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. G&#243;mez as well as by Carlos III Institute of Health grants awarded to Dr R G&#243;mez [PI14/00547 and PI17/02329] and to Prof A. Cano [PI12/02582].

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...

<ol>
	<li>Nap, A. W., Groothuis, P. G., Demir, A. Y., Evers, J. L., Dunselman, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+endometriosis.">Pathogenesis of endometriosis.</a> Best Practice &amp; Research: Clinical Obstetrics &amp; Gynaecology. 18, 233-244 (2004).</li><li>Holoch, K. J., Lessey, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endometriosis+and+infertility.">Endometriosis and infertility.</a> Clinical Obstetrics and Gynecology. 53, 429-438 (2010).</li><li>Eskenazi, B., Warner, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+endometriosis.">Epidemiology of endometriosis.</a> Obstetrics and Gynecology Clinics of North America. 24 (2), 235-258 (1997).</li><li>Giudice, L. C., Kao, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endometriosis.">Endometriosis.</a> Lancet. 364 (9447), 1789-1799 (2004).</li><li>Donnez, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficacy+of+medical+and+surgical+treatment+of+endometriosis-associated+infertility+and+pelvic+pain.">The efficacy of medical and surgical treatment of endometriosis-associated infertility and pelvic pain.</a> Gynecologic and Obstetric Investigation. 54, 2-7 (2002).</li><li>D'Hooghe, T. M., Bambra, C. S., Cornillie, F. J., Isahakia, M., Koninckx, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+and+laparoscopic+appearance+of+spontaneous+endometriosis+in+the+baboon+(Papio+anubis,+Papio+cynocephalus).">Prevalence and laparoscopic appearance of spontaneous endometriosis in the baboon (Papio anubis, Papio cynocephalus).</a> Biology of Reproduction. 45 (3), 411-416 (1991).</li><li>Dick, E. J., Hubbard, G. B., Martin, L. J., Leland, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Record+review+of+baboons+with+histologically+confirmed+endometriosis+in+a+large+established+colony.">Record review of baboons with histologically confirmed endometriosis in a large established colony.</a> Journal of Medical Primatology. 32 (1), 39-47 (2003).</li><li>Donnez, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+endometriotic+nodules+in+an+experimental+baboon+modelmimicking+human+deep+nodular+lesions.">Induction of endometriotic nodules in an experimental baboon modelmimicking human deep nodular lesions.</a> Fertility &amp; Sterility. 99 (3), 783-789 (2013).</li><li>Grummer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+in+endometriosis+research.">Animal models in endometriosis research.</a> Human Reproduction Update. 12 (5), 641-649 (2006).</li><li>Rossi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+aspects+of+endometriosis+in+a+mouse+model+through+analysis+of+implantation+and+progression.">Dynamic aspects of endometriosis in a mouse model through analysis of implantation and progression.</a> Archives of Gynecology and Obstetrics. 263 (3), 102-107 (2000).</li><li>Grummer, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peritoneal+endometriosis:+validation+of+an+in-vivo+model.">Peritoneal endometriosis: validation of an in-vivo model.