We would like to thank the members of the Reizes laboratory for their critical review and insights during preparation of the manuscript, as well as the Imaging and Histology cores at Lerner Research Institute for their assistance in data collection and data analysis. This work was supported through an internal grant funding through the Research Program Committee at Cleveland Clinic and by an external grant through the Society for Reproductive Investigation and Bayer. Research in the Reizes Laboratory is also funded through VeloSano Bike to Cure, Center of Research Excellence in Gynecologic Cancer, and through The Laura J. Fogarty Endowed Chair for Uterine Cancer Research. Cleveland Clinic owns the copyright permission for&#160;Figure 1&#160;and&#160;Figure 2.

NOTE: The use of animals in this study was approved by the Institutional Animal Care and Use Committee (IACUC) at the Cleveland Clinic Lerner Research Institute. All publicly available animal care and use standards were performed following guidelines by the National Institutes of Health.&#160;This procedure is utilizing aseptic techniques. The Petri dish is sterile. The PBS/saline used is sterile. The surgical instruments for necropsy and tissue dissection are sterilized via autoclave. We use 70% EtOH on the instruments between animal cases (if more than one per session) to decrease contamination.
1.&#160;Preparation of donor and recipient mice&#160;(see Figure 1)
<ol>
	<li>Ensure that appropriate approval is in place to work with laboratory animals.</li>
	<li>Determine the strain of mouse. In these experiments, wildtype and mutant mice having a C57BL/6J background were utilized, but investigators using similar models report success with other mouse strains such as BALB/c mice10. In addition, donor and/or recipient mice can utilize reporter systems, such as GFP or RFP mice (see&#160;Figure 2&#160;and&#160;Figure 3).</li>
	<li>For timing of the endometrial tissue transplant, ensure that the donor mice are between 22-24 days old at the time of gonadotropin injection. This ensures that they are reproductively na&#239;ve, e.g., not yet begun estrous cycling.</li>
	<li>Ensure that the recipient mice are reproductively intact (no prior ovariectomy) between 2 to 4 months of age. While not required, it is recommended to place urine-soaked bedding from a male mice cage periodically in the recipient&#39;s cage to facilitate ongoing estrous cycling and again at 72 h prior to endometrial tissue transplant. 
	NOTE: This will not ensure synchronization of estrous cycle of the recipients, as the placement urine-soaked bedding is highly dependent on the amount of male bedding used and has variable results. If synchronization of the estrous cycle is desired, three consecutive subcutaneous injections of 100 ng/100 &#181;L estradiol will bring all animals to the estrous phase. While we found no difference in lesion induction based upon phase of estrous cycle, other groups have found the cycle phase to be important10.</li>
	<li>Subcutaneously inject Pregnant Mare Serum Gonadotropin (PMSG, 2 IU diluted into 200 &#181;L) into donor mice subcutaneously using a fine needle (25-27 G recommended).Do not give PMSG to recipient mice, as it will trigger ovulation and subsequent high progesterone environment, which is less receptive to endometriosis formation. 
	NOTE: Prior mouse models have utilized either PMSG or estrogen to stimulate endometrial proliferation in the donor mice prior to endometrial harvest23. PMSG is recommended for the following reasons: The half-life of PMSG is 40 h whereas the half-life of 17-beta-estradiol is only 2 h. Utilizing a single subcutaneous injection of PMSG in the donors 40 h prior to procurement of endometrial tissue provides a sustained duration of exposure to gonadotropin stimulation, which acts to stimulate endogenous estrogen. Many preparations of exogenous estrogen require multiple injections to adequately prime the endometrium.</li>
	<li>Following PMSG injection, schedule necropsy, procurement, and transplant into the recipient between 38-42 h. Time the endometrial tissue harvest following gonadotropin injection to ensure collection of tissue before ovulation, which occurs at 42 h post injection. Ovulation produces a high progesterone (P4) environment, which would reduce lesion establishment.</li>
</ol>
2.&#160;Procurement of donor mouse endometrial tissue
<ol>
	<li>After euthanasia of donor mouse (using CO2&#160;chamber followed by cervical dislocation), spray the abdomen with 70% ethanol solution; this serves to reduce contamination of the procured tissue from skin flora and from dislodged hairs. With dissecting scissors, make a shallow transverse snip at midline through the skin and subcutaneous tissue. Then grasping each side of the skin incision, use blunt traction to open the abdomen.</li>
	<li>Identify the uterus. Before removing the uterus, trim away adjacent connective tissue. Transect each uterine horn just below their respective fallopian tube and then transect the cervix to remove the entire uterus en bloc.</li>
	<li>After removal from the abdominal cavity, inspect the uterus carefully and remove any additional peripheral fat or connective tissue. Place the uterus in a droplet of cold PBS on a Petri dish. Determine and document the combined mass of the entire uterus.</li>
