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

우리는 원고를 준비하는 동안 중요한 검토와 통찰력에 대한 Reizes 실험실의 구성원뿐만 아니라 데이터 수집 및 데이터 분석에 대한 지원을 Lerner 연구소의 이미징 및 히스토로지 코어에 감사드립니다. 이 사업은 클리블랜드 클리닉의 연구 프로그램 위원회를 통해 내부 보조금 기금을 통해 지원되었으며 생식 조사 및 바이엘 협회를 통한 외부 보조금으로 지원되었습니다. Reizes 실험실에 있는 연구는 또한 치료벨로사노 자전거를 통해 투자됩니다, 부인과 암에 있는 연구 우수성의 센터, 그리고 Uterine 암 연구를 위한 로라 J. Fogarty 부여 의자를 통해. 클리블랜드 클리닉은 그림 1 및 그림 2에대한 저작권 권한을 소유하고 있습니다.

참고 : 이 연구에서 동물의 사용은 클리블랜드 클리닉 러너 연구소에서 기관 동물 관리 및 사용위원회 (IACUC)에 의해 승인되었다. 공개적으로 이용 가능한 모든 동물 관리 및 사용 기준은 국립 보건원의 지침에 따라 수행되었습니다. 이 절차는 무균 기술을 활용하고 있습니다. 페트리 접시는 멸균입니다. 사용되는 PBS/식염수는 멸균입니다. 부검 및 조직 해부에 대한 수술 기구는 오토 클레이브를 ?...

<ol>
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H., 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=Pathophysiology+and+immune+dysfunction+in+endometriosis.">Pathophysiology and immune dysfunction in endometriosis.</a> BioMed Research International. 2015, (2015).</li><li>Falcone, T., Flyckt, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+management+of+endometriosis.">Clinical management of endometriosis.</a> Obstetrics and Gynecology. 131 (3), 557-571 (2018).</li><li>Vercellini, P., 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=Association+between+endometriosis+stage,+lesion+type,+patient+characteristics+and+severity+of+pelvic+pain+symptoms:+a+multivariate+analysis+of+over+1000+patients.">Association between endometriosis stage, lesion type, patient characteristics and severity of pelvic pain symptoms: a multivariate analysis of over 1000 patients.</a> Human Reproduction. 22 (1), 266-271 (2007).</li><li>Bulun, S. 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lesion+development+is+modulated+by+the+natural+estrous+cycle+and+mouse+strain+in+a+minimally+invasive+model+of+endometriosis.">Lesion development is modulated by the natural estrous cycle and mouse strain in a minimally invasive model of endometriosis.</a> Biology of Reproduction. 97 (6), 810-821 (2017).</li><li>Martinez, J., Bisbal, V., Marin, N., Cano, A., G&#243;mez, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+monitoring+of+lesion+size+in+a+heterologous+mouse+model+of+endometriosis.">Noninvasive monitoring of lesion size in a heterologous mouse model of endometriosis.</a> Journal of Visualized Experiments: JoVE. (144), (2019).</li><li>Pelch, K. E., Sharpe-Timms, K. L., Nagel, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+of+surgically-induced+endometriosis+by+auto-transplantation+of+uterine+tissue.">Mouse model of surgically-induced endometriosis by auto-transplantation of uterine tissue.</a> Journal of Visualized Experiments: JoVE. (59), e3396 (2012).</li><li>Nishimoto-Kakiuchi, 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=Spontaneous+endometriosis+in+cynomolgus+monkeys+as+a+clinically+relevant+experimental+model.">Spontaneous endometriosis in cynomolgus monkeys as a clinically relevant experimental model.</a> Human Reproduction. 33 (7), 1228-1236 (2018).</li><li>Nair, H. 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. 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=KLF11+is+an+epigenetic+mediator+of+DRD2/dopaminergic+signaling+in+endometriosis.">KLF11 is an epigenetic mediator of DRD2/dopaminergic signaling in endometriosis.</a> Reproductive Sciences. 224 (8), 1129-1138 (2017).</li><li>Jones, R. L., Lang, S. A., Kendziorski, J. A., Greene, A. D., Burns, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+Mouse+Model+of+Experimentally+Induced+Endometriosis+to+Evaluate+and+Compare+the+Effects+of+Bisphenol+A+and+Bisphenol+AF+Exposure.">Use of a Mouse Model of Experimentally Induced Endometriosis to Evaluate and Compare the Effects of Bisphenol A and Bisphenol AF Exposure.</a> Environmental Health Perspectives. 126 (12), 127004 (2018).</li><li>Greaves, 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=A+novel+mouse+model+of+endometriosis+mimics+human+phenotype+and+reveals+insights+into+the+inflammatory+contribution+of+shed+endometrium.">A novel mouse model of endometriosis mimics human phenotype and reveals insights into the inflammatory contribution of shed endometrium.</a> The American Journal of Pathology. 184 (7), 1930-1939 (2014).</li><li>Nothnick, W. B., Graham, A., Holbert, J., Weiss, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-451+deficiency+is+associated+with+altered+endometrial+fibrinogen+alpha+chain+expression+and+reduced+endometriotic+implant+establishment+in+an+experimental+mouse+model.">miR-451 deficiency is associated with altered endometrial fibrinogen alpha chain expression and reduced endometriotic implant establishment in an experimental mouse model.</a> PloS One. 9 (6), 100336 (2014).</li></ol>