</a> Human Reproduction. 16 (8), 1736-1743 (2001).</li><li>Becker, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+non-invasive+model+of+endometriosis+for+monitoring+the+efficacy+of+antiangiogenic+therapy.">A novel non-invasive model of endometriosis for monitoring the efficacy of antiangiogenic therapy.</a> The American Journal of Pathology. 168 (6), 2074-2084 (2006).</li><li>Laschke, M. W., Giebels, C., Nickels, R. M., Scheuer, C., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+progenitor+cells+contribute+to+the+vascularization+of+endometriotic+lesions.">Endothelial progenitor cells contribute to the vascularization of endometriotic lesions.</a> The American Journal of Pathology. 178 (1), 442-450 (2011).</li><li>Nap, A. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiangiogenesis+therapy+for+endometriosis.">Antiangiogenesis therapy for endometriosis.</a> The Journal of Clinical Endocrinology &amp; Metabolism. 89 (3), 1089-1095 (2004).</li><li>Wang, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prodrug+of+green+tea+epigallocatechin-3-gallate+(Pro-EGCG)+as+a+potent+anti-angiogenesis+agent+for+endometriosis+in+mice.">Prodrug of green tea epigallocatechin-3-gallate (Pro-EGCG) as a potent anti-angiogenesis agent for endometriosis in mice.</a> Angiogenesis. 16 (1), 59-69 (2013).</li><li>Delgado-Rosas, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+ergot+and+non-ergot-derived+dopamine+agonists+in+an+experimental+mouse+model+of+endometriosis.">The effects of ergot and non-ergot-derived dopamine agonists in an experimental mouse model of endometriosis.</a> Reproduction. 142 (5), 745-755 (2011).</li><li>Al-Jefout, M., Andreadis, N., Tokushige, N., Markham, R., Fraser, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pilot+study+to+evaluate+the+relative+efficacy+of+endometrial+biopsy+and+full+curettage+in+making+a+diagnosis+of+endometriosis+by+the+detection+of+endometrial+nerve+fibers.">A pilot study to evaluate the relative efficacy of endometrial biopsy and full curettage in making a diagnosis of endometriosis by the detection of endometrial nerve fibers.</a> American Journal of Obstetrics &amp; Gynecology. 197 (6), 578 (2007).</li><li>Fortin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+human+endometriotic+tissue+maintenance+and+regression+in+a+noninvasive+mouse+model+of+endometriosis.">Quantitative assessment of human endometriotic tissue maintenance and regression in a noninvasive mouse model of endometriosis.</a> Molecular Therapy. 9 (4), 540-547 (2004).</li><li>Liu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+nude+mouse+models+for+green+fluorescence+human+endometriosis.">Improved nude mouse models for green fluorescence human endometriosis.</a> Journal of Obstetrics and Gynaecology Research. 36 (6), 1214-1221 (2010).</li><li>Wang, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+red+fluorescent+nude+mouse+model+of+human+endometriosis:+advantages+of+a+non-invasive+imaging+method.">A red fluorescent nude mouse model of human endometriosis: advantages of a non-invasive imaging method.</a> European Journal of Obstetric Gynecologic and Reproductive Biology. 176, 25-30 (2014).</li></ol>