	<li>Transect each horn from the uterus fundus, making the transection as close to the fundus as possible so as to maximize the length of each horn. Using the aid of a dissecting microscope, place one blade of the dissecting scissors inside the lumen of the first horn, then cut along the major axis of the tube. Carefully open the tube, keeping in mind which side is the serosa and which side is the epithelium.</li>
	<li>Put 500 cc of saline or PBS on a new Petri dish; the liquid will stay together due to surface tension.</li>
	<li>Then, perform fragmentation of the uterus in a uniform manner. It is better to have fewer larger lesions than many smaller lesions. Begin by separating off the epithelium from the myometrium by grasping the endometrial layer and peeling it away.
	<ol>
		<li>Alternately, simply fragment the tissue without separating off the myometrium (as long as the epithelial side is fully exposed), but retaining the myometrium lessens the physiologic relevance of this model to human disease. Ensure that fragments are as large as possible but small enough to pass through an 18 G needle; (1 mm x 1 mm is recommended).</li>
		<li>Collect 10-12 of these 1 mm x 1 mm fragments from the first horn (which roughly corresponds to 40 mg tissue in the C57BL/6J mice). Place collected fragments into the liquid collection.</li>
	</ol>
	</li>
	<li>Perform the same steps for the other uterine horn for a total of about 24 fragments per mouse. Document the total number of fragments.</li>
</ol>
3.&#160;Peritoneal injection of tissue fragments into recipient mouse
<ol>
	<li>Aspirate the suspended 1 mm x 1 mm fragments using the blunt end of a 1 cc syringe; the total volume should be 1 mL.</li>
	<li>Attach an 18 G needle to the full syringe and load the fluid into the needle. Consider a mock injection back into the Petri dish to ensure that all the tissue will pass through the needle.</li>
	<li>Take the recipient mouse. Either before or after IP injection, obtain a vaginal smear of the recipient mouse for estrous cycle documentation (10 &#181;L of saline using the bulb syringe at the vaginal orifice and plated on the glass slide with the coverslip).</li>
	<li>Perform intraperitoneal injection of the fragments with the syringe at a 45 degree angle, taking care to not inject subcutaneously.</li>
	<li>If fragments remain after injection, draw an additional 200 &#181;L of the fluid into the syringe for injection to ensure that all the fragments are successfully injected intraperitoneally.</li>
	<li>Once assured of no bleeding or complications, place the recipient mice back in their cages and feed a normal diet.</li>
</ol>
4.&#160;Harvest of endometriotic lesions
<ol>
	<li>Euthanize recipient mice at approximately 21 days following fragment injection post-transplant. 
	NOTE: From experience and from discussion with collaborators using similar models, the maximum lesion size and number occur at approximately 3 weeks post-transplant; after 3 weeks, lesions begin to regress in size.&#160;The IACUC approved method of euthanasia is used.&#160;</li>
	<li>Following euthanasia and cervical dislocation, spray the animal&#39;s abdomen with 70% ethanol and tent the skin to cut superficially with dissecting scissors. Incise the skin and subcutaneous space with scissors to bluntly open the abdomen.</li>
	<li>Before any further dissection is undertaken, a complete survey for gross lesions is performed, with size measured by calipers and documented as to their anatomical region (see below).</li>
	<li>Perform complete collection of three distinct anatomic regions en bloc (regardless of whether or not lesions can be appreciated grossly). If lesions are seen in other regions (e.g., bowels), these should be ignored and not collected unless the lesion traverses one of the three areas below.
	<ol>
		<li>A = abdominal wall/peritoneum (can either be flattened into a cassette or rolled up, as long as consistent between samples).</li>
		<li>B = pancreas and mesenteric fat.</li>
		<li>C = parauterine connective tissue and fat (white glistening tissue that surrounds the uterus but does not involve any organs other than the bladder; take care to not mistake the bladder for a lesion).</li>
	</ol>
	</li>
	<li>Place each dissected area in a cassette, appropriately labeled, and place it into formalin and process as per lab protocol for histologic sectioning.</li>
	<li>Section formalin blocks in two slides (D1 and D2) per tissue area at two uniform depths.</li>
</ol>
5.&#160;Scoring of endometriotic lesions
<ol>
	<li>Scan (at 40x magnification) and archive slides.</li>
	<li>Use digital slide reading software, define the longest distance (X) between edges of an endometriotic lesion-whether the edge consists of glands or stroma-and mark it. A continuous lesion is defined by glands surrounded by stroma; the line does not necessarily traverse only endometriotic tissue (as in the case of determining endpoints on an irregularly shaped or undulating focus of endometriosis) but the two end points need to be connected by continuous stroma and/or glands (see&#160;Figure 4).</li>
	<li>Make a second line (Y) 90 degrees across the first line, with the length of the second line determined following the rules above.</li>
	<li>If multiple non-contiguous lesions are encountered, give each their own X and Y measurements.</li>
	<li>Calculate the final score for each slide as the summation of areas (X*Y) of each lesion.</li>
	<li>Take the larger of the scores from the two slides (D1 vs D2) as the final score for that region (A, B, C).</li>
	<li>Total the scores from each region to give the final microscopic score for that animal.</li>
</ol>

