Stress During Development of Experimental Endometriosis Influences Nerve Growth and Disease Progression

Abstract

Purpose: We have previously shown that stress prior to induction worsens clinical presentation and inflammatory parameters in a rat model of endometriosis. This study was designed to examine whether stress during the development of endometriosis can affect the growth of endometriotic implants through nerve growth and immune alterations.

Methods: Endometriosis was surgically induced in female Sprague-Dawley rats by suturing uterine horn implants onto the small intestine mesentery. Two weeks later, one group of rats (endo-stress) was subjected to a 10-day swim stress protocol. Controls had no stress (endo-no stress) or sutures only and stress (sham-stress). On day 60, all rats were killed and examined for the presence of endometriotic vesicles. The size of each vesicle was measured. The uterus and colon were removed and assessed for damage, cell infiltration, and expression of nerve growth factor (NGF), its receptors (p75 and Tropomyosin receptor kinase A (Trk-A)/pTrk-A), and calcitonin gene-related peptide, a sensory fiber marker. A differential analysis of peritoneal fluid white blood cell count was performed.

Results: Stress significantly increased endometriotic vesicle size but not colonic damage and increased infiltration of mast cells. Significantly increased expression of NGF and its receptors was found in the uterus of animals with endometriosis receiving stress.

Conclusions: Stress stimulates the development of ectopic endometrial vesicles in an animal model of endometriosis and increases inflammatory cell recruitment to the peritoneum. In addition, stress promotes nerve fiber growth in the uterus.

Keywords: Trk; endometriosis; nerve growth factor; p75; stress.

Figure 1.

A, Experimental design. Animals were subjected to swim stress for 10 consecutive days. All animals were checked for regular cycling by analysis of smears during the protocol and killed 60 days after surgery, with a smear at the time of killing. B, Stress increases anxiety levels. Both the sham-stress and endo-stress groups had increased fecal pellet counts compared to endo-no stress or normal indicating increased anxiety levels (n = 10-11 [SEM]; *P <.05, **P <.01, ***P <.001 vs endo-no stress or normal). C, Stress increases implant severity. None of the normal or sham-stress animals developed vesicles. All of the endo animals developed vesicles in at least one of the implant sites. The endo-stress animals had significantly bigger vesicles (n = 10-11 [SEM]; *P <.05). SEM indicates standard error of the mean.

Figure 2.

Effect of stress on inflammatory parameters. The endo-stress animals had (A) the highest MPO levels although this did not reach statistical significance and (B) increased numbers of mast cells compared to normal (*P< .05). (C) No significant differences were observed in numbers of white blood cells in the peritoneal fluid (n = 8-11 [SEM]). MPO indicates myeloperoxidase; SEM, standard error of the mean.

Figure 3.

Immunohistochemical staining in rat uterus. (A) Representative pictures of NGF and Trk-A expression in uterus (×40). Stress significantly increased the expression of (B) NGF and (C) its receptor Trk-A in uterus (n = 6-8 animals per group [SEM], **P <.01 vs normal, ^## P <.01, ^### P <.001 vs sham-stress, ^$$ P <.01 vs endo). NGF indicates nerve growth factor; SEM, standard error of the mean.

Figure 4.

Immunohistochemical staining in rat colon. (A) Representative pictures of NGF and Trk-A expression in colon (×40). Stress significantly increased the expression of (B) NGF and (C) its receptor Trk-A in colon (n = 6-8 animals per group [SEM], **P <.01 vs normal; ^# P <.05, ^## P <.01 vs sham-stress). NGF indicates nerve growth factor; SEM, standard error of the mean.