作者感谢杰罗姆·瓦莱罗（Jerome Valero）编辑了这份手稿。 图1 和 图3I，J，L 是用 BioRender.com 创建的。

所有动物均按照加拿大动物护理委员会在《实验动物护理和使用指南》中规定的指南进行治疗。该实验方案已获得多伦多综合医院研究所动物护理委员会、大学健康网络的批准。
1. 供体手术
注意：BALB / c小鼠被用作实验供体的一个例子。所有程序都必须使用无菌技术进行。
<ol><li>在操作之前，记录每只鼠标的重量。</li>
	<li>使用CO2 室对小鼠实施安乐死。</li>
	<li>确认死亡后，将鼠标置于仰卧位并用胶带固定四肢。</li>
	<li>用 70% 异丙醇对手术区域进行消毒，为手术区域做好准备。 注意：如果需要并按照当地动物伦理委员会的建议，从切口部位剪下毛皮。</li>
	<li>在皮肤上做一个中线切口，从中腹部开始，延伸到颈前区。</li>
	<li>通过小心地缩回脂肪垫、横向移动带状肌肉并将气管与周围的结缔组织分开来进入气管。使用镊子在气管和食道之间创造空间。</li>
	<li>抬起剑突并切断横膈膜。</li>
	<li>抬高胸骨，通过插入止血钳确保从胸骨到颈部区域的畅通路径。夹住两侧的肋骨，切开胸骨，向上延伸至颈部肌肉。</li>
	<li>去除胸腺和任何阻塞气管的脂肪或肌肉，以暴露气管分叉处。</li>
	<li>切开主支气管，小心地将气道与食道分开。</li>
	<li>切开喉部并将其取出。</li>
	<li>用无菌盐水溶液或用浸泡在无菌盐水或保存溶液中的无菌纱布的保存液喷洒解剖的气管，并将其放在冰上以保持其活力。</li>
</ol>2. 受体手术
注意：C57BL / 6小鼠用作实验受体的示例。
<ol><li>在手术当天早上以 1 mg/kg 的剂量皮下注射缓释丁丙诺啡。</li>
	<li>使用5%异氟烷在诱导室中诱导麻醉。</li>
	<li>一旦小鼠被轻度麻醉，腹膜内注射由（0.1mg / g）甲苯噻嗪和（0.01mg / g）氯胺酮组成的混合物。</li>
	<li>将小鼠放回诱导室，保持2-3%异氟烷。</li>
	<li>在手术部位剃掉皮毛。此外，以 7 mg/kg 的剂量沿计划的注射部位皮下注射布比卡因作为线块。</li>
	<li>在经口气管插管前确认对脚趾捏合没有反射反应。使用20G静脉导管经口气管插管小鼠，并将其连接到潮气量为500μL，呼吸频率为120bpm，100%氧气和2%异氟烷的呼吸机。使用带有夹子的支架夹在舌头上，将动物保持在垂直位置，颈部伸直，以方便此过程。</li>
	<li>激活加热垫并将鼠标放置在加热垫顶部的右侧位置，头部远离外科医生，尾巴朝向外科医生（图 2）。用胶带固定四肢。在眼睛上涂上兽药膏，以防止麻醉时干燥。</li>
	<li>用7.5%聚维酮碘擦洗手术区域，用70%异丙醇消毒，用10%聚维酮碘重新擦洗。使用无菌手术布覆盖手术区域。</li>
	<li>在此期间，将供体气管加载到 16 G 静脉导管中（图 3C，D）。</li>
