter were determined by averaging the number of stained nerve fibers (minimum length 50 mm, determined through a micrometer eyepiece) in 17 randomly selected high power fields of view. We controlled the positive nerve fiber staining by incubating the tissue with respective control antibodies which always yielded a negative result. Statistical analysis was performed by using the Mann-Whitney U test. Results: Specimens were obtained from 9 women. A significant loss of sympathetic nerve fibers in the bowel muscularis propria was observed in the proximity of the endometriotic nodule when com- pared with the specimen excised in healthy bowel tissue (260%, P 1⁄4 0.045). The density of the sensory nerve fibers in the muscle wall near to the lesions tended to be higher compared to the same region far away from the nodule, but there was no significant difference (P 1⁄4 0.297). Conclusions: This study demonstrates that there is a loss of the sympathetic innervation around bowel endometriotic nodule; future studies should determine whether proinflammatory sensory nerves are up-regulated by the presence of endometriosis. Understanding of the effects of endometriosis on intestinal innervation may be useful to improve its treatment.