我们要感谢我们的资金来源：再生医学临床研究员培训资助（AN）的加州理工学院，美国国家科学基金会（兆瓦），艾琳Perstein奖（TCM），美国外科医生学院（中医），美国小儿外科协会（中医），三月优生优育基金会（中医）。

1。制备注射移液器<ol><li>校准移液器拉马等玻璃吸管分离发生在15秒内（请参见制造商的说明，关于校准）。吸管将有锥度中分离出来。 </li><li>切断的吸管等，从开始锥度到距离吸管年底1.04厘米到1.05厘米年底。吸管的长度是成反比的吸管孔的口径。要知道，一个较长的吸管也处于弱势的提示，注射期间更容易破损。 </li><li>移液器的下一个步骤是创建一个光滑的斜面锐化冰山上的钻石磨刀轮。移液器移液器切割到适当的大小时，往往打破了自然锥轮上放置的吸管时，必须使用在其尖端。在这个过程中，重要的是锐化轮吸管轻轻休息，并定期重新评估，以确保移液器仍然是触摸轮（斜角变得顺畅，它就会失去与锐化轮接触）。 </li><li>一旦斜面是光滑，枪头必须削尖（图1）。提升吸管等，它不再是触摸锐化轮，10-50度顺时针旋转，旋转锐化〜10秒的车轮放在吸管回。吸管从磨砺轮撤出，现在研究的边缘。这一步通常需要反复与一些小的调整，创建一个尖锐的边缘。如果移液器是放置时间过长优势，它更有可能发展不平坦的表面（图1）。一侧完成后，您可以重复上述步骤相反的边缘进行锐化。 </li><li>使用永久性标记每4毫米开始移液器锥度开始绘制一个圆周线。这些界限对应量5uL。 </li></ol> 2， 在子宫移植<ol><li>准备注射的材料（如细胞，病毒等）后，您可以设置注射。我们使用以下设置连接压缩空气一个微量：（压力设置：输入60-80磅，填补40 PSI，注入8-12 PSI，余额0 PSI，按住0 PSI;注射时间100毫秒）。 </li><li>消毒的注射针，其次是与无菌1X PBS两次70％的乙醇清洁两次。由于吸管尖是很脆弱的的，我们不建议高压灭菌注射移液器。 </li><li>注射可以使用2.5X - 3.5X放大倍率的放大镜或解剖显微镜。准备好你的程序区与变暖的毯子，照明，和必要的手术器械。 </li><li>麻醉孕鼠（我们利用异氟醚和氧气通过连续流麻醉单位提供）。在仰卧位，并加盖加热垫每个磁带​​的肢体，以确保在鼠标放在鼠标。 </li><li>剪贴的皮毛。然后，穿无菌手术手套，准备腹部，其次是酒精，10％的碘伏和注入丁丙诺啡镇痛等。 </li><li>请在小腹（最下方的切口应约1厘米优越的introitus）1厘米切口。切开皮肤和筋膜。一定要小心，不要伤及腹部器官（肠子和膀胱）立即下薄薄的一层筋膜。 </li><li>用棉签，轻轻地伸展筋膜，并提供通过妊娠子宫切口。统计先确定左，右卵巢，以确保您可视化整个子宫对胎儿的总数。放置在腹腔的子宫，然后再继续使胎儿保持温暖，而你准备注射。 </li><li>制定适当的材料体积为计划注入胎儿的数目。却使你的样品针，重要的是要保持淹没枪头，以避免吸进微量管你的样品。由于一般电池体积小，我们通常把他们在一个小的离心管（如：0.5ML），或直接上的封口膜，能够不打破枪头的情况下，以填补针。 </li><li>轻轻握住每个胎儿和确定胎儿计划注入（如肝，腹腔，腿部等）的一部分。用拇指和食指，就必须稳定羊膜腔内的胎儿，以便它不会旋转，而您正在执行注射。重要的是要举行的胎儿牢牢足够稳定轻轻足够，以避免损坏。 </li><li>小心地插入吸管，子宫，羊膜，胎儿皮肤和进入靶器官。如果移液器是尖锐的，它应该很容易通过这些组织通过。如果枪头是沉闷的，你的Wi<html>会发现子宫和羊水膜的露营。注入到每一个胎儿所需的量。