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 efficient therapies requires of the refinement of the existing animal models of endometriosis in such a way that human lesions can be appropriately mimicked, and the effects of compounds on lesion size among others can be closely assessed.

Primate models have been used to mimic endometriosis by implanting ectopic lesions histologically identical and at similar sites as in humans^6,7,8; however, ethical concerns and the high economic costs related to experimentation with primates limit their use⁹. Consequently, the use of small animals, especially rodents, for the implementation of in-vivo models of endometriosis continues to be favored as it allows studies with larger numbers of individuals^10,11. Endometriosis can be induced in these animals by transplanting either pieces of rodent uterine horns ("homologous models")^12,13 or human endometrial/endometriotic tissue to ectopic sites (heterologous models)¹⁴. In contrast to humans, rodents do not shed their endometrial tissue and thereby endometriosis can not be developed spontaneously in these species. Therefore, homologous mouse models of endometriosis have been criticized due to the fact that implanted ectopic mouse uterine tissue does not reflect the characteristics of human endometriotic lesions¹⁵.

Appropriate physiology of endometriosis can be mimicked in the heterologous models of endometriosis where fresh human endometrial fragments are implanted into immunodeficient animals. In conventional heterologous models, the therapeutic effects of compounds of interest are commonly assessed at the end point by the assessment of lesion size with the use of calipers¹⁶. An obvious limitation is that, as such, endpoint animal models do not allow studying implantation dynamics or endometriotic lesion development over time. An additional limitation is that the use of calipers does not allow accurate measurements of lesion size. Indeed, the standard error provided by calipers is in the same range (i.e., millimeters) as the size of the lesions implanted in mice, thus restricting the capacity of these tools to detect actual variations in size.

In order to overcome such limitations, herein, we describe the generation of a heterologous mouse model of endometriosis in which implanted human tissue is engineered to express a reporter m-Cherry fluorescent protein. Detection of the fluorescent signal with an appropriate image system enables non-invasive monitoring of lesion status with simultaneous quantification of its size in real time. Thus, our model provides clear advantages when compared to conventional endpoint models as it brings the opportunity of real-time non-invasive monitoring and the possibility to perform more objective and accurate estimation of variations in lesion size.

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 Pre-processing

1. Obtain a good quality biopsy of human endometrial aspirate by using a cannula attached to a suction device. Pour the biopsy into a flask containing 10 mL of sterile saline and wash with gentle manual agitation.
   NOTE: Procedures on how to obtain good quality biopsies have been previously described¹⁷.
2. Wash the biopsy of any remaining blood or mucus. Repeat the process by pouring the tissue to flasks containing fresh saline as many tines as required until tissue is observed clean.
3. Transfer biopsy fragments with a healthy appearance into a solution containing 10 mL of complete Dulbecco's Modified Eagle's medium (DMEM) medium with 10% Fetal Bovine Serum (FBS) and 1% antibiotic-antimycotic solution.
4. Pour the content in a 10 cm Petri dish and proceed to chop the tissue into 5–10 mm³ pieces with a pair of scalpels.

2. Adenoviral Transfection of Endometrial Fragments

NOTE: All the materials that are going to be employed in the process should be introduced in the hood in advance. Take out everything that is not going to be used in the process and place a flask with bleach. All material that comes into contact with the adenoviral vector must be disinfected with the bleach before discarding it in the biohazard container.

1. Once the biopsy has been chopped, take a new Petri dish. Pipette multiple 30–50 µL DMEM drops of spread throughout the whole dish. Leave enough space between drops so they do not come into contact.
2. Aided with a needle attached to syringe, place a single piece of fragment inside each of the medium drops.
   NOTE: Each drop should contain a single piece of endometrium.
3. Prepare the Ad-mCherry working solution by diluting 1:20 of the mCherry adenoviral stock solution [1 x 10¹⁰ pfu/mL]) into DMEM medium WITHOUT antibiotics (DMEM + 10% filtered FBS).
4. Dispense 100–200 μL of the Ad-mCherry solution per well on a 96-well plate, filling as many wells as fragments are available in the Petri dish drops (step 2.2). In addition, fill at least 3 wells with adenovirus free DMEM medium as a negative control.
5. Aided with a needle attached to syringe, transfer each of the fragments in the Petri dish (step 2.2) to each of the wells containing ad-mCherry solution in the 96-well plate.
6. Place the 96 well-plate containing the endometrial fragments with Ad-mCherry solution into an incubator at 37 °C with 5% CO₂ for 16 h.
7. Wash out the remaining adenoviruses in the medium by transferring tissues into a new 96-well plate filled with complete DMEM medium (WITH antibiotics-antimycotics, free of adenovirus) and incubate for 37 °C with 5% CO₂ for 24–48 h.
   NOTE: Remember to cover with bleach all well plates and tips that came in contact with Ad-virus before discarding into the biohazard container.
8. Take out the plate from the incubator and place it under a fluorescence microscope at 568 nm (red channel) to test for optimal labeling.
9. Choose the most brilliant fragments and place them into a new 96-well plate with fresh complete DMEM medium (WITH antibiotics-antimycotics, free of adenovirus).
10. Seal the plate well with a plastic paraffin film and transport it to into the specific-pathogen-free animal area for implantation of endometrial fragments into recipient animals.