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The authors have no conflicts of interest to disclose.

Our study demonstrates that endometriosis can be reliably induced in mice without requiring use of ovariectomy and/or survival surgery, and that ectopic endometrial lesions can be identified and quantified using a standardized survey of the abdomen and histologic analysis.
Many murine studies of endometriosis utilize surgically induced endometriosis in which donor endometrium is sutured in place to the bowel, abdominal wall, or other intraperitoneal location12,20. This has the advantage of standardizing the size and location of the transplanted tissue. However, in addition to added logistical challenges of survival surgery, this may introduce confounding variables from the surgery itself, given that endometriosis is an inflammatory disease and that in a clinical setting, endometriosis typically develops prior to any surgical procedure. Intraperitoneal injection, on the other hand, more accurately models the retrograde menstruation that is thought to cause the majority of endometriosis lesions.
Ovariectomy is often performed in rodent models to reduce variability introduced by the estrous cycle; and as endometriosis is a hormone-dependent disease, supraphysiologic doses of estradiol are then administered in these cases. This practice arguably limits the applicability of a mouse model to human disease. Our study demonstrates that ovariectomy is not necessary to reliably create endometriosis lesions and that phase of estrous cycle does not impact the ability to establish lesions.
A shortfall of other intraperitoneal injection models is the reliance on subjective measures of lesion size or burden. While use of calipers or other instruments may provide objective measures, these measurements are recorded at the time of lesion harvest and therefore not able to be verified later and may be subject to intra-observer variability. In our model, histologic quantification can be performed and verified long after necropsy, meaning that those who perform the necropsy do not need to have expertise in identification of gross lesions and are more easily blinded as to the intervention received. Furthermore, as our work illustrates, a proportion of disease may be missed when only relying on gross disease; in addition, many suspected lesions may actually be physiologic (e.g., lymphoid tissue) or fibrosis. Finally, taking standardized sections of entire anatomic regions in each mouse further reduces variability. This approach of procuring the same amount of tissue per mouse prevents the inevitable underscoring that would occur with small samples and the over-scoring of large samples, especially since microscopic disease may be present.
Ultimately, these refinements serve to both standardize and simplify the approach, resulting in a mouse model that can study endometriosis in a high throughput fashion. This model is an assayable method to screen for pathways and drug targets, and our lab is currently utilizing this model for a drug validation study. This model is particularly helpful when attempting to determine the relative importance of aberrant gene expression (or drug treatment) in the donor endometrium versus the recipient peritoneal environment. It also can also be utilized with reporter mice (as we have shown) and immunocompromised and knockout mice.
In summary, we provide several important innovations that provide a murine model with high reliability, reproducibility, and objectivity in studying endometriosis. Our approach advances the field by not only providing a straightforward, streamlined process for lesion induction but also a standardized way of measuring and reporting data.