우리의 연구는 자궁 내막증이 난소 절제술 및 / 또는 생존 수술의 사용을 요구하지 않고 마우스에서 안정적으로 유도 될 수 있음을 보여 주며, 자궁 내막 병변은 복부 및 역사학 분석의 표준화 된 설문 조사를 사용하여 식별하고 정량화 할 수 있음을 보여줍니다.
자궁내막증의 많은 뮤린 연구는 기증자 자?...

자궁내막증은 여성생식기관의수수께끼 질환으로, 여성1,2에상당한 재정적, 건강부담이 있다. 자궁내막증의 병인은 완전히 이해되지 않으며, 코엘로믹 대사증, 배아 뮐레리안 휴식, 골수 유래 전구세포의 모집, 역행 생리3을포함한 여러 가지 설명이 제안되었다. 이러한 제안된 기계장치의 다중 양상이 관련될 수 있고, 단 하나 설명이 질병의 모든 양식을 설명할 수 있는 동안, 자궁내막증 병인의 주요 모형은 역행생리입니다. 역행 생리는 나팔관을 통해 복막 구멍으로 생리 유출물의 통로입니다. 생리 여성의 90 %가 정기적으로 역행 생리4,5을겪는 것으로 추정됩니다. 역행 생리의 이 일반적인 현상을 감안할 때, 왜 자궁내막증은 여자의 부세트에서만 발전하는 것은불분명5. 이 질병의 병인학을 더 잘 이해하기 위해 직접적인 인간 연구는 실현 가능하지 않으며 동물 연구가 보증됩니다.
자궁내막증은 치료와 연구 모두에 도전이다. 질병의 보급은 알려지지 않았지만 10%1로추정된다. 자궁내막증의 몇몇 향상된 모형이 비침습적인 화상 진찰을 통해 정확하게 확인될 수 있는 동안, 결정적인 진단은 외과적으로 취득한 생검 견본의 조직병리학 분석을 통해서만 달성됩니다; 시각적으로 병에 걸린 것으로 보이는 병변은 실제로 다른 원인6에서 섬유증또는 흉터일 수 있다. 질병의 심각도 및 정도는 증상과 상관 관계가 없습니다7.
자궁내막증 병변은 마이크로환경 내에서 복잡한 방식으로 상호작용하는 이질성 세포 유형 및 집단으로 구성되므로, 따라서 세포모델의유용성을제한8,9. 생체 내에서 모델이 존재하지만, 이들은 고유의 도전과한계가 10,11,12. 영장류 모델은 이상적이지만종종13,14,15가가능하지 않습니다. 몇몇 비 영장류 포유동물은 생리하고 자발적으로 자궁내막증을개발16. 자궁 내막증의 설치류 모델은 존재하지만 각각은한계가 17. 이들 모델의 대부분은 기증자 수혜자 벽 또는 창자에 자궁내막 조직을 봉합하거나 이식하는 생존 수술을 요구하며, 기술적 복잡성을 추가하고 마취가 필요하며 수술 자체로부터 면역 인자를혼동18,19,20. 또한, 많은 모델 은 ovariectomy 및 에스트로겐 보충을 필요로; 병변 수율을 증가하는 동안, 이것은 시간, 비용 및 추가 생존 수술을 추가합니다. 인트라피오날(IP) 주사 모델은 마취 또는 생존 수술을 필요로 하지 않으며, 이들 모델은 논리적으로 역행 생리를 시뮬레이션하여 모델을 봉합하는모델(21,22,23)보다더 잘 한다. 그러나 대부분의 IP 모델은 주입 후 자궁내막 단편의 무작위 분산으로 인해 병변 위치에서 더 많은 가변성을 가지므로 병변 식별 및 측정에 더 많은 편견이 있습니다.
여기서 우리는 주관적인 채점 대신 현미경 정량화에 의존하는 새로운, 단순화되고 효율적인 시스템으로 기존 자궁 내막증 모델의 여러 기능을 통합하는 자궁 내막증의 뮤린 모델을 제시합니다.