当务之急是，你的动作，在插入，注射，并撤回移液器时的稳定。 </li><li>一旦注入所需体积，仔细撤回移液器。如果处理得当，材料不应该泄漏出去羊膜腔或子宫。如果一个大洞，在羊膜羊水泄漏，哪一个可以识别的创建，然后危及生存。最后，研究涉及产后收获，所有的胎儿必须接受技术上完美的注射，因为是没有办法辨别胎儿出生后注射和uninjected的。 </li><li>如果您正在执行注射进入胎儿的肝脏，您可能偶尔会看到血枪头撤回后，这证实了你在正确的位置，不应影响生存。如果进行腹腔注射，略低于胎儿肝脏的目的。肌肉注射，您可能胎儿的位置，以确定髋关节和股骨和注入臀肌。最后，脑室注射，它是很容易看到日冕的缝线直接吸管适当的目标。对于这些注射，稍大口径移液器没有打破吸管穿透头骨。 </li><li>接下来，小心地将子宫进入腹腔。确保它不是本身或周围血管供应扭曲。提供1ML的无菌1X PBS在母亲的腹腔，以取代在手术过程中丢失的任何流体。在2层5-0编织可吸收缝合线（例如：薇乔）关闭切口。首先关闭而不伤及底层肠或膀胱筋膜，然后关闭皮肤。 </li><li>在这个时候，可以管理程序后药物镇痛。返回每个注入的女性，以自己的个人笼笼变暖毯地方。监视鼠标，直到它是直立的。放置在笼子里的床上用品和鼠标放置在一个安静的区域，它不应该受到干扰，直到分娩后数天（即无笼清洗）鼠标设施。最大限度地减少对动物的任何额外的压力会减少早产的机会。 </li><li>一旦你已经完成了注射，两次清洁你的吸管，用无菌的1 × PBS，一旦有70％的乙醇。 </li><li>注入女性应每天观察，以确保没有困难，他们是从程序恢复。切口应监测红肿，发炎，伤口分离，和其他感染的迹象。手术后止痛药，可管理的需要。 </li></ol> 3。代表性的成果： 注入胎儿足月分娩的生存是主要限制因素，以实现这一技术的成功。根据材料注入和应变正在使用的鼠标，存活率可以有所不同。在一般情况下，注射到E14，在野生型小鼠的造血细胞，导致至少50％的活产幼仔。生存的高利率是可能的，这取决于注射技术问题，以及被注入小鼠的特点。 尽量减少子宫和羊水膜的创伤，这个协议是最重要的的技​​术方面。夏普，小口径吸液管将在最短的期间注入子宫创伤的结果。我们不建议使用预制针口径标准注射针头过大，会导致在子宫内的一个大洞。手工制作的玻璃注射移液器是唯一在子宫内注射，可利用小口径针。认真细致的手术技术，包括温和的子宫和一个短的麻醉处理，以达到最佳的效果也非常重要。 如小鼠的遗传背景，胎龄和产仔数，收件人的具体特点，还可以影响生存。某些菌株的小鼠更容易早产和流产，根据他们的遗传背景。肌肉或神经退行性缺陷的15个转基因动物，可以有能力受损提供胎儿，阴道经过中线剖腹探查。8这些怀孕的女性可能需要交付剖宫产。我们还发现，在注射时的胎龄，可能会影响可行性。年轻比胚胎12天的胎儿，比旧的胎儿存活率较低。最后，我们发现，大量的枯枝落叶尺寸（&gt; 10胎儿），往往有注射后胎儿死亡的比率较高。注意该技术的技术方面，可以最大限度地注入胎儿的生存注入小鼠的具体特点。 ve_content“&gt;当这些方法都正确执行，可以预期，所有的胎儿暴露注射材料。产后的设置类似， 但是，在子宫内的细胞或病毒的成功交付并不总是导致供体细胞植入或基因表达，干细胞的植入，例如，是依赖于几个因素，如剂量和移植的细胞来源。同样，病毒转导的成功，部分是，所使用的病毒载体类型决定。我们必须了解小狗生存的影响，细胞植入病毒转导的因素很多，要实现这一协议的成功。 <img src="/files/ftp_upload/2303/2303fig1.jpg" alt="图1" /> 图1，图描绘适当锐化注射移液器（步骤1.4）