Endometriosis is an enigmatic disease of the female reproductive tract with significant financial and health burdens on women1,2. The etiology of endometriosis is not completely understood, and multiple explanations have been proposed including coelomic metaplasia, embryonic M&#252;llerian rests, recruitment of bone marrow derived progenitor cells, and retrograde menstruation3. While multiple aspects of these proposed mechanisms may be involved, and no single explanation can account for all forms of the disease, the leading model of endometriosis pathogenesis is retrograde menstruation. Retrograde menstruation is the passage of menstrual effluent through the fallopian tubes and into the peritoneal cavity; it is estimated that 90% of menstruating women regularly undergo retrograde menstruation4,5. Given this commonplace phenomenon of retrograde menstruation, why endometriosis develops only in a subset of women is unclear5. To better understand the etiology of this disease, direct human studies are not feasible and animal studies are warranted.
Endometriosis is a challenge both to treat and to study. Prevalence of the disease is not known but estimated to be 10%1. While some advanced types of endometriosis may be accurately identified through noninvasive imaging, a definitive diagnosis is only achieved through histopathological analysis of surgically obtained biopsy specimens; lesions that visually appear to be diseased, may in fact be fibrosis or scarring from other causes6. Severity and extent of disease does not correlate with symptomatology7.
Endometriosis lesions consist of heterogeneous cell types and populations which interact in complex ways within the microenvironment, therefore, limiting the usefulness of cellular models8,9. In vivo models exist, but these have inherent challenges and limitations10,11,12. Primate models are ideal but are often not feasible13,14,15. Few non-primate mammals menstruate and develop endometriosis spontaneously16. Rodent models of endometriosis exist but each have limitations17. Many of these models require survival surgery to suture or implant endometrial tissue into the donor recipient wall or bowel, adding technical complexity, need for anesthesia, and confounding immune factors from the surgery itself18,19,20. In addition, many models require ovariectomy and estrogen supplementation; while increasing lesion yield, this adds time, expense, and additional survival surgery. Intraperitoneal (IP) injection models do not require anesthesia or survival surgery, and these models logically simulate retrograde menstruation better than suturing models21,22,23. Most IP models, however, are subject to more variability in lesion location due to the random dispersal of endometrial fragments following injection and therefore to more bias in lesion identification and measurement.
Here we present a murine model of endometriosis that integrates several features of existing endometriosis models into a novel, simplified, and efficient system that relies on microscopic quantification in lieu of subjective grading.