	<li>使用手术刀在接受者的皮肤上切开一个切口，烧灼肌肉和结缔组织。</li>
	<li>打开第五或第六肋间隙，并使用两个牵开器保持肋骨腔打开。</li>
	<li>用棉签和剪刀解剖肺下韧带。</li>
	<li>模拟供体气管通路的创建（图3G，H）。</li>
	<li>用三通旋塞阀部分闭塞呼吸机流出管，以促进左肺充气，从而固定呼吸机流出管。</li>
	<li>通过用 20 G 针刺穿左肺来创建通路。确保穿刺深度大致等于气管同种异体移植物的长度。选择肺边缘的穿刺部位（如 图3I所示），确保通路平行于桌面（ 如图3J中的蓝色圆圈标记）。 注意：向上的插入角度将导致胸膜层的穿透，而较深的角度可能会导致主要血管出血（ 如图3J中的红十字标记）。</li>
	<li>将 16 G 静脉导管插入左肺，并将供体气管挤压到左肺中。插入气管同种异体移植物后，松开三通旋塞阀，使呼气流畅通无阻地通过流出管。</li>
	<li>用夹子关闭胸膜注射部位（图3K，L）。将夹子精确定位在穿刺部位，其边缘对齐以匹配肺边缘的轮廓（由图3L中的蓝色圆圈表示）。 注意： 夹子部位的位置不正确会导致密封无效和漏气，而夹子深度不足可能会导致夹子在手术后脱落（如 图 3L 中的红叉所示）。</li>
	<li>用生理盐水填充胸腔，并用纱布吸收生理盐水。</li>
	<li>使用运行缝合技术对左肺进行再充气并闭合肋骨。</li>
	<li>用中断的缝合线闭合肌肉和皮肤。</li>
	<li>手术结束时皮下注射美洛昔康镇痛药，剂量为 5 mg/kg。</li>
	<li>观察接收鼠标，直到它醒来。然后，取出气管插管并将受体小鼠放入笼子中。 注意：受体小鼠应单独饲养。</li>
	<li>美洛昔康（5mg/kg）每日一次皮下注射，术后24小时开始，术后持续3天。</li>
</ol>3.从受体小鼠收集样本
<ol><li>使用5%异氟烷在诱导室中诱导麻醉。</li>
	<li>在口气管插管前确认对脚趾捏合没有反射反应。插管方法和呼吸器设置与受体手术相同。</li>
	<li>将鼠标置于仰卧位并固定四肢。</li>
	<li>用 70% 异丙醇对手术区域进行消毒，为手术区域做好准备。</li>
	<li>在皮肤上做一个中线切口，从中腹部开始，延伸到颈前区。</li>
	<li>使用连接到25G针头的1ml注射器通过下腔静脉对小鼠进行放血，导致安乐死。</li>
	<li>打开胸部，以与供体小鼠相同的方式进入气管。用 7-0 丝绸将气管绑在插管周围。</li>
	<li>去除胸腺、脂肪和肌肉，露出心脏。</li>
	<li>切开左心房、右心房和下腔静脉。用 3 mL 无菌生理盐水通过右心室灌注肺部。</li>
	<li>对于组织学分析，通过插管用 10% 福尔马林充气肺部。</li>
	<li>拔管通气管并用 7-0 丝绸绑住气管。</li>
	<li>将喉部和食道分开。将它们拉向下部，然后提取心脏和肺阻滞，将其放入10%福尔马林中。</li>
</ol>