<ol>
	<li>Flake, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+X-linked+severe+combined+immunodeficiency+by+in+utero+transplantation+of+paternal+bone+marrow.">Treatment of X-linked severe combined immunodeficiency by in utero transplantation of paternal bone marrow.</a> N Engl J Med. 335, 1806-1810 (1996).</li><li>Wengler, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-utero+transplantation+of+parental+CD34+haematopoietic+progenitor+cells+in+a+patient+with+X-linked+severe+combined+immunodeficiency+(SCIDXI).">In-utero transplantation of parental CD34 haematopoietic progenitor cells in a patient with X-linked severe combined immunodeficiency (SCIDXI).</a> Lancet. 348, 1484-1487 (1996).</li><li>Flake, A. W., Zanjani, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+utero+hematopoietic+stem+cell+transplantation:+ontogenic+opportunities+and+biologic+barriers.">in utero hematopoietic stem cell transplantation: ontogenic opportunities and biologic barriers.</a> Blood. 94, 2179-2191 (1999).</li><li>Merianos, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+alloantibodies+induce+a+postnatal+immune+response+that+limits+engraftment+following+in+utero+hematopoietic+cell+transplantation+in+mice.">Maternal alloantibodies induce a postnatal immune response that limits engraftment following in utero hematopoietic cell transplantation in mice.</a> J Clin Invest. 119, 2590-2600 (2009).</li><li>Peranteau, W. H., Endo, M., Adibe, O. O., Flake, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+an+immune+barrier+after+in+utero+hematopoietic-cell+transplantation.">Evidence for an immune barrier after in utero hematopoietic-cell transplantation.</a> Blood. 109, 1331-1333 (2007).</li><li>Kim, H. B., Shaaban, A. F., Yang, E. Y., Liechty, K. W., Flake, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microchimerism+and+tolerance+after+in+utero+bone+marrow+transplantation+in+mice.">Microchimerism and tolerance after in utero bone marrow transplantation in mice.</a> J Surg Res. 77, 1-5 (1998).</li><li>Durkin, E. T., Jones, K. A., Rajesh, D., Shaaban, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+chimerism+threshold+predicts+sustained+engraftment+and+NK-cell+tolerance+in+prenatal+allogeneic+chimeras.">Early chimerism threshold predicts sustained engraftment and NK-cell tolerance in prenatal allogeneic chimeras.</a> Blood. 112, 5245-5253 (2008).</li><li>Mackenzie, T. C., Shaaban, A. F., Radu, A., Flake, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engraftment+of+bone+marrow+and+fetal+liver+cells+after+in+utero+transplantation+in+MDX+mice.">Engraftment of bone marrow and fetal liver cells after in utero transplantation in MDX mice.</a> J Pediatr Surg. 37, 1058-1064 (2002).</li><li>Hayashi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mixed+chimerism+following+in+utero+hematopoietic+stem+cell+transplantation+in+murine+models+of+hemoglobinopathy.">Mixed chimerism following in utero hematopoietic stem cell transplantation in murine models of hemoglobinopathy.</a> Exp Hematol. 31, 176-184 (2003).</li><li>Bouchard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+transgene+expression+in+cardiac+and+skeletal+muscle+following+fetal+administration+of+adenoviral+or+adeno-associated+viral+vectors+in+mice.">Long-term transgene expression in cardiac and skeletal muscle following fetal administration of adenoviral or adeno-associated viral vectors in mice.</a> J Gene Med. 5, 941-950 (2003).</li><li>Meza, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+pyruvate+kinase+deficiency+in+mice+by+gene+therapy+using+the+human+isoenzyme.">Rescue of pyruvate kinase deficiency in mice by gene therapy using the human isoenzyme.</a> Mol Ther. 17, 2000-2009 (2009).</li><li>MacKenzie, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transduction+of+liver+and+muscle+after+in+utero+injection+of+lentiviral+vectors+with+different+pseudotypes.">Efficient transduction of liver and muscle after in utero injection of lentiviral vectors with different pseudotypes.</a> Mol Ther. 6, 349-358 (2002).</li><li>MacKenzie, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transduction+of+satellite+cells+after+prenatal+intramuscular+administration+of+lentiviral+vectors.">Transduction of satellite cells after prenatal intramuscular administration of lentiviral vectors.</a> J Gene Med. 7, 50-58 (2005).</li><li>Sabatino, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+expression+of+hF.IX+After+tolerance+induction+by+in+utero+or+neonatal+administration+of+AAV-1-F.IX+in+hemophilia+B+mice.">Persistent expression of hF.IX After tolerance induction by in utero or neonatal administration of AAV-1-F.IX in hemophilia B mice.</a> Mol Ther. 15, 1677-1685 (2007).</li><li>Mellor, A. L., Munn, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunology+at+the+maternal-fetal+interface:+lessons+for+T+cell+tolerance+and+suppression.">Immunology at the maternal-fetal interface: lessons for T cell tolerance and suppression.</a> Annu Rev Immunol. 18, 367-391 (2000).</li><li>Billingham, R. E., Brent, L., Medawar, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actively+acquired+tolerance+of+foreign+cells.">Actively acquired tolerance of foreign cells.</a> Nature. 172, 603-606 (1953).</li><li>Endo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+to+ocular+stem+cells+by+early+gestational+intraamniotic+injection+of+lentiviral+vector.">Gene transfer to ocular stem cells by early gestational intraamniotic injection of lentiviral vector.</a> Mol Ther. 15, 579-587 (2007).</li><li>Waddington, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+transgene+expression+by+administration+of+a+lentivirus-based+vector+to+the+fetal+circulation+of+immuno-competent+mice.">Long-term transgene expression by administration of a lentivirus-based vector to the fetal circulation of immuno-competent mice.</a> Gene Ther. 10, 1234-1240 (2003).</li><li>Schachtner, S., Buck, C., Bergelson, J., Baldwin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally+regulated+expression+patterns+following+in+utero+adenovirus-mediated+gene+transfer.">Temporally regulated expression patterns following in utero adenovirus-mediated gene transfer.</a> Gene Ther. 6, 1249-1257 (1999).</li></ol>