<table><tbody><tr><td>&lt;em&gt;&lt;strong&gt;Supplies for injecting PMSG into donor mouse&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>1 mL Tuberculin syringe with 27G needle</td><td>Fisher Scientific</td><td>14-826-87</td><td></td></tr><tr><td>Pregnant mare serum gonadotropin</td><td>Sigma-Aldrich</td><td>9002-70-4</td><td></td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Supplies for necropsy of donor mouse and tissue processing&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>6&amp;rdquo; serrated forceps, curved tip</td><td>Electron Microscopy Sciences</td><td>72993-6C</td><td></td></tr><tr><td>70% ethanol solution</td><td>Pharmco</td><td>33000HPLCCS4L</td><td>70% solution dilute ethyl acetate 200 proof</td></tr><tr><td>Analytical balance</td><td>Mettler Toledo</td><td>ME54TE</td><td></td></tr><tr><td>Carbon dioxide</td><td>TriGas Supplier</td><td></td><td></td></tr><tr><td>Dissecting tray</td><td>Fisher Scientific</td><td>S14000</td><td></td></tr><tr><td>No. 10 disposable scalpel</td><td>Fisher Scientific</td><td>NC9999403</td><td></td></tr><tr><td>Scissors, curved</td><td>Electron Microscopy Sciences</td><td>72941</td><td></td></tr><tr><td>Scissors, straight</td><td>Electron Microscopy Sciences</td><td>72940</td><td></td></tr><tr><td>Stereo microscope</td><td>Leica Microsystems</td><td>Leica SE 4</td><td>For tissue dissection</td></tr><tr><td>Sterile phosphate buffered saline (PBS)</td><td>Institutional core facility supplies</td><td></td><td></td></tr><tr><td>Surgical instrument sterilization tray</td><td>Electron Microscopy Sciences</td><td>66112-02</td><td></td></tr><tr><td>Tissue culture dishes</td><td>Fisher Scientific</td><td>08-772E</td><td></td></tr><tr><td>Weighing dishes</td><td>Fisher Scientific</td><td>02-202-103</td><td></td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Supplies for injecting into recipient mouse&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>1 cc syringe</td><td>BD Biosciences</td><td>301025</td><td></td></tr><tr><td>18 G needle</td><td>Fisher Scientific</td><td>148265d</td><td></td></tr><tr><td>200 uL pipette tip</td><td>Fisher Scientific</td><td>02-707-422</td><td></td></tr><tr><td>Double distilled water</td><td>Institutional core facility supplies</td><td></td><td></td></tr><tr><td>Latex bulb</td><td>Fisher Scientific</td><td>03-448-21</td><td></td></tr><tr><td>Micro cover glass slip</td><td>VWR</td><td>48366-067</td><td></td></tr><tr><td>Microscope slide</td><td>Fisher Scientific</td><td>12-544-7</td><td></td></tr><tr><td>Standard light microscope</td><td>Leica Microsystems</td><td>DM IL</td><td>For evaluating vaginal cytology smears</td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Supplies for harvesting tissue from recipient mouse&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>10% Buffered formalin</td><td>Fisher Scientific</td><td>SF100-4</td><td></td></tr><tr><td>Biopsy foam pads</td><td>Fisher Scientific</td><td>22-038-222</td><td></td></tr><tr><td>Precision Digital Calipers</td><td>Electron Microscopy Sciences</td><td>62065-40</td><td></td></tr><tr><td>Processing/embedding cassettes</td><td>Fisher Scientific</td><td>22-272416</td><td></td></tr></tbody></table>

a syngeneic murine model of endometriosis using naturally cycling mice

Endometriosis is a leading cause of pelvic pain and infertility. It is defined by the presence of endometrial tissue in extrauterine locations. The development of novel therapies and diagnostic tools for endometriosis has been limited due in part to challenges in studying the disease. Outside of primates, few mammals menstruate, and none develop spontaneous endometriosis. Rodent models are popular but require artificial induction of endometriosis, with many utilizing either immunocompromised mice or surgically induced disease. Recently, more attention has been given to models involving intraperitoneal injection. We present a murine model of endometriosis that integrates several features of existing endometriosis models into a novel, simplified system that relies on microscopic quantification in lieu of subjective grading. In this model, we perform hormonal stimulation of donor mice, intraperitoneal injection, systematic abdominal survey and tissue harvest, and histologic quantification that can be performed and verified at any time after necropsy. This model requires minimal resources and training; does not require expertise by lab technicians in murine survival surgery or in the identification of gross endometriotic lesions; can be used in immunocompromised, immunocompetent, and/or mutant mice; and reliably creates endometriotic lesions that are histologically consistent with human endometriotic disease.

