Name: a syngeneic murine model of endometriosis using naturally cycling mice
Uploaded: 2020-11-24T15:00:00
Description: Watch this Scientific Journal Video about A Syngeneic Murine Model of Endometriosis using Naturally Cycling Mice at JoVE.com

我们要感谢Reizes实验室的成员在编写手稿期间所做的批判性审查和见解，以及Lerner研究所的成像和组织学核心在数据收集和数据分析方面给予的帮助。这项工作通过克利夫兰诊所的研究计划委员会提供内部赠款资金，并通过生殖调查协会和拜耳获得外部赠款。Reizes实验室的研究也通过维洛萨诺自行车治疗，妇科癌症研究中心，并通过劳拉J.福加蒂赋予子宫癌研究主席资助。克利夫兰诊所拥有图1和图2的版权许可。

注：在这项研究中使用动物得到了克利夫兰诊所勒纳研究所的机构动物护理和使用委员会（IACUC）的批准。所有公开的动物护理和使用标准都是根据国家卫生研究院的指导方针执行的。此过程采用无菌技术。佩特里菜是无菌的。使用的PBS/盐水是无菌的。用于尸检和组织解剖的手术器械通过高压灭菌器进行灭菌。我们在动物病例之间的仪器上使用 70% 的 EtOH（如果每节会话超过一次），以减少污染。...

<ol>
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E., 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=Endometriosis.">Endometriosis.</a> Endocrine Reviews. 40 (4), 1048-1079 (2019).</li><li>Brueggmann, D., 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=Novel+three-dimensional+in+vitro+models+of+ovarian+endometriosis.">Novel three-dimensional in vitro models of ovarian endometriosis.</a> Journal of Ovarian Research. 7, 17 (2014).</li><li>Dodds, K. N., Beckett, E. A. H., Evans, S. F., Hutchinson, M. 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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=An+efficient+model+of+human+endometriosis+by+induced+unopposed+estrogenicity+in+baboons.">An efficient model of human endometriosis by induced unopposed estrogenicity in baboons.</a> Oncotarget. 7 (10), 10857-10869 (2016).</li><li>Lagan&#224;, A. S., 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=Translational+animal+models+for+endometriosis+research:+a+long+and+windy+road.">Translational animal models for endometriosis research: a long and windy road.</a> Annals of Translational Medicine. 6 (22), 431 (2018).</li><li>Bellofiore, 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=First+evidence+of+a+menstruating+rodent:+the+spiny+mouse+(Acomys+cahirinus).">First evidence of a menstruating rodent: the spiny mouse (Acomys cahirinus).</a> Amercian Journal of Obstetrics and Gynecology. 216 (1), 1-11 (2017).</li><li>Bruner-Tran, K. L., Mokshagundam, S., Herington, J. L., Ding, T., Osteen, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+experimental+endometriosis:+identifying+mechanisms+of+disease+and+therapeutic+targets.">Rodent models of experimental endometriosis: identifying mechanisms of disease and therapeutic targets.</a> Current Women's Health Reviews. 14 (2), 173-188 (2018).</li><li>Bilotas, M. A., 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=Interplay+between+endometriosis+and+pregnancy+in+a+mouse+model.">Interplay between endometriosis and pregnancy in a mouse model.</a> PloS One. 10 (4), 0124900 (2015).</li><li>Peterse, D., 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=Of+mice+and+women:+a+laparoscopic+mouse+model+for+endometriosis.">Of mice and women: a laparoscopic mouse model for endometriosis.</a> Journal of Minimally Invasive Gynecology. 25 (4), 578-579 (2018).</li><li>Richards, E. 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我们的研究表明，子宫内膜异位症可以在小鼠中可靠诱导，而无需使用卵巢切除术和/或生存手术，并且通过腹部的标准化调查和组织分析可以识别和量化异位子宫内膜病变。
许多子宫内膜异位症的研究利用手术诱发的子宫内膜异位症，其中供体子宫内膜被缝合到位到肠道，腹壁，或其他腹腔内位置<sup cla...