3. Generation of the Endometriosis Mouse Model

NOTE: Use 6–8 week-old athymic nude (or similar immunocompromised strains) female mice housed in specific pathogen–free conditions, as recipient animals. To avoid hormonal cycle-dependent variations and simultaneously fuel lesion growth with estradiol, animals are ovariectomized and placed with 60-day release capsules containing 18 mg of 17 βeta-Estradiol (17β-E₂). Oophorectomy and pellet placement have to be performed at least one week in advance of grafting endometrial fragments into the recipient animals.

1. Oophorectomy
   NOTE: Prepare surgical sterilized material in a hood. Prepare anesthesia equipment and a post-surgical recovery zone ready in the same room.
   1. Perform a subcutaneous injection of morphine derivative at a dose of 5 mg/kg per mouse. Let the mice rest for 30 min after injection so as analgesics effects of drug can be manifested.
   2. Connect the inhalation anesthesia equipment and let oxygen and isoflurane (2% mg/kg) flow for a few min into a sealed anesthesia chamber.
   3. Introduce the animal into the isoflurane anesthesia chamber. Wait for 3–5 min and check that animals are fully anesthetized by pressing one of its paws. Transfer the animal to the surgery area and maintain anesthesia by placing a mask with continuous flow of isoflurane gas covering the respiratory airways.
   4. After disinfecting the area with chlorohexidine, perform a transverse 0.5 cm costal incision approximately at the height of the hip with sharp scissors.
   5. Separate the skin from the muscle to get access to the abdominal cavity, identify the white fat pad that surrounds the ovary and retract the ovary with dissection forceps.
   6. Tie a knot around the oviduct with absorbable suture and tighten it to ensure appropriate hemostasis before excising the ovary.
   7. Close the muscular layer with 6-0 absorbable suture, and then close the skin with 6-0 non-absorbable suture. Clean the area again with antiseptic solution.
   8. Repeat the procedure to remove the contra lateral ovary.
2. Estradiol pellet implant
   NOTE: Take care that the animal is anesthetized during the oophorectomy surgery to place pellets at that point.
   1. Immediately after the completion of the oophorectomy procedure, clean the skin with antiseptic solution surrounding the neck and make a transverse subcutaneous small (0.5 cm) incision with sharp scissors in the nape.
   2. Use the scissors to dissect the skin from the muscle, making a pocket large enough to allow placing the pellets.
   3. Insert the pellet containing 18 mg of 17β-E₂ and suture the skin with a 6-0 non-absorbable suture. Clean the area again with antiseptic solution.
   4. Place the animal in the recovery zone and administer an optimal dose of long-lasting analgesia to ease the recovery period.
3. Endometrial implant surgery
   NOTE: Allow at least a period of seven days quarantine to allow full recovery of animals after oophorectomy before starting endometrial fragment implantation surgery. For optimal synchronization with labeling of tissue, collect the biopsy 2–3 days before implantation surgery so as to avoid long-term culture of explants.
   1. Get the surgical room in the specific pathogen free zone ready in advance. Prepare the hood with all required surgical material, the anesthesia equipment and the post-surgical recovery zone also.
   2. Bring the animals to the room, perform a subcutaneous injection of a morphine derivative at a dose of 5 (mg/kg) in each mouse. Let the mice rest for 30 min after injection so analgesics effects of drug can be manifested.
   3. Connect the inhalation anesthesia equipment and let oxygen and isoflurane (2% mg/kg) flow for a few min into a sealed anesthesia chamber.
   4. Before starting surgery, move the plate containing fluorescently labeled fragments (from step 2.10) into the hood, unseal it and pour the fragments into a Petri dish for easier handling.
   5. Introduce the animal into the isoflurane anesthesia chamber. Wait for 3–5 min and check that animals are fully anesthetized by pressing one of its paws. Transfer the animal to the surgery area and maintain anesthesia by placing a mask with continuous flow of isoflurane gas covering the respiratory airways.
   6. Place the anesthetized animal face up. Disinfect the ventral area. Perform a longitudinal 1.5 cm incision in abdomen with sharp scissors and separate the skin from the muscle. Then, perform a longitudinal 1.5 cm incision in the muscle to access the peritoneal cavity.
   7. Hold the left edge of the abdomen muscular wall with mini-forceps and fold it trying to expose the inner face of the peritoneum on the outside.
   8. Take an endometrial implant with mini tweezers, soak it briefly in an n-butyl-ester cyanoacrylate adhesive and place it to the peritoneum where it will get attached. Let it dry for a few seconds. Repeat steps 3.3.6 and 3.3.7 to place an implant on the contralateral side of the peritoneum.
   9. Close the muscular layer with an absorbable 6-0 suture, and then close the skin with a non-absorbable 6-0 suture. Clean the area again with antiseptic solution.
   10. Place the animal in the recovery zone and administer an optimal dose of long-lasting analgesia.