For an initial proof of concept experiment, donor endometrium from RFP mice was injected into wildtype recipient mice. H&#38;E staining revealed histopathologic confirmation of classic architecture of endometriosis lesion (Figure 3A). Fluorescent microscopy confirmed that the observed lesion in question originated from the donor (Figure 3B).
The second experiment was performed using 10 wildtype C57BL/6J donors and 10 recipients. An additional 5 recipients received sham treatment (injected with PBS and not endometrial fragments) and assigned a random identification number; reviewers were blinded prior to necropsy and histopathologic review.
All recipients received donor endometrial tissue within 42 h of PSMG donor treatment. There was no correlation between this time interval and final uterine weight or lesion size. At the time of donor procurement, average total uterine weight was 54 &#177; 9.5 mg. Average fragment number was 22.4 &#177; 5.2, resulting in average fragment weight of 2.5 &#177; 0.5 mg. Endpoint surgery occurred at either day 20 or day 22 for all recipients.
With an average number of lesions of 1.5 and average gross total lesion diameter of 3.7 mm, these end points are similar to other studies utilizing mouse models with a similar weight of injected endometrial fragments (Figure 2)10. The prevalence of lesions for all mice was 80%. The mice without lesions had significantly greater endometrial fractioning compared to mice with lesions (30.5 total fragments versus 20.3, respectively; average total fragments for all mice was 22.4) resulting in below average fragment size at the time of injection (1.9 mg vs 2.6 mg). As expected, sham controls (injected with saline and not endometrial fragments) had no gross or microscopic disease. In a subsequent study using an expanded number of mice, estrous phase of the recipient mouse was not associated with lesion number or microscopic disease score.
Gross lesion number appears to be a poor surrogate marker for total lesion burden, as there was discordance between the macroscopic and the microscopic disease present in the tissues. In 60% (n = 6) of the cases, there was agreement between the macroscopic and microscopic scoring (agreement defined as both showing presence or absence and difference in total size was within 3 mm). However, in 40% (n = 4) of the cases, there was disagreement, with 10% (n = 1) of the time macroscopic disease was absent but microscopic disease was present by histology, 10% (n = 1) macroscopic disease was seen despite no absence of endometriosis on confirmatory histology (Figure 5), and the remaining 20% (n = 2) there was agreement in presence but not magnitude of lesion size. Thus, macroscopic examination for lesions alone is not sufficient for the quantification of endometriosis disease burden.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61960/61960fig1v2.jpg" /> 
Figure 1: Overview of the model.&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61960/61960fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61960/61960fig02.jpg" /> 
Figure 2: Surveyed regions for lesion quantification. While the bowel and other intraperitoneal locations can harbor lesions, it is more challenging to distinguish these grossly and an exhaustive survey of the abdomen would be more time intensive with diminishing returns. Therefore, a consistent, systematic approach of harvesting the complete tissue (regardless of whether gross disease is present) is performed in the three most common regions of endometriosis formation: the abdominal wall/peritoneum (A), the pancreas and mesenteric fat (B), and the parauterine fat (C). Labeled are representative images of microscopic findings of lesions from each of the three regions. 40x magnification.&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61960/61960fig02large.jpg" style="font-size: 14px;" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61960/61960fig03.jpg" /> 
Figure 3: Induction of endometriosis using donor mice endometrium expressing red fluorescent protein. (A) H&#38;E section of the lesion. (B) Fluorescent microscopy with DAPI staining. 40x magnification.&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61960/61960fig03large.jpg" style="font-size: 14px;" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61960/61960fig04.jpg" /> 
Figure 4: Representative images of the software used to measure the dimensions of the lesion dimensions and quantify lesion burden.&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61960/61960fig04large.jpg" style="font-size: 14px;" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61960/61960fig05.jpg" /> 
Figure 5: Representative samples of gross &#34;lesions&#34; that are not endometriosis by histopathologic examination. 40x magnification.&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61960/61960fig05large.jpg" style="font-size: 14px;" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Syngeneic Murine Model of Endometriosis using Naturally Cycling Mice at JoVE.com