探索肺移植中的闭塞性气道疾病

<ol>
	<li>Chambers, D. C., 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=The+International+Thoracic+Organ+Transplant+Registry+of+the+International+Society+for+Heart+and+Lung+Transplantation:+Thirty-fifth+adult+lung+and+heart-lung+transplant+report-2018;+Focus+theme:+Multiorgan+Transplantation.">The International Thoracic Organ Transplant Registry of the International Society for Heart and Lung Transplantation: Thirty-fifth adult lung and heart-lung transplant report-2018; Focus theme: Multiorgan Transplantation.</a> J Heart Lung Transplant. 37 (10), 1169-1183 (2018).</li><li>Hertz, M. I., Jessurun, J., King, M. B., Savik, S. K., Murray, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproduction+of+the+obliterative+bronchiolitis+lesion+after+heterotopic+transplantation+of+mouse+airways.">Reproduction of the obliterative bronchiolitis lesion after heterotopic transplantation of mouse airways.</a> American J Pathol. 142 (6), 1945-1951 (1993).</li><li>Ikonen, T. S., Brazelton, T. R., Berry, G. J., Shorthouse, R. S., Morris, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+re-growth+is+associated+with+inhibition+of+obliterative+airway+disease+in+orthotopic+tracheal+allografts+in+non-immunosuppressed+rats.">Epithelial re-growth is associated with inhibition of obliterative airway disease in orthotopic tracheal allografts in non-immunosuppressed rats.</a> Transplantation. 70 (6), 857 (2000).</li><li>Dutly, A. 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+model+for+post-transplant+obliterative+airway+disease+reveals+angiogenesis+from+the+pulmonary+circulation.">A novel model for post-transplant obliterative airway disease reveals angiogenesis from the pulmonary circulation.</a> Am J Transplant. 5 (2), 248-254 (2005).</li><li>Wagnetz, 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=Rejection+of+tracheal+allograft+by+intrapulmonary+lymphoid+neogenesis+in+the+absence+of+secondary+lymphoid+organs.">Rejection of tracheal allograft by intrapulmonary lymphoid neogenesis in the absence of secondary lymphoid organs.</a> Transplantation. 93 (12), 1212-1220 (2012).</li><li>Hirayama, 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=Local+long-term+expression+of+lentivirally+delivered+IL-10+in+the+lung+attenuates+obliteration+of+intrapulmonary+allograft+airways.">Local long-term expression of lentivirally delivered IL-10 in the lung attenuates obliteration of intrapulmonary allograft airways.</a> Hum Gene Ther. 22 (11), 1453-1460 (2011).</li><li>Watanabe, T., 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=Recipient+bone+marrow-derived+IL-17+receptor+A-positive+cells+drive+allograft+fibrosis+in+a+mouse+intrapulmonary+tracheal+transplantation+model.">Recipient bone marrow-derived IL-17 receptor A-positive cells drive allograft fibrosis in a mouse intrapulmonary tracheal transplantation model.</a> Transpl Immunol. 69, 101467 (2021).</li><li>Matsuda, Y., 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=Spleen+tyrosine+kinase+modulates+fibrous+airway+obliteration+and+associated+lymphoid+neogenesis+after+transplantation.">Spleen tyrosine kinase modulates fibrous airway obliteration and associated lymphoid neogenesis after transplantation.</a> Am J Transplant. 16 (1), 342-352 (2016).</li><li>Suzuki, Y., 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=Effect+of+CTLA4-Ig+on+Obliterative+bronchiolitis+in+a+mouse+intrapulmonary+tracheal+transplantation+model.">Effect of CTLA4-Ig on Obliterative bronchiolitis in a mouse intrapulmonary tracheal transplantation model.</a> Ann Thorac Cardiovasc Surg. 27 (6), 355-365 (2021).</li><li>Watanabe, T., 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+potent+anti-angiogenic+factor,+vasohibin-1,+ameliorates+experimental+bronchiolitis+obliterans.">A potent anti-angiogenic factor, vasohibin-1, ameliorates experimental bronchiolitis obliterans.</a> Transplant Proc. 44 (4), 1155-1157 (2012).</li><li>Aloisi, F., Pujol-Borrell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphoid+neogenesis+in+chronic+inflammatory+diseases.">Lymphoid neogenesis in chronic inflammatory diseases.</a> Nat Rev Immunol. 6 (3), 205-217 (2006).</li><li>Cupedo, T., Jansen, W., Kraal, G., Mebius, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+secondary+and+tertiary+lymphoid+structures+in+the+skin.">Induction of secondary and tertiary lymphoid structures in the skin.</a> Immunity. 21 (5), 655-667 (2004).</li><li>Sato, M., 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=The+role+of+intrapulmonary+de+novo+lymphoid+tissue+in+obliterative+bronchiolitis+after+lung+transplantation.">The role of intrapulmonary de novo lymphoid tissue in obliterative bronchiolitis after lung transplantation.</a> J Immunol. 182 (11), 7307-7316 (2009).</li><li>Okazaki, M., 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=Maintenance+of+airway+epithelium+in+acutely+rejection+orthotopic+vascularized+mouse+lung+transplants.">Maintenance of airway epithelium in acutely rejection orthotopic vascularized mouse lung transplants.</a> A J Resp Cell Mol Biol. 37 (6), 625-630 (2007).</li><li>Yamada, Y., 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=Chronic+airway+fibrosis+in+orthotopic+mouse+lung+transplantation+models-an+experimental+reappraisal.">Chronic airway fibrosis in orthotopic mouse lung transplantation models-an experimental reappraisal.</a> Transplantation. 102 (2), e49-e58 (2018).</li><li>Watanabe, T., 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+B+cell-dependent+pathway+drives+chronic+lung+allograft+rejection+after+ischemia-reperfusion+injury+in+mice.">A B cell-dependent pathway drives chronic lung allograft rejection after ischemia-reperfusion injury in mice.</a> Am J Transplant. 19 (12), 3377-3389 (2019).</li><li>Guo, Y., 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=Vendor-specific+microbiome+controls+both+acute+and+chronic+murine+lung+allograft+rejection+by+altering+CD4+Foxp3++regulatory+T+cell+levels.">Vendor-specific microbiome controls both acute and chronic murine lung allograft rejection by altering CD4+Foxp3+ regulatory T cell levels.</a> Am J Transplant. 19 (10), 2705-2718 (2019).</li></ol>