4. In Vivo Fluorescent Imaging with an In Vivo Imaging System

1. Turn on the in vivo imaging system device, initialize the program and allow the CCD camera to cool down for a few min.
2. Prepare the inhalation anesthesia equipment. Open the isoflurane flow at 2% for a couple of min to fill the anesthetic chamber. Prepare the post-anesthesia recovery zone.
3. Once the program has started and the CCD camera have cooled down, click the Imaging Wizard tool: A tutorial starts with a series of consecutive windows displayed, each one corresponding to a parameter of interest with several options available to be chosen by clicking in the corresponding box. Move forward through the tutorial by selecting the appropriate boxes in each window and click the OK button to move to the next set of parameters with the following sequence.
   1. Select the Epiluminescence box for the fluorescence parameter, select the mCherry box for the filter pairs parameter. Check the Photograph mode and Confirm Focus boxes and select the Automatic box for the exposure parameter.
4. Once the instrument has been set up, move one animal inside the anesthesia chamber. When fully anesthetized, transfer the animal inside the in vivo imaging system cage and place it side up with its head inside a tubule connected to the anesthesia machine. Close the lid and click Acquire for monitoring.
5. Acquire the images appearing (a total of five images, one image for each pair of filters selected) and save data by clicking the Save As button. Move the mouse to the area of post-anesthesia recovery. Repeat the process with the remaining animals.
6. Repeat monitoring two or three times a week to follow-up the signal appropriately during the time course.
7. Proceed to sacrifice the animal at the end of the time course by CO₂ asphyxiation.

5. Quantification of In Vivo Fluorescence Images

1. Segregation of actual fluorescence through image unmixing:
   1. Open the In Vivo Imaging Analysis Coupled Software Program.
   2. Choose the “Sequenceinfo” file to start the analysis. Two windows will appear: “Sequence view” and “Tool Palette”. Choose Tool Palette and once the menu has been displayed select the following options.
   3. Select Corrections and click on the box Adaptive FL Background Subtraction to remove the undesirable fluorescent signals from the luminescent image data. Choose the threshold of greatest interest and click Set.
   4. Click Spectral Unmixing, select the wavelengths of interest, the method chosen for unmixing (library, guided, automatic or manual) and then click Start Unmix.
   5. Select the Unmixed image corresponding to mCherry signal and double click. A new window will appear with the final image of the signal of interest.
   6. Repeat step 5.1.3.
   7. OPTIONAL: If a representative image (JPEG) of the unmix result is needed, choose the desired settings in Tool Palette | Image Adjust (color table, binning, contrast, etc.), and then click on Export Graphics in the unmix window to export the current image view as an image.
   8. Save the unmixed file: File | Save As | Choose Folder and Ok.
   9. Repeat the process with the rest of the monitoring days and with all animals.
2. ROIs set up and signal quantification
   1. Click Browse and select the Unmixed file (see step 5.1.8) of interest to be analyzed. A new window will appear.
   2. Click Add To List to include all the unmixed files from each animal at different time points and then click on Load As A Group. All images must appear as a single sequence.
   3. Go to Tool Palette window: Click off the box Individual scale to obtain all images on the same scale.
   4. Double click in one image of the sequence and create a ROI on the zone of interest using the following sequence. Go to ROI Tools and select Countour and Auto 1 option, click on the circle shape appearing, place it on the center the fluorescent signal and then click Create on the displayed window.
      Note: This automatically highlights pixels with an intensity of fluorescence above background values (i.e., lesion) and generates a shape whose area embraces the outlined pixels.
   5. Copy the created ROI shape and paste on a background zone where there is no signal.
   6. Click on Measure ROIs | Select All to display values of fluorescence intensity. Proceed to select data values with the mouse, click right button, press copy and paste on a spreadsheet.

6. Data (fluorescent signal) Normalization

1. Select the initial time point at which signal intensity is maximal. Proceed to normalize signal at each time point by using the formula:
   Signal intensity at each time point / Maximal signal intensity observed during the time course) x 100.

Results

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...

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