Noninvasive Monitoring of Lesion Size in a Heterologous Mouse Model of Endometriosis

Summary

Here, we present a protocol for live-imaging of fluorescently labeled human endometrial fragments grafted in mice. The method allows studying the effects of drugs of choice on endometriotic lesion size through monitoring and quantification of fluorescence emitted by the fluorescent reporter on real time

Abstract

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time through noninvasive monitoring of fluorescence emitted by implanted ectopic human endometrial tissue. For this purpose, biopsies of human endometrium are obtained from donor women ongoing oocyte donation. Human endometrial fragments are cultured in the presence of adenoviruses engineered to express cDNA for the reporter fluorescent protein mCherry. Upon visualization, labeled tissues with an optimal rate of fluorescence after infection are subsequently chosen for the implantation in recipient mice. One week prior to the implantation surgery, recipient mice are oophorectomized, and estradiol pellets are placed subcutaneously to sustain the survival and growth of lesions. On the day of surgery mice are anesthetized, and peritoneal cavity accessed through a small (1.5 cm) incision by the linea-alba. Fluorescently labeled implants are tweezed, briefly soaked in glue and attached to the peritoneal layer. Incisions are sutured, and animals left to recover for a couple of days. Fluorescence emitted by endometriotic implants is usually non-invasively monitored every 3 days for 4 weeks with an in vivo imaging system. Variations in the size of endometriotic implants can be estimated in real time by quantification of the mCherry signal and normalization against the initial time-point showing maximal fluorescence intensity.

Traditional preclinical rodents of models of endometriosis do not allow non-invasive monitoring of lesion in real time but rather allow evaluation of the effects of drugs assayed at the end point. This protocol allows one to track lesions in real time and is more useful to explore the therapeutic potential of drugs in preclinical models of endometriosis. The main limitation of the model thus generated is that non-invasive monitoring is not possible over long periods of time due to the episomal expression of Ad-virus.

Introduction

Endometriosis is a chronic gynecologic disorder initiated by the implantation of the functional endometrium outside the uterine cavity. Ectopic lesions grow and induce inflammatory processes leading to chronic pelvic pain and infertility¹. It is estimated that up to 10–15% of women of reproductive age are affected by endometriosis2, and it is present in approximately 40–50% of infertile women³. Current pharmacological treatments for endometriosis are unable to completely eradicate lesions and not free of side effects^4,5. The research for more efficien....

Protocol

The use of human tissue specimens was approved by the Institutional Review Board and Ethics Committee of the Hospital Universitario La Fe. All patients provided written informed consent. The study involving animals was approved by the Institutional Animal Care Committee at the Centro de Investigacion Principe Felipe de Valencia, and all procedures were performed following the guidelines for the care and use of mammals from the National Institutes of Health.

1. Endometrial Tissue Collection and P.......

Discussion

The protocol herein detailed describes the implementation of an animal model of endometriosis in which the architecture of implanting lesions architecture is preserved whilst simultaneously allowing real time assessment of fluorescence emitted by mCherry labeled endometrial tissue. In this protocol, we describe the use of a specific in vivo imaging system and related software to non-invasively assess fluorescence emitted by the labeled lesion. Each user should adapt the protocol depending on the specific imaging device a.......

Materials

Name	Company	Catalog Number	Comments
Endosampler™	Medgyn	22720	Cannula for sampling the uterine endometrium
DMEM Medium	VWR	HYCLSH30285.FS	Medium
Ad-mCherry	Vector Biolabs	1767	Adenoviral vector expressing mCherry
PBS, 1X solution, sterile, pH 7,4	VWR	E504-500ML	Buffer for washes
Pellets 17-B-Estradiol 18 mg/ 60 days	Innovative Research of America	SE-121	Hormone pellets for rodents
Vetbond™ Tissue Adhesive	3M	780-680	Tissue adhesive
Petri dishes in polystyrene crystal	Levantina	367-P101VR20	Petri dishes
Penicillin-Streptomicin	Sigma	P4333-100ML	Antibiotics
Syringes, medical 10 ml 0,5 ml	VWR	CODA626616	Syringes
Nitrile gloves, powder-free	VWR	112-2754	Gloves
Soft swiss nude mice	Charles River	SNUSSFE05S	Mice for animal experiment
Ivis Spectrum In vivo Imaging system	Perkin Elmer	124262	In vivo Monitoring equipment
Living Image® (Ivis software)	Perkin Elmer	---	In vivo monitoring software
Fetal Bovine Serum	Gibco	10082147	Enrichment serum
96-well cell culture treated plates	Life technologies	167008	Culture plates
Urine flasks	Summedical	4004-248-001	Flasks for washes
Sterile surgical blades	(Aesculap Division) Sanycare	1609022-0008	Surgical blades
Isovet 1000 mg/g	B-BRAUN	---	Isoflurane (Anesthetic)
Buprex® 0.3 mg	Schering Plough S.A.	---	Buprenorphine (Analgesic solution)
Injectable morphine solution 10 mg/mL	B BRAUN	---	Morphine (Analgesic solution)
Monofyl® Absorbable Sutures	COVIDIEN	---	Sutures
Desinclor chlorhexidine	Promedic SA	---	Antiseptic solution
Microscopy DMi8	Leica Mycrosystems	---	fluorescence microscope
Hera Cell 150 Incubator	Thermo Scientific	51026282	Incubator