本手稿的作者没有利益冲突需要披露。

小鼠IPTT程序包括关键步骤。关于麻醉，第一个关键步骤是气管插管。必须将鼠标保持在适当的高度，双腿放在桌子上，以可视化声带并促进立即插管。此外，还需要仔细调整呼吸量和呼气末正压 （PEEP）。通常，500μL的呼吸容积和2cmH2O的PEEP足以使体重为25-30g的小鼠满意。然而，较大的受体小鼠可能会经历“打嗝”发作，表明缺氧。在这种情况下，可能需要增加呼吸量，PEEP，并暂时夹住呼...

肺移植已被确定为终末期呼吸系统疾病患者的有效治疗方法。然而，人肺移植受者的中位生存率仅为 6 年左右，闭塞性细支气管炎 （OB） 是一种阻塞性气道疾病 （OAD），是移植后第一年后死亡的主要原因1。
已经利用几种动物模型来研究OAD的潜在机制。其中一种模型是异位气管移植 （HTT） 模型2。在该模型中，气管移植物被植入接受者的皮下组织或网膜中。发生缺血诱导的气管移植物上皮细胞丢失，随后发生同种异体淋巴细胞浸润和供体上皮细胞凋亡。成纤维细胞和肌成纤维细胞在气管周围迁移，产生细胞外基质。最后，气道管腔完全纤维性闭塞。HTT模型技术简单，提供 体内 环境，并提供高可重复性。
研究 OAD 的另一种模型是大鼠原位气管移植 （OTT） 模型，其中气管移植物插入受体的气管以维持生理通气3。在该模型中，缺血诱导的供体上皮细胞耗竭导致它们被气管内受体上皮细胞取代，形成通畅的气道并伴有中度纤维化。尽管这些模型有助于理解肺移植后气道闭塞，但它们在肺实质微环境的概括方面存在局限性。
我们的研究小组介绍了大鼠肺内气管移植（IPTT）模型，其中气管移植物植入受体肺4（图1）。IPTT 模型表现出肺微环境中发生的气道管腔纤维性闭塞。此外，它已成功应用于技术上比大鼠IPTT 5,6,7,8,9,10更具挑战性的小鼠。小鼠IPTT模型的这种适应使我们能够更深入地研究使用转基因小鼠进行肺移植后OAD肺免疫环境的复杂细节。
IPTT 模型具有一些独特的功能。一种是新血管生成，它由肺循环促进，在气道闭塞中起着至关重要的作用 4,10。此外，IPTT 模型表现出淋巴样聚集体，其中一些具有表达外周淋巴结地址蛋白的高内皮小静脉，表明它们是三级淋巴器官 （TLO）7,8。TLO 类似于淋巴结，由 T 细胞、B 细胞组成，通常由滤泡树突状细胞伴生的生发中心组成 11,12。TLOs在各种慢性炎症性疾病（包括气道闭塞）中已有报道，这使得IPTT模型适用于研究TLOs在气道闭塞中的作用7,8,11,12,13。本文介绍了小鼠 IPTT 模型的方法，目的是让研究人员熟悉该模型，并促进对肺移植后气道闭塞的进一步研究。

