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Biology

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

Published: February 26th, 2019

DOI:

10.3791/58358

1Instituto de Investigación Sanitaria, 2Unidad de Animalario del Centro de Investigación Príncipe Felipe (CIPF), 3Departamento de Pediatría, Obstetricia y Ginecología, Universidad de Valencia, 4Servicio de Obstetricia y Ginecología, Hospital Clínico Universitario de Valencia

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

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.

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 effects4,5. The research for more efficien....

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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.......

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Here, we describe the process for creating a heterologous model of endometriosis in which the architecture of lesions is preserved by implanting fluorescently labeled pieces of human endometrium into immunocompromised mice, thus allowing non-invasive monitoring of lesion progression. Labeling of endometrial fragments is achieved by infection with adenovirus engineered to express mCherry, a protein emitting fluorescence in the near infrared region. In Figure 1.......

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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.......

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This work was supported by Spanish Ministry of Economy and Competitiveness through the Miguel Servet Program [CP13/00077] cofounded by FEDER (European Regional Development Fund) and awarded to Dr R. Gómez as well as by Carlos III Institute of Health grants awarded to Dr R Gómez [PI14/00547 and PI17/02329] and to Prof A. Cano [PI12/02582].

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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

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