比林汉姆，布伦特和梅达沃在50年前， 在子宫内移植在小鼠体内诱导外源蛋白的免疫耐受。从那时起，16几个变化，这种技术已被用于解决在免疫学和干细胞生物学的问题。 这里详细的协议是IUT的最方便的方法之一。胎肝提供了一个轻松的可视化的目标，并提供通过门户网站和肝静脉全身血液循环的访问。然而，一些已修改，以优化的时间和所需注射的位置。?...

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		<th>Catalogue Number</th>
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<td>Pipettes</td>
<td>&nbsp;</td>
<td>Kimble</td>
<td>71900-100</td>
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<td>Pipette puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments Company</td>
<td>Model P-30</td>
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<td>Microinjector</td>
<td>&nbsp;</td>
<td>Narishige</td>
<td>IM-300</td>
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<td>Pipette sharpener</td>
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<td>Sutter Instruments Company</td>
<td>Model BV-10</td>
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a mouse model of in utero transplantation

在子宫内移植的干细胞和病毒，用于治疗人类胎儿先天性疾病的巨大潜力。例如， 在子宫内的造血干细胞移植（IUT）已成功地用于治疗重症联合免疫缺陷。1,2在其他几个条件的患者，然而，IUT的已经尝试没有成功。鉴于这些好坏参半的结果，可用性一个有效率的非​​人性化的模型来研究干细胞移植和基因治疗的生物后遗症，是推进这一领域的关键。我们和其他人使用IUT的研究在野生型小鼠4-7和遗传性疾病的影响因素在宫内移植造血干细胞成功植入小鼠模型。8,9胎儿环境也提供了相当大的优势在子宫内基因治疗的成功。例如，交付10的腺病毒，腺相关病毒，逆转录病毒11和慢病毒载体进入胎儿体内12,13 在子宫内导致在远离注射网站长期的基因表达的多器官的转导。因此，基因疗法可能被视为一个单基因疾病，如肌肉萎缩症或囊性纤维化的可能的治疗策略。 IUT的另一个潜在优势是能够到一个特定的抗原诱导免疫耐受。由于血友病因子IX的宽容早在发展成果引入到这种蛋白质的小鼠 。14 除了其使用在调查潜在的人类疗法，对IUT的小鼠模型可以是一个强大的工具，在发育和干细胞生物学研究的基本问题。例如，一个可以提供各种小分子诱导或抑制特定基因的表达在妊娠阶段的定义和操作的发展途径。这些改动的影响，可以在不同的时间点进行评估后，最初的移植。此外，还有一个可以移植到胎儿的环境，研究在非照射和泰然自若的主机环境中的干细胞分化的多能干或宗族的祖细胞。 在免疫学领域，发育和干细胞生物学内的IUT的小鼠模型已经提供了许多见解。在此视频为基础的协议，我们描述了一个步骤，循序渐进的方式执行IUT的小鼠胎儿，并概述这项技术的关键步骤和潜在的隐患。

Watch this Scientific Journal Video about A Mouse Model of in Utero Transplantation at JoVE.com