The authors have nothing to disclose.

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...

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&#8211;15% of women of reproductive age are affected by endometriosis2, and it is present in approximately 40&#8211;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 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 humans6,7,8; 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 individuals10,11. Endometriosis can be induced in these animals by transplanting either pieces of rodent uterine horns (&#34;homologous models&#34;)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.

<table>
	<tbody>
		<tr>
			<td>Endosampler&#8482;</td>
			<td>Medgyn</td>
			<td>22720</td>
			<td>Cannula for sampling the uterine endometrium</td>
		</tr>
		<tr>
			<td>DMEM Medium</td>
			<td>VWR</td>
			<td>HYCLSH30285.FS</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>Ad-mCherry</td>
			<td>Vector Biolabs</td>
			<td>1767</td>
			<td>Adenoviral vector expressing mCherry</td>
		</tr>
		<tr>
			<td>PBS, 1X solution, sterile, pH 7,4</td>
			<td>VWR</td>
			<td>E504-500ML</td>
			<td>Buffer for washes</td>
		</tr>
		<tr>
			<td>Pellets 17-B-Estradiol 18 mg/ 60 days</td>
			<td>Innovative Research of America</td>
			<td>SE-121</td>
			<td>Hormone pellets for rodents</td>
		</tr>
		<tr>
			<td>Vetbond&#8482; Tissue Adhesive</td>
			<td>3M</td>
			<td>780-680</td>
			<td>Tissue adhesive</td>
		</tr>
		<tr>
			<td>Petri dishes in polystyrene crystal</td>
			<td>Levantina</td>
			<td>367-P101VR20</td>
			<td>Petri dishes</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomicin</td>
			<td>Sigma</td>
			<td>P4333-100ML</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Syringes, medical 10 ml 0,5 ml</td>
			<td>VWR</td>
			<td>CODA626616</td>
			<td>Syringes</td>
		</tr>
		<tr>
			<td>Nitrile gloves, powder-free</td>
			<td>VWR</td>
			<td>112-2754</td>
			<td>Gloves</td>
		</tr>
		<tr>
			<td>Soft swiss nude mice</td>
			<td>Charles River</td>
			<td>SNUSSFE05S</td>
			<td>Mice for animal experiment</td>
		</tr>
		<tr>
			<td>Ivis Spectrum In vivo Imaging system</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td>In vivo Monitoring equipment</td>
		</tr>
		<tr>
			<td>Living Image&#174; (Ivis software)</td>
			<td>Perkin Elmer</td>
			<td>---</td>
			<td>In vivo monitoring software</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10082147</td>
			<td>Enrichment serum</td>
		</tr>
		<tr>
			<td>96-well cell culture treated plates</td>
			<td>Life technologies</td>
			<td>167008</td>
			<td>Culture plates</td>
		</tr>
		<tr>
			<td>Urine flasks</td>
			<td>Summedical</td>
			<td>4004-248-001</td>
			<td>Flasks for washes</td>
		</tr>
		<tr>
			<td>Sterile surgical blades</td>
			<td>(Aesculap Division) Sanycare</td>
			<td>1609022-0008</td>
			<td>Surgical blades</td>
		</tr>
		<tr>
			<td>Isovet 1000 mg/g</td>
			<td>B-BRAUN</td>
			<td>---</td>
			<td>Isoflurane (Anesthetic)</td>
		</tr>
		<tr>
			<td>Buprex&#174; 0.3 mg</td>
			<td>Schering Plough S.A.</td>
			<td>---</td>
			<td>Buprenorphine (Analgesic solution)</td>
		</tr>
		<tr>
			<td>Injectable morphine solution 10 mg/mL</td>
			<td>B BRAUN</td>
			<td>---</td>
			<td>Morphine (Analgesic solution)</td>
		</tr>
		<tr>
			<td>Monofyl&#174; Absorbable Sutures</td>
			<td>COVIDIEN</td>
			<td>---</td>
			<td>Sutures</td>
		</tr>
		<tr>
			<td>Desinclor chlorhexidine</td>
			<td>Promedic SA</td>
			<td>---</td>
			<td>Antiseptic solution</td>
		</tr>
		<tr>
			<td>Microscopy DMi8</td>
			<td>Leica Mycrosystems</td>
			<td>---</td>
			<td>fluorescence microscope</td>
		</tr>
		<tr>
			<td>Hera Cell 150 Incubator</td>
			<td>Thermo Scientific</td>
			<td>51026282</td>
			<td>Incubator</td>
		</tr>
	</tbody>
</table>

noninvasive monitoring of lesion size in a heterologous mouse model of endometriosis

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.

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...

Watch this Scientific Journal Video about Noninvasive Monitoring of Lesion Size in a Heterologous Mouse Model of Endometriosis at JoVE.com

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

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 ...

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.

noninvasive-monitoring-lesion-size-heterologous-mouse-model

Research

JoVE Journal

Biology

8.0K visualizações.  Instituto de Investigación Sanitaria. Este método pode ajudar a responder a perguntas-chave na avaliação dos efeitos de drogas terapêuticas no campo da endometriose, como os efeitos de compostos anti-androgênicos no tamanho das lesões endometriotes. A principal vantagem dessa técnica é que os efeitos das drogas específicas podem ser avaliados em modelos animais de endometriose em tempo real, e não no ponto final. O procedimento será Jessica Martinez, que é técnica do meu laboratório, assim como Viviana Bisbal e Nere...