<table><tbody><tr><td>BALB/cJ</td><td>The Jackson Laboratory</td><td>8-10 weeks 25-30 g</td><td>Male, Donor</td></tr><tr><td>BD 1 mL Syringe</td><td>Becton Dickinson</td><td>309659</td><td></td></tr><tr><td>BD PrecisionGlide Needle Aiguile BD&lt;br/&gt; PrecisionGlide</td><td>Becton Dickinson</td><td>305122</td><td></td></tr><tr><td>Bovie Change-A-Tip Deluxe High-Temperture</td><td>Bovie</td><td>DEL1</td><td></td></tr><tr><td>C57BL/6J</td><td>The Jackson Laboratory</td><td>8-10 weeks 25-30 g</td><td>Male, Recipient</td></tr><tr><td>Dumont #5/45 Forceps</td><td>F&amp;middot;S&amp;middot;T</td><td>11251-35</td><td></td></tr><tr><td>Ethicon Ligaclip Multiple -Clip Appliers-</td><td>Ethicon</td><td>LX107</td><td></td></tr><tr><td>Extra Fine Graefe Forceps</td><td>F&amp;middot;S&amp;middot;T</td><td>11150-10</td><td></td></tr><tr><td>Glover Bulldog Clamp</td><td>Integra</td><td>320-127</td><td></td></tr><tr><td>Halsted-Mosquito Hemostats</td><td>F&amp;middot;S&amp;middot;T</td><td>13009-12</td><td></td></tr><tr><td>Horizon Titanium Ligating Clips</td><td>Teleflex</td><td>001201</td><td></td></tr><tr><td>Leica M651 Manual surgical microscope for microsurgical procedures</td><td>Leica</td><td></td><td></td></tr><tr><td>Magnetix Fixator with spring lock</td><td>CD+ LABS</td><td>ACD-001</td><td></td></tr><tr><td>Microsurgical Scissor</td><td>Jarit</td><td>277-051</td><td></td></tr><tr><td>Mouse and Perinatal Rat Ventilator Model 687</td><td>Harvard</td><td>55-0001</td><td></td></tr><tr><td>Perfadex Plus</td><td>XVIVO</td><td>19850</td><td></td></tr><tr><td>Retractor Tip Blunt - 2.5 mm</td><td>CD+ LABS</td><td>ACD-011</td><td></td></tr><tr><td>small animal table</td><td>CD+ LABS</td><td>ACD-003</td><td></td></tr><tr><td>Surgipro Blue 24&quot; CV-1 Taper, Double Armed</td><td>Covidien</td><td>VP702X</td><td></td></tr><tr><td>Systane ointment</td><td>Alconn</td><td>1444062</td><td></td></tr><tr><td>System Elastomer</td><td>CD+ LABS</td><td>ACD-007</td><td></td></tr><tr><td>Terumo Surflo IV Catheter, 20 G x 1 in</td><td>Terumo Medical Corporation</td><td>SR-OX2025CA</td><td></td></tr><tr><td>VMT table Top</td><td>benson</td><td>91803300</td><td></td></tr></tbody></table>

murine intrapulmonary tracheal transplantation: a model for investigating obliterative airway disease after lung transplantation

小鼠肺内气管移植 （IPTT） 用作肺移植后闭塞性气道疾病 （OAD） 的模型。该模型最初由我们的团队报道，由于其高技术可重复性和研究免疫行为和治疗干预的适用性，该模型已用于 OAD 的研究。
在 IPTT 模型中，啮齿动物气管移植物通过胸膜直接插入受体的肺部。该模型不同于异位气管移植 （HTT） 模型，其中移植物移植到皮下或网膜部位，也不同于原位气管移植 （OTT） 模型，其中供体气管取代受体的气管。 
成功实施 IPTT 模型需要先进的麻醉和手术技能。麻醉技能包括接受者的气管插管、设置适当的通气参数以及麻醉恢复后适当定时拔管。手术技巧对于在肺内精确放置移植物以及确保有效密封脏胸膜以防止漏气和出血至关重要。一般来说，学习过程大约需要 2 个月。
与 HTT 和 OTT 模型相比，在 IPTT 模型中，同种异体移植气道在相关肺微环境中发生气道闭塞。这使研究人员能够研究肺移植后气道闭塞所涉及的肺特异性免疫学和血管生成过程。此外，该模型的独特之处在于它表现出三级淋巴器官 （TLO），这在人类肺同种异体移植物中也可见。TLO 由 T 细胞群和 B 细胞群组成，其特征是存在指导免疫细胞募集的高内皮小静脉;因此，它们很可能在移植物的接受和排斥中起着至关重要的作用。我们得出的结论是，IPTT 模型是研究肺移植同种异体移植物气道闭塞发展中涉及的肺内免疫和促纤维化途径的有用工具。

根据我们的经验，熟练掌握此模型通常需要大约 2 个月的培训。一旦达到熟练程度，供体程序通常需要 15 分钟，而接受者程序大约需要 30 分钟。训练有素的操作员的预期死亡率为 0%。
在 图4A中，气管同种异体移植物与成纤维细胞组织完全阻塞，上皮细胞明显被破坏。相反，在 图4B中，气管同工移植物保持通畅，上皮细胞被保留。...