A Syngeneic Murine Model of Endometriosis using Naturally Cycling Mice

Many rodent models of endometriosis are limited by technical complexity, reproducibility, and/or need for immunocompromised animals or special reporter mice. We present a simplified system of lesion induction using any experimental mouse with an independently verifiable, objective scoring system and with no requirement for ovariectomy or survival surgery.

Endometriosis is a leading cause of pelvic pain and infertility. It is defined by the presence of endometrial tissue in extrauterine locations. The ...

a-syngeneic-murine-model-of-endometriosis-using-naturally-cycling-mice

Universidad San Sebastian

Research

JoVE Journal

Medicine

5.1K Views.  Cleveland Clinic. Many rodent models of endometriosis are limited by technical complexity, reproducibility, and/or need for immunocompromised animals or special reporter mice. We present a simplified system of lesion induction using any experimental mouse with an independently verifiable, objective scoring system and with no requirement for ovariectomy or survival surgery.

Video: A Syngeneic Murine Model of Endometriosis using Naturally Cycling Mice

Mouse Model of Surgically-induced Endometriosis by Auto-transplantation of Uterine Tissue

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of lesions, and/or chronic pharmaceutical management of pain and infertility symptoms. The cost associated with endometriosis has been estimated at 22 billion dollars per year in the United States1. To further our understanding of mechanisms underlying this enigmatic disease, animal models have been employed. Primates spontaneously develop endometriosis and therefore primate models most closely resemble the disease in women. Rodent models, however, are more cost effective and readily available2. The model that we describe here involves an autologous transfer of uterine tissue to the intestinal mesentery (Figure 1) and was first developed in the rat3 and later transferred to the mouse4. The goal of the autologous rodent model of surgically-induced endometriosis is to mimic the disease in women. We and others have previously shown that the altered gene expression pattern observed in endometriotic lesions from mice or rats mirrors that observed in women with the disease5,6. One advantage of performing the surgery in the mouse is that the abundance of transgenic mouse strains available can aid researchers in determining the role of specific components important in the establishment and growth of endometriosis. An alternative model in which excised human endometrial fragments are introduced to the peritoneum of immunocompromised mice is also widely used but is limited by the lack of a normal immune system which is thought to be important in endometriosis2,7. Importantly, the mouse model of surgically induced endometriosis is a versatile model that has been used to study how the immune system8, hormones9,10 and environmental factors11,12 affect endometriosis as well as the effects of endometriosis on fertility13 and pain14.

A description of the surgical induction of endometriosis in mice and rats by auto-transplantation of uterine tissue to the arterial cascade of the intestinal mesentery.

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a lean PCOS-like mouse model that mimics women with PCOS is clinically meaningful. In this protocol, we describe such a model. By inserting a 4 mm length of DHT (dihydrotestosterone) crystal powder pellet (total length of pellet is 8 mm), and replacing it monthly, we are able to produce a PCOS-like mouse model with serum DHT levels 2 fold higher than mice not implanted with DHT (no-DHT). We observed reproductive and metabolic dysfunction without changing body weight and body composition. While exhibiting a high degree of infertility, a small subset of these PCOS-like female mice can get pregnant and their offspring show delayed puberty and increased testosterone as adults. This PCOS-like lean mouse model is a useful tool to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

We describe the development of a lean PCOS-like mouse model with&#160;dihydrotestosterone pellet to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a ...