<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

자궁내막증은 골반 통증과 불임의 주요 원인입니다. 그것은 외피 위치에 자궁 내막 조직의 존재에 의해 정의된다. 자궁 내막증을 위한 새로운 치료 및 진단 공구의 발달은 질병을 공부에 있는 도전 때문에 부분적으로 제한되었습니다. 영장류 의 외부, 몇 포유동물 생리, 아무도 자발적인 자궁 내막증을 개발하지 않습니다. 설치류 모델은 인기가 있지만 많은 사람들이 면역 손상 된 마우스 또는 외과적으로 유도 된 질병을 활용하여 자궁 내막증의 인공 유도가 필요합니다. 최근에는 복막 주사와 관련된 모델에 더 많은 관심을 기울이고 있습니다. 기존 자궁내막증 모델의 여러 기능을 주관적인 채점 대신 미세한 정량화에 의존하는 새로운 단순화 된 시스템으로 통합하는 자궁 내막증의 뮤린 모델을 제시합니다. 이 모델에서는, 우리는 기증자 마우스의 호르몬 자극, 복막 주사, 체계적인 복부 조사 및 조직 수확, 및 부검 후에 언제든지 수행되고 검증될 수 있는 조직적 정량화를 능력을 발휘합니다. 이 모델에는 최소한의 리소스와 교육이 필요합니다. 뮤린 생존 수술이나 총 자궁 내막증 병변의 식별에 실험실 기술자에 의해 전문 지식을 필요로하지 않습니다; 면역 손상, 면역 무능, 및 돌연변이 마우스에서 사용할 수 있습니다. 안정적으로 인간 자궁 내막증 질환과 조직학적으로 일치하는 자궁 내막증 병변을 만듭니다.

개념 실험의 초기 증거를 위해, RFP 마우스에서 기증자 내메트리움은 야생 형 받는 사람 마우스로 주입되었다. H&E 염색은 자궁내막증 병변의 고전적인 건축에 대한 조직병리학적 확인을공개했다(그림 3A). 형광 현미경 검사는 문제의 관찰 된 병변이 기증자(도 3B)로부터유래것을 확인했다.
두 번째 실험은 10개의 야생형 C57BL/6J 기증자...

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

자궁내막증의 많은 설치류 모델은 기술적 복잡성, 재현성 및/또는 면역 손상된 동물 또는 특수 리포터 마우스에 대한 필요성에 의해 제한됩니다. 우리는 독립적으로 검증 가능하고 객관적인 점수 매기기 시스템을 갖춘 실험용 마우스를 사용하여 병변 유도의 단순화 된 시스템을 제시하며, 난소 절제술 이나 생존 수술에 대한 요구 사항이 없습니다.

자연적으로 사이클링 마우스를 사용하는 자궁내막증의 신제닉 뮤린 모델

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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