子宫内膜异位症是女性生殖道的一种神秘疾病，给1、2岁女性带来沉重的经济和健康负担。子宫内膜异位症的病因尚未完全了解，并提出了多种解释，包括宇宙代谢、胚胎穆勒安息、骨髓衍生祖细胞的招募和逆行月经3。虽然这些拟议机制的多个方面可能涉及，而且没有单一的解释可以解释该疾病的所有形式，但子宫内膜异位症发病机制的主要模式是逆行月经。逆行月经是月经污水通过输卵管进入腹腔：据估计，90%的月经妇女定期接受逆行月经4，5。鉴于这种常见的月经逆行现象，为什么子宫内膜异位症只在一部分妇女中发展尚不清楚。为了更好地了解这种疾病的病因，直接的人类研究是不可行的，动物研究是有道理的。
子宫内膜异位症是治疗和研究的挑战。该病的流行率尚不清楚，但估计为10%1。虽然一些先进的子宫内膜异位症可以通过非侵入性成像准确识别，但只有通过对手术获得的活检标本进行组织病理分析才能得出明确的诊断：在视觉上看起来是病变的病变，实际上可能是纤维化或疤痕从其他原因6。疾病的严重程度和程度与症状7无关。
子宫内膜异位症病变由异质细胞类型和群体组成，这些细胞类型和群体在微环境内以复杂的方式相互作用，因此限制了细胞模型8、9的用处。在体内模型存在，但这些有内在的挑战和局限性10，11，12。灵长类动物模型是理想的，但往往不可行13，14，15。很少有非灵长类哺乳动物月经并自发地发展为子宫内膜异位症。子宫内膜异位症的啮齿动物模型存在，但每个模型都有限制17。其中许多模型需要生存手术缝合或植入子宫内膜组织到捐赠者接受壁或肠道，增加了技术复杂性，需要麻醉，并混淆免疫因素从手术本身18，19，20。此外，许多模型需要卵巢切除术和雌激素补充：在增加病变产量的同时，这增加了时间、费用和额外的生存手术。腹膜（IP）注射模型不需要麻醉或生存手术，这些模型逻辑上模拟逆行月经比缝合模型21，22，23更好。然而，由于注射后子宫内膜片段的随机分散，以及因此在病变识别和测量方面更加偏差，大多数 IP 模型在病变位置的变异性更大。
在这里，我们提出了子宫内膜异位症的穆林模型，将现有子宫内膜异位症模型的几个特征整合到一个新颖、简化和高效的系统中，该系统依赖于微观量化来代替主观分级。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Supplies for injecting PMSG into donor mouse</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>Supplies for necropsy of donor mouse and tissue processing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6&#8221; 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>Supplies for injecting into recipient mouse</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>Supplies for harvesting tissue from recipient mouse</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

子宫内膜异位症是骨盆疼痛和不孕症的主要原因。它是由子宫外部位的子宫内膜组织的存在来定义的。子宫内膜异位症新疗法和诊断工具的开发受到限制，部分原因是在研究这种疾病方面面临挑战。除灵长类动物外，很少有哺乳动物月经，也没有一种会出现自发性子宫内膜异位症。啮齿动物模型很受欢迎，但需要人工诱导子宫内膜异位症，许多模型利用免疫功能低下的小鼠或手术诱发的疾病。最近，人们更加关注涉及腹内注射的模型。我们提出了子宫内膜异位症的模糊模型，将现有子宫内膜异位症模型的几个特征整合到一个新的简化系统中，该系统依靠微观量化代替主观分级。在这个模型中，我们执行激素刺激的捐赠小鼠，腹腔注射，系统的腹部调查和组织收获，和组织定量，可以在坏死后的任何时间进行和验证。这种模式需要最少的资源和培训：不需要实验室技术人员在穆林生存手术或确定子宫内膜病变方面的专业知识;可用于免疫功能低下、免疫能力和/或突变小鼠：并可靠地创建与人类子宫内膜疾病在组织学上一致的子宫内膜病变。

为了概念实验的初步证明，从RFP小鼠的供体子宫内膜被注射到野生型受体小鼠。H&E染色揭示了子宫内膜异位症病变经典结构的组织病理学确认（图3A）。荧光显微镜证实，观察到的病变来自供体（图3B）。
第二个实验使用10个野生类型C57BL/6J捐赠者和10个接受者进行。另有5名接受者接受了虚假治疗（注射了PBS，而不是子宫内膜片段）...