Developmental Biology

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

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 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 ...

Biology

Development of a Novel Endometrioma-Simulating Mouse Model

Establishment of an Experimental Mouse Model of Endometrioma to Study its Related Infertility

Endometrioma (OMA), a subtype of endometriosis characterized by the formation of endometriotic cysts in the ovaries, affects 17-44% of individuals diagnosed with endometriosis. Women with OMA often experience compromised fertility, yet the exact mechanisms underlying OMA-associated infertility remain unclear. Notably, existing animal models simulate superficial peritoneal endometriosis (SUP) and deep infiltrating endometriosis (DIE), leaving a notable gap in research focused on OMA. In response to the gap of knowledge, this paper introduces a pioneering OMA-simulating mouse model and provides a comprehensive description of the techniques and procedures employed in the model. With a high success rate of 83% and ovarian lesion specificity, this model holds significant promise for advancing our understanding of OMA, particularly in the context of infertility. It offers a valuable platform for conducting targeted research into OMA-associated fertility challenges, potentially paving the way for improved diagnostic and therapeutic strategies in the field of reproductive medicine.

This study introduces a novel mouse model specifically designed to simulate endometrioma, a clinically significant subtype of endometriosis. By utilizing C57BL/6J mice, the model aims to elucidate the pathophysiological mechanisms underlying endometrioma-related infertility, offering a refined tool to bridge the current knowledge gap in reproductive medicine.

Endometrioma (OMA), a subtype of endometriosis characterized by the formation of endometriotic cysts in the ovaries, affects 17-44% of individuals ...

Establishing 3D Endometrial Organoids from the Mouse Uterus

Endometrial tissue lines the inner cavity of the uterus and is under the cyclical control of estrogen and progesterone. It is a tissue that is composed of luminal and glandular epithelium, a stromal compartment, a vascular network, and a complex immune cell population. Mouse models have been a powerful tool to study the endometrium, revealing critical mechanisms that control implantation, placentation, and cancer. The recent development of 3D endometrial organoid cultures presents a state-of-the-art model to dissect the signaling pathways that underlie endometrial biology. Establishing endometrial organoids from genetically engineered mouse models, analyzing their transcriptomes, and visualizing their morphology at a single-cell resolution are crucial tools for the study of endometrial diseases. This paper outlines methods to establish 3D cultures of endometrial epithelium from mice and describes techniques to quantify gene expression and analyze the histology of the organoids. The goal is to provide a resource that can be used to establish, culture, and study the gene expression and morphological characteristics of endometrial epithelial organoids.

This protocol describes methodologies to establish mouse endometrial epithelial organoids for gene expression and histological analyses.

Endometrial tissue lines the inner cavity of the uterus and is under the cyclical control of estrogen and progesterone. It is a tissue that is composed of ...

Optimized Thin Endometrium Mouse Model with Low Mortality and Minimal Adhesions

Establishing a Mouse Model of Thin Endometrium

Thin endometrium (TE) has been widely recognized as a critical cause of infertility. However, the pathogenesis of TE remains unclear, and satisfactory treatment options are still urgently needed. Several animal models of TE have been developed, but the mouse model involving abdominal surgery and injection of 95% ethanol presents a formidable challenge due to the high mortality rate and risk of intrauterine adhesions if not performed correctly. Here, we describe a detailed protocol that produces reliable and reproducible TE with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus with varying infusion times. The results showed that all of the mice successfully developed TE with infusion times ranging from 1-3 min, characterized by a typical reduction in endometrial thickness and the number of glands, as well as excessive endometrial fibrosis. These findings suggest that this mouse model is suitable for studying thin endometrium and can serve as a platform for developing future TE treatments.

This article describes a detailed protocol for producing a reliable and reproducible thin endometrium with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus within 1-3 min.

Thin endometrium (TE) has been widely recognized as a critical cause of infertility. However, the pathogenesis of TE remains unclear, and satisfactory ...