This method can help answer key questions in evaluating therapeutic drug effects in the endometriosis field, such as the effects of anti-androgenic compounds on the size of endometriotic lesions. The main advantage of this technique is that the effects of the specific drugs can be assessed in animal models of endometriosis in real-time, rather than at the endpoint. Demonstrating the procedure will be Jessica Martinez, which is a technician from my lab, as well Viviana Bisbal and Nerea Marin, which are veterinarians from Centro de Investigacion Principe Felipe and which are close collaborators with our team.
Two to three days before the implant surgery, transfer rinsed, healthy endometrial tissue biopsy fragments into a 10 centimeter Petri dish containing complete medium, and use scalpels to mince the tissue into five to 10 millimeter cubed pieces. Using a micropipette, add 30 to 50 microliter drops of DMEM across the bottom of a new 10 centimeter culture dish, leaving enough space between each drop so that they do not come into contact with each other. When all of the drops have been placed, use a syringe needle to place a single piece of biopsy tissue fragment into each drop.
Next, add 100 to 200 microliters of freshly prepared adeno-mCherry to one well of a 96 well plate per tissue fragment, plus three wells with adenovirus free DMEM as a negative control. Then use the needle to transfer one tissue sample into each well of adeno-mCherry solution for an overnight incubation at 37 degrees celsius and 5%CO2. The next morning place the plate on a fluorescence microscope stage and use a 568 nanometer laser to check the fragments for an optimal labeling.
Using the appropriate dilution of infecting mCherry adenovirus is key for an efficient labeling of the tissue. So to determine the appropriate concentration, several for mCherry dilution, for each individual experiment, have to be evaluated. Then transfer the most brilliant fragments into individual wells of a new 96 well plate containing fresh, complete medium.
At least seven days after oophorectomy, decant the endometrial tissue fragments into a Petri dish, and confirm a lack of response to toe pinch in an anesthetized, oophorectomized animal. With the mouse in the supine position disinfect the ventral area and make a 1.5 centimeter longitudinal incision in the abdomen. Separate the skin from the muscle and make a second 1.5 centimeter incision in the muscle to access the peritoneal cavity.
Holding the left edge of the abdominal muscular wall with mini-forceps, fold the muscle to expose the inner face of the peritoneum on the outside of the animal. Use mini-tweezers to briefly soak one endometrial implant in n-butyl ester cyanoacrylate adhesive and attach the fragment to the peritoneum. When the adhesive is dried, place a second implant on the contralateral side of the peritoneum as just demonstrated.
Use an absorbable 6-0 suture to close the muscle layer and a non-absorbable 6-0 suture to close the skin. Then clean the skin incision with antiseptic solution and place the animal in the recovery zone with monitoring until full recumbency. For in-vivo fluorescent imaging of the implanted tissue fragments, 24 to 48 hours after the implantation surgery turn on the in-vivo imaging system, initialize the software, and allow the CCD camera to cool down for a few minutes.
Once the instrument is ready place the first anesthetized mouse into the in-vivo imaging system cage, in the supine position, with the head inside a tubule connected to the anesthesia machine, and close the lid. Then click acquire and save the five resulting images. To quantify the in-vivo fluorescence, in the appropriate image analysis software click browse and select the unmixed file of interest to be analyzed.
A new window will appear. Click add to list to include all of the unmixed files from each animal at different time points and click load as a group. All of the images should appear in a single sequence.
In the tool palette window, unselect the individual scale checkbox to adjust all of the images to the same scale, and double click on one image of the sequence. To create a region of interest, in the region of interest tools select the contour and auto one options, and click on the circle shape to place the circle in the center of the fluorescent signal. Click create to create the region of interest and copy and paste the region of interest on a background where there is no signal.
Then click measure regions of interest and select all to display values for the fluorescence intensity of each region and copy and paste these values into a spreadsheet. The labeling of the endometrial fragments is achieved by infection with adenovirus engineered to express mCherry, a protein that emits fluorescence in the near-infrared section that is significantly more robust than the non-infected endometrial tissue autofluorescence. During monitoring, in addition to the reference wavelength for mCherry, fluorescent images are taken with different pairs of excitation emission wavelength filters.
To define the characteristic fluorescent tissue emission profiles of the lesions from the background, and autofluorescence emitted by the host tissues, the monitoring images containing raw fluorescence emitted by the animals during each time point are brought together, unnormalized, in a single file. Next, the fluorescence is unmixed, normalized, and represented as a false color image, and the regions of interests corresponding to specific legion and background signals are automatically recognized by the program and quantified. The background region of interest signaling is subtracted from the legion region of interest signaling.
And the results of the intensity at each time point are normalized against the time point at which intensity is maximal. After several weeks of monitoring, the viable implants can be recovered for further downstream analysis. While attempting this procedure it's important to remember to select the concentration of Ad-mCherry that provides the most brilliant signal without compromising the viability of the tissue.
Following this procedure, the lesions can be recovered and used to study additional distinctive parameters. This procedure is optimized for the non-invasive monitoring of xenograft human endometriosis lesions. However, it can be easily adapt for assessing the effects of a specific drugs of interest on the sites of a xenograft in real-time.
