Luminescent mouse model of endometriosis: three-dimensional morphology of lesions and cytokine profiles

Abstract

The pathophysiology of endometriosis remains incompletely understood, necessitating the development of effective animal models for research. We generated and characterized a luminescent endometriosis mouse model utilizing luminescent B6-CAG-ELuc transgenic mice as uterine tissue donors and B6.Cg-c/c-hr/hr mice as recipients, enabling non-invasive in vivo imaging. Following transplantation of minced uterine tissue fragments into the peritoneal cavity of recipients, we monitored lesion growth via in vivo imaging system on 0, 14, 28, 42 days post transplantation. Morphology of the lesion was observed by dissecting microscopy, X-ray micro-computed tomography, and conventional histology. Inflammation-related serum cytokines were quantified using multiplex immunobeads assay. The growth of endometriotic lesions was efficiently observed by bioluminescence from day 0 through 42 days post transplantation. Comprehensive morphological observations revealed typical endometriotic lesions consisted of multiple fluid-filled cysts lined with single-layered epithelium, associated with glandular epithelial tissues and interstitial stroma. The level of IL-1β, IL-2, IL-6, IL-10, IL-12p70, IFN-γ, and TNF-α was quantified simultaneously in each serum sample to evaluate the temporal changes of each cytokine, showing four distinct patterns: IFN-γ and TNF-α showed continuous increase, IL-12p70 and IL-1β demonstrated gradual increase followed by marked elevation, IL-6 and IL-2 exhibited dramatic increase in later stages, while IL-10 showed transient increase followed by gradual decrease. In conclusion, this luminescent endometriosis mouse model using B6 luminescent transgenic mice as uterine tissue donor and B6.Cg-c/c-hr/hr recipient could be used to investigate comprehensive cytokine profiling in the development of endometriosis.

Keywords: bioluminescence; cytokine; endometriosis; in vivo imaging; micro-CT.

Fig. 1.

Generation of the endometriosis mouse. (A) An experimental work flow for the preparation of donor tissue fragments, surgical transplantation of the donor uterine tissues to the recipient, and observation of luminescent signals by IVIS. (B) Experimental time schedule. Recipient mouse was injected subcutaneously with 0.5 µg/mouse of 17β estradiol (E2) weekly. Endometriotic lesions were collected on 14, 28 and 42 days post transplantation (d.p.t.). n=10 mice per group in each time point.

Fig. 2.

Longitudinal observation of endometriosis mouse model by non-invasive in vivo imaging. The ELuc light emission from the lesion in the abdominal cavity was detected by IVIS on 0, 14, 28, 42 d.p.t. in the same recipient mouse administered with E2 (A) and without E2 (B). The exposure time was 30 seconds. (C) Temporal changes of the total luminescent flux (photon/second) with E2 (A) and without E2 (B) at four time points. E2(+): n=10 at each time point, E2(−): n=6. Bars: mean ± SEM. Significant P-values by Welch’s t-test are indicated: *P<0.05, **P<0.01, ***P<0.001.

Fig. 3.

Representative images of endometriotic lesions by dissecting microscopy. (A) A dome-shaped tissue mass mainly consisted of two cystic structures (*) engrafted at the suture site on the peritoneum on 14 d.p.t. The recipient’s epigastric vessels are indicated with arrowheads. White arrow: a part of black suture thread is faintly visible through the tissue. (B) An engrafted tissue mass at the suture site (white arrow) on the peritoneum on 28 d.p.t. The engrafted tissue consisted of multiple cysts containing pale yellow to transparent fluid (*). (C) An expanded cystic mass (*) found near the pancreas. The engrafted tissue was partly covered with the peripancreatic adipose tissue (pat).

Fig. 4.

Representative images of endometriotic lesions by 3D mCT imaging and conventional histology. (A1) A 3D reconstructed mCT image of a dome-shape engrafted tissue on 14 d.p.t. (see Supplementary Movie 1A for 360° view). (A2) A digital cross-section of A1 to visualize the internal structure of the cyst. (B1) A cystic engrafted lesion associated with a pancreatic tissue (see Supplementary Movie 1B for 360° view) and (B2) a digital cross-section of the cystic engrafted lesion (*) and the surrounding capillaries on 28 d.p.t. pnc: pancreatic tissue. (C1) An expanded cystic lesion on the peritoneum with a dense capillary network extended on the surface of the cystic lesion on 42 d.p.t. (see Supplementary Movie 1C for 360° view). (C2) Two tissue cross-sections 1 and 2 cut at different positions and angles, created from three-dimensional μCT data. Expanded cysts (*) fused to form the lesion. (D) H&E-stained tissue section on 42 d.p.t. prepared by embedding in standard paraffin blocks after mCT image acquisition. An expanded cyst (*) with a fluid-filled luminal space was lined with single-layered epithelium and surrounded by uterine gland-like (gl) structures and interstitial stroma.

Fig. 5.

Change in serum level of cytokines overtime. Serum levels of IFN-γ (A), TNF-α (B), IL-12p70 (C), IL-1β (D), IL-6 (E), IL-2 (F), IL-10 (G) in the recipient mice with endometriotic lesions (n=10 per group) and non-treated control as 0 d.p.t. (n=5). Bars: mean ± SEM. Significant P-values are indicated as follows: *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.