A Mouse Model of in Utero Transplantation

在小鼠模型<em&gt;在子宫内</em&gt;移植是一种多用途的工具，可用于胎儿干细胞移植和基因治疗研究的潜在的临床应用。在这个协议中，我们提出了一个执行此技术的一般方法

一个小鼠模型在子宫内移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

a-mouse-model-of-in-utero-transplantation

Research

JoVE Journal

Medicine

A Mouse Model of in Utero Transplantation

19.3K 次观看.  University of California. 在小鼠模型在子宫内移植是一种多用途的工具，可用于胎儿干细胞移植和基因治疗研究的潜在的临床应用。在这个协议中，我们提出了一个执行此技术的一般方法

视频: A Mouse Model of in Utero Transplantation

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

肺动脉瓣使用异位心脏移植小鼠模型移植

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

科研

医学

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

小鼠角膜移植模型，研究实体器官移植的最常见形式

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

移植到小鼠卵巢脂肪垫

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

套筒技术在主动脉移植小鼠模型中的应用-教学视频

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

应用改良袖套技术的小鼠胰腺移植模型

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions between cellular and non-cellular blood components during thrombus formation, reliable studies of the physiology and pathophysiology of thrombosis can only be performed in vivo. Therefore, this article presents an ear model in hairless mice and focuses on the in vivo analysis of microcirculation, thrombus formation, and thrombus evolution. By using intravital fluorescence microscopy and the intravenous (iv) application of the respective fluorescent dyes, a repetitive analysis of microcirculation in the auricle can easily be performed, without the need for surgical preparation. Furthermore, this model can be adapted for in vivo studies of different issues, including wound healing, reperfusion injury, or angiogenesis. In summary, the ear of hairless mice is an ideal model for the in vivo study of skin microcirculation in physiological or pathophysiological conditions and for the evaluation of its reaction to different systemic or topical treatments.

The ear model of the hairless SKH1-Hrhr mouse enables intravital fluorescence microscopy of microcirculation and phototoxic thrombus induction without prior surgical preparation in the examined microvascular bed. Therefore, the ear of the hairless mouse is an excellent in vivo model to study the complex interactions during microvascular thrombus formation, thrombus evolution, and thrombolysis.

活体显微镜及血栓诱导在无毛小鼠的耳垂

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions ...

Cardiopulmonary Bypass in a Mouse Model: A Novel Approach

As prolonged cardiopulmonary bypass becomes more essential during cardiac interventions, an increasing clinical demand arises for procedure optimization and for minimizing organ damage resulting from prolonged extracorporal circulation. The goal of this paper was to demonstrate a fully functional and clinically relevant model of cardiopulmonary bypass in a mouse. We report on the device design, perfusion circuit optimization, and microsurgical techniques. This model is an acute model, which is not compatible with survival due to the need for multiple blood drawings. Because of the range of tools available for mice (e.g., markers, knockouts, etc.), this model will facilitate investigation into the molecular mechanisms of organ damage and the effect of cardiopulmonary bypass in relation to other comorbidities.

This paper describes how to perform cardiopulmonary bypass in mice. This novel model will facilitate the investigation of the molecular mechanisms involved in organ damage.

小鼠体外循环模型: 一种新方法

As prolonged cardiopulmonary bypass becomes more essential during cardiac interventions, an increasing clinical demand arises for procedure optimization ...

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

胰岛门静脉移植小鼠模型的研究

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Veno-Venous Extracorporeal Membrane Oxygenation in a Mouse

The use of extracorporeal membrane oxygenation (ECMO) has increased substantially in recent years. ECMO has become a reliable and effective therapy for acute as well as end-stage lung diseases. With the increase in clinical demand and prolonged use of ECMO, procedural optimization and prevention of multi-organ damage are of critical importance. The aim of this protocol is to present a detailed technique of veno-venous ECMO in a non-intubated, spontaneously breathing mouse. This protocol demonstrates the technical design of the ECMO and surgical steps. This murine ECMO model will facilitate the study of pathophysiology related to ECMO (e.g., inflammation,bleeding and thromboembolic events). Due to the abundance of genetically modified mice, the molecular mechanisms involved in ECMO-related complications can also be dissected.

Here we present a protocol describing the technique of veno-venous extracorporeal membrane oxygenation (ECMO) in a non-intubated, spontaneously breathing mouse. This murine model of ECMO can be effectively implemented in experimental studies of acute and end-stage lung diseases.

小鼠静脉内膜氧化术

The use of extracorporeal membrane oxygenation (ECMO) has increased substantially in recent years. ECMO has become a reliable and effective therapy for ...