Fig 1.
Diagram of experimental protocols.
Rats in Experiment 1 received endometriosis or sham surgery and were allowed to progress for 7 days. Rats in Experiment 2 received sham surgery or endometriosis. Then the rats from the endometriosis group were injected with either vehicle (10% Tween 80) or antalarmin (i.p., once daily) for seven consecutive days after surgery and allowed to progress for 53 additional days. During the endometriosis progression period, animals were undisturbed except for weekly weighing done at the same time of cage changing.
Fig 2.
Behavioral assessment for anxiety at 7 days after sham or endometriosis surgery.
(A) Total distance traveled during the first 5 minutes of the open field test and (B) time spent in the center of the arena. (C) Total distance traveled in the zero maze during the 5 min duration of the test and the amount of time spent in the open segments of the maze (D). * represents p< 0.05 compared to sham group. n = 8 in each group.
Table 1.
Characteristics of endometriosis vesicles at seven days after auto-transplantation surgery.
Fig 3.
Adrenocorticotropic hormone (ACTH) and corticosterone levels in serum.
(A) ACTH and (B) corticosterone were quantified in the same animals that underwent behavioral measurements (n = 8 per group). No significant differences between groups were noted for either parameter.
Fig 4.
qRT-PCR and Western Blot analysis of CRHR1 within endometriosis vesicles and uterus of the endometriosis rats and sham rats.
(A) At 7 days after the autotransplantation surgery to induce endometriosis, we observed a significant two-fold increase in CRHR1 mRNA within endometriosis vesicles only (n = 7 per group). (B) Western blot analysis confirmed the qRT-PCR observation showing a significant increase in CRHR1 within the endometriosis vesicles only. (C) Sample Western blot bands with the respective marker. CRHR1 was observed just above the 50 kDa marker and β-actin above the 37kDa marker. * represents p< 0.05 compared to sham group. ** represents p< 0.01 on a post-hoc analysis compared to the two other groups. Sham group n = 7, endo uterus, n = 7, endo vesicles n = 9.
Fig 5.
Behavioral assessment for anxiety at 60 days after sham or endometriosis surgery.
Rats that received sham surgery did not receive any treatment (n = 10). Rats in the endometriosis groups received antalarmin (n = 20) or vehicle treatment (n = 20) during one week after surgery until 53 days before behavioral testing. (A and B) All animals were tested in the open field or (C and D) the elevated zero maze. (A) In comparison to sham, we observed a significant decrease in locomotor activity of the group that received antalarmin. (B) However, time spent in the center of the open field was not different between groups. (C) In the elevated zero maze, a significant decrease in locomotion was observed for rats that had endometriosis as compared to sham, regardless of the drug treatment. (D) Rats with endometriosis, regardless of drug treatment, showed a trend towards spending less time in the open segment of the zero maze as compared to sham group. * represents p< 0.05 compared to sham.
Fig 6.
Morphological characteristics of endometriosis vesicles.
(A) The percent of implants that developed into vesicles was significantly lower in the antalarmin treated group (n = 20) compared to the vehicle control group (n = 20). (B) The total weight of all vesicles per rat was smaller for the antalarmin treated rats. (C) The average vesicle volume per rat was significantly smaller for the antalarmin treated group compared to the vehicle control group. (D) The average vesicle area per rat was significantly smaller in the antalarmin group compared to the vehicle group. (E) Vesicles that developed were classified by grade based on a scale by size showing that the individual vesicles in the antalarmin treated group tended to be smaller (grade 2) or not develop at all (grade 1). * p< 0.05, ** p< 0.01.
Fig 7.
Serum corticosterone, ACTH and adrenal weight.
We used ELISA to measure (A) corticosterone and (B) adrenocorticotropic hormone (ACTH) in the serum of rats. There was no significant difference between groups in serum corticosterone levels at the time of sacrifice. However, a significantly higher level of ACTH was observed for rats that received antalarmin compared to the two other groups. (C) The wet weight of adrenal glands was collected and normalized for body weight for each animal. Significantly lighter adrenal glands were noted in rats with prior antalarmin treatment compared to sham group. * represents p< 0.05.
Fig 8.
mRNA levels measured by qRT-PCR from the uterus and endometriosis vesicles.
(A) corticotropin releasing hormone (CRH), n = 14 vehicle and n = 9 antalarmin. (B) Urocortin 1 peptide, n = 9 in both groups. Data normalized to the uterus of sham rats. * represents p< 0.05 compared to sham rats’ uterus.
Fig 9.
mRNA levels measured by qRT-PCR from the uterus and endometriosis vesicles.
(A) corticotropin releasing hormone receptor type 1 (CRHR1). (B) corticotropin releasing hormone receptor type 2 (CRHR2). (C) Glucocorticoid receptor (GR). Data normalized to the uterus of sham rats. * represents p< 0.05 compared to sham rats’ uterus. For all panels, n = 9 in vehicle and antalarmin groups.
Fig 10.
Percent increase in weight for rats with endometriosis treated with antalarmin or vehicle.
(A) After seven days of treatment, antalarmin treatment significantly decreased the weight of the rats and this difference persisted for two additional days after the drug treatment has stopped. (B) During the subsequent weeks, the antalarmin group remained weighing less than control group and by weeks 6 and 7 this difference reached statistical significance. * p< 0.05, ** p< 0.01.