Watch this Scientific Journal Video about Author Spotlight: Investigating the Key Factors of Obliterative Bronchiolitis After Lung Transplantation at JoVE.com

作者聚焦：调查肺移植后闭塞性毛细支气管炎的关键因素

小鼠肺内气管移植 （IPTT） 模型对于研究肺移植后的闭塞性气道疾病 （OAD） 很有价值。它以高可重复性提供了对同种异体移植后气道闭塞中肺特异性免疫学和血管生成行为的见解。在这里，我们描述了 IPTT 程序及其预期结果。

小鼠肺内气管移植：肺移植后闭塞性气道疾病研究模型

Murine intrapulmonary tracheal transplantation (IPTT) is used as a model of obliterative airway disease (OAD) following lung transplantation. Initially ...

我们的研究正在检查肺移植后的慢性同种异体肺移植功能障碍。当您移植肺时，超过一半的患者会出现纤维性闭塞或气道异常。了解这一点确实是了解肺移植后长期生存的关键。这种肺内气管移植模型的重要性在于，移植的气管被放置在肺本身的环境中，因此您可以根据移植后的反应来查看同种异体移植物与同种异体移植物。最令人着迷的是，即使异种移植物具有相同的缺血和相同的损伤，也可以完全恢复并具有正常的解剖结构，但同种异体移植物会发生纤维性闭塞，了解为什么在同种异体移植物中发生这种情况是了解肺移植中纤维化的潜在机制的关键。

murine-intrapulmonary-tracheal-transplantation-model-for

Research

JoVE Journal

Medicine

Murine Intrapulmonary Tracheal Transplantation: A Model for Investigating Obliterative Airway Disease After Lung Transplantation

771 次观看.  University Health Network. 我们的研究正在检查肺移植后的慢性同种异体肺移植功能障碍。当您移植肺时，超过一半的患者会出现纤维性闭塞或气道异常。了解这一点确实是了解肺移植后长期生存的关键。这种肺内气管移植模型的重要性在于，移植的气管被放置在肺本身的环境中，因此您可以根据移植后的反应来查看同种异体移植物与同种异体移植物。最令人着迷的是，即使异种移植物具有相同?...

视频: 作者聚焦：调查肺移植后闭塞性毛细支气管炎的关键因素

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and standardized not only for the study of allo-rejection but also between syngeneic strains for examining mechanisms of ischemia-reperfusion injury after lung transplantation. Although the application of the rat and other large animal model 2 contributed significantly to the elucidation of these studies, the scope of those investigations is limited by the scarcity of knockout and transgenic rats. Due to no effective therapies for obliterative bronchiolitis, the leading cause of death in lung transplant patients, there has been an intensive search for pre-clinical models that replicate obliterative bronchiolitis. The tracheal allograft model is the most widely used and may reproduce some of the histopathologic features of obliterative bronchiolitis 3. However, the lack of an intact vasculature with no connection to the recipient's conducting airways, and incomplete pathologic features of obliterative bronchiolitis limit the utility of this model 4. Unlike transplantation of other solid organs, vascularized mouse lung transplants have only recently been reported by Okazaki and colleagues for the first time in 2007 5. Applying the basic principles of the rat lung transplant, our lab initiated the obliterative bronchiolitis model using minor histoincompatible antigen murine orthotopic single-left lung transplants which allows the further study of obliterative bronchiolitis immunopathogenesis6.

Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.

闭塞性细支气管炎在原位肺移植小鼠模型的发展

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

科研

医学

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

无创性气管内插管，以研究小鼠肺的病理生理

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

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.

用改进的袖套法小鼠颈部心脏移植模型

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

Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of  Obliterative Airway Disease

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the pathophysiology, treatment