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

许多啮齿动物子宫内膜异位症模型受技术复杂性、可重复性和/或免疫功能低下的动物或特殊记者小鼠的需求的限制。我们使用任何实验鼠使用独立可验证的客观评分系统，无需进行卵巢切除术或生存手术，即可提供简化的病变诱导系统。

使用自然循环小鼠的子宫内膜异位症的合成穆林模型

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

Our mirroring model of endometriosis is a simplified and efficient system that integrates the best features of existing models. And perhaps most importantly, relies on microscopic quantification in lieu of subjective grading. Modeling endometriosis can be difficult because there can be many confounding factors and sources of bias.In our approach, endometriosis is induced without ovariectomy or survival surgery. And the endometrial lesions are objectively quantified. These Innovations provide a highly reliable and reproducible animal model of endometriosis that can be used to further endometriosis disease research.To facilitate ongoing estrous cycling, place urine soaked bedding from a male mouse cage into the recipient female mouse cage periodically throughout development, and again at 72 hours before endometrial tissue transplant. When the donor female mice are between 22 to 24 days old, use a one milliliter syringe equipped with a 25 to 27 gauge needle to subcutaneously inject two international units of pregnant mare serum gonadotropin in 200 microliters of sterile saline into the lower mouse abdomen. 38 to 42 hours after injection, use dissecting scissors to make a shallow transverse incision through the skin and subcutaneous tissue of the euthanized mouse at the midline and use blunt traction to open the skin on either side of the incision.Before removing the uterus, trim the adjacent connective tissue and transact each uterine horn just below its respective fallopian tubes, then transect the cervix to allow removal of the entire uterus and block. Inspect the uterus carefully, removing any additional peripheral fat or connective tissue and place the uterus in a droplet of cold PBS on a Petri dish for weighing. Transect each horn across the uterine fundus as close to the fundus as possible and place the dish under a dissecting microscope.Placing one blade of the dissecting scissors inside the lumen of the first horn, cut along the major axis of the tube keeping in mind, which side is this serosa and which side is the epithelium. Then use forceps to grasp and peel away the endometrial layer and mince the tissue into 10 to 12, one by one millimeter fragments. Alternatively, mince the tissue into one by one millimeter pieces without separating off the myometrium.Then place the tissues into 500 microliters of saline in a new Petri dish and harvest the endometrium from the second uterine horn as demonstrated. For peritoneal delivery of the tissue fragments into the recipient animal, use the blunt end of a one milliliter syringe to collect the tissue pieces. A total volume of one milliliter of tissue and saline should be collected.Attach an 18 gauge needle to the syringe and gently depress the plunger to load the fluid into the needle. A mock injection into the Petri dish can be performed to ensure that all of the tissue will pass through the needle tip. Either before or after the injection, use a bulb syringe to administer 10 microliters of saline to obtain a vaginal smear of the recipient animal for estrous cycle documentation.Holding the syringe at a 45 degree angle, intraperitoneally inject the fragments. If fragments remain after the injection, draw an additional 200 microliters of saline into the syringe to allow the rest of the pragmatists to be injected. Once you're assured of no bleeding or complications, place the mouse back in the home cage with a normal diet.Approximately 21 days after fragment injection, spray the recipient mouse abdomen was 70%ethanol, intent the skin to allow creation of a superficial subcutaneous incision to bluntly open the abdomen. Perform a complete survey of the abdominal wall and peritoneum, pancreas and mesenteric fat and para uterine connective fat and tissue for gross lesions. Harvest each region of tissue entirely and place the dissected tissue samples into an appropriate labeled cassette for formalin fixation and processing according to standard protocols.Then section the formalin blocks onto two slides per tissue area at two uniform depths. To score the endometriosis lesions, scan the tissue sections at a 40 times magnification and archive the slides. After scanning, use an appropriate digital slide reading software program to define and mark the longest distance between the edges of each endometriosis lesion.A continuous lesion is defined by glands surrounded by stroma. The line does not necessarily traverse only the endometriosis tissue, but the two end points must be connected by continuous stroma and or glands. Make a second line, 90 degrees across the first line as demonstrated.If multiple non-contiguous lesions are encountered, give each their own X and Y measurements. Calculate the final score for each slide as the summation of the areas of each lesion and use the larger of the scores from the two sections on each slide as the final score for that tissue region. Then total the scores from each region to give the final microscopic score for that animal.In this representative sample, histopathologic analysis after donor endometrium injection revealed a classic architecture of endometriosis lesion. Fluorescent microscopy of the tissue sample confirmed that the lesion originated from the donor. It is important to note that the macroscopic examination for lesions alone is not sufficient for the quantification of the endometriosis disease burden as the gross lesions observed in these representative samples were ultimately determined not to be endometriosis upon histological examination.Collection of all the tissue within the designated regions reduces bias by allowing for a systematic, objective approach to lesion quantification. Once this reliable and reproducible model has been achieved, researchers can use it to investigate a multitude of endometriosis pathways and therapies.

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