使用自然循环小鼠的子宫内膜异位症的合成穆林模型
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Supplies for injecting PMSG into donor mouse | |||
| 1 mL Tuberculin syringe with 27G needle | Fisher Scientific | 14-826-87 | |
| Pregnant mare serum gonadotropin | Sigma-Aldrich | 9002-70-4 | |
| Supplies for necropsy of donor mouse and tissue processing | |||
| 6” serrated forceps, curved tip | Electron Microscopy Sciences | 72993-6C | |
| 70% ethanol solution | Pharmco | 33000HPLCCS4L | 70% solution dilute ethyl acetate 200 proof |
| Analytical balance | Mettler Toledo | ME54TE | |
| Carbon dioxide | TriGas Supplier | ||
| Dissecting tray | Fisher Scientific | S14000 | |
| No. 10 disposable scalpel | Fisher Scientific | NC9999403 | |
| Scissors, curved | Electron Microscopy Sciences | 72941 | |
| Scissors, straight | Electron Microscopy Sciences | 72940 | |
| Stereo microscope | Leica Microsystems | Leica SE 4 | For tissue dissection |
| Sterile phosphate buffered saline (PBS) | Institutional core facility supplies | ||
| Surgical instrument sterilization tray | Electron Microscopy Sciences | 66112-02 | |
| Tissue culture dishes | Fisher Scientific | 08-772E | |
| Weighing dishes | Fisher Scientific | 02-202-103 | |
| Supplies for injecting into recipient mouse | |||
| 1 cc syringe | BD Biosciences | 301025 | |
| 18 G needle | Fisher Scientific | 148265d | |
| 200 uL pipette tip | Fisher Scientific | 02-707-422 | |
| Double distilled water | Institutional core facility supplies | ||
| Latex bulb | Fisher Scientific | 03-448-21 | |
| Micro cover glass slip | VWR | 48366-067 | |
| Microscope slide | Fisher Scientific | 12-544-7 | |
| Standard light microscope | Leica Microsystems | DM IL | For evaluating vaginal cytology smears |
| Supplies for harvesting tissue from recipient mouse | |||
| 10% Buffered formalin | Fisher Scientific | SF100-4 | |
| Biopsy foam pads | Fisher Scientific | 22-038-222 | |
| Precision Digital Calipers | Electron Microscopy Sciences | 62065-40 | |
| Processing/embedding cassettes | Fisher Scientific | 22-272416 |
References
- Zondervan, K. T., Becker, C. M., Missmer, S. A. Endometriosis. England Journal of Medicine. 382 (13), 1244-1256 (2020).
- Schwartz, K., Llarena, N. C., Rehmer, J. M., Richards, E. G., Falcone, T. The role of pharmacotherapy in the treatment of endometriosis across the lifespan. Expert Opinion on Pharmacotherapy. 21 (8), 893-903 (2020).
- Giudice, L. C. Clinical practice. Endometriosis. New England Journal of Medicine. 362 (25), 2389-2398 (2010).
- D’Hooghe, T. M., Debrock, S. Endometriosis, retrograde menstruation and peritoneal inflammation in women and in baboons. Human Reproduction Update. 8 (1), 84-88 (2002).
- Ahn, S. H., et al. Pathophysiology and immune dysfunction in endometriosis. BioMed Research International. 2015, (2015).
- Falcone, T., Flyckt, R. Clinical management of endometriosis. Obstetrics and Gynecology. 131 (3), 557-571 (2018).
- Vercellini, P., et al. Association between endometriosis stage, lesion type, patient characteristics and severity of pelvic pain symptoms: a multivariate analysis of over 1000 patients. Human Reproduction. 22 (1), 266-271 (2007).
- Bulun, S. E., et al. Endometriosis. Endocrine Reviews. 40 (4), 1048-1079 (2019).
- Brueggmann, D., et al. Novel three-dimensional in vitro models of ovarian endometriosis. Journal of Ovarian Research. 7, 17 (2014).
- Dodds, K. N., Beckett, E. A. H., Evans, S. F., Hutchinson, M. R. Lesion development is modulated by the natural estrous cycle and mouse strain in a minimally invasive model of endometriosis. Biology of Reproduction. 97 (6), 810-821 (2017).
- Martinez, J., Bisbal, V., Marin, N., Cano, A., Gómez, R. Noninvasive monitoring of lesion size in a heterologous mouse model of endometriosis. Journal of Visualized Experiments: JoVE. (144), (2019).
- Pelch, K. E., Sharpe-Timms, K. L., Nagel, S. C. Mouse model of surgically-induced endometriosis by auto-transplantation of uterine tissue. Journal of Visualized Experiments: JoVE. (59), e3396 (2012).
- Nishimoto-Kakiuchi, A., et al. Spontaneous endometriosis in cynomolgus monkeys as a clinically relevant experimental model. Human Reproduction. 33 (7), 1228-1236 (2018).
- Nair, H. B., et al. An efficient model of human endometriosis by induced unopposed estrogenicity in baboons. Oncotarget. 7 (10), 10857-10869 (2016).
- Laganà, A. S., et al. Translational animal models for endometriosis research: a long and windy road. Annals of Translational Medicine. 6 (22), 431 (2018).
- Bellofiore, N., et al. First evidence of a menstruating rodent: the spiny mouse (Acomys cahirinus). Amercian Journal of Obstetrics and Gynecology. 216 (1), 1-11 (2017).
- Bruner-Tran, K. L., Mokshagundam, S., Herington, J. L., Ding, T., Osteen, K. G. Rodent models of experimental endometriosis: identifying mechanisms of disease and therapeutic targets. Current Women’s Health Reviews. 14 (2), 173-188 (2018).
- Bilotas, M. A., et al. Interplay between endometriosis and pregnancy in a mouse model. PloS One. 10 (4), 0124900 (2015).
- Peterse, D., et al. Of mice and women: a laparoscopic mouse model for endometriosis. Journal of Minimally Invasive Gynecology. 25 (4), 578-579 (2018).
- Richards, E. G., et al. KLF11 is an epigenetic mediator of DRD2/dopaminergic signaling in endometriosis. Reproductive Sciences. 224 (8), 1129-1138 (2017).
- Jones, R. L., Lang, S. A., Kendziorski, J. A., Greene, A. D., Burns, K. A. Use of a Mouse Model of Experimentally Induced Endometriosis to Evaluate and Compare the Effects of Bisphenol A and Bisphenol AF Exposure. Environmental Health Perspectives. 126 (12), 127004 (2018).
- Greaves, E., et al. A novel mouse model of endometriosis mimics human phenotype and reveals insights into the inflammatory contribution of shed endometrium. The American Journal of Pathology. 184 (7), 1930-1939 (2014).
- Nothnick, W. B., Graham, A., Holbert, J., Weiss, M. J. miR-451 deficiency is associated with altered endometrial fibrinogen alpha chain expression and reduced endometriotic implant establishment in an experimental mouse model. PloS One. 9 (6), 100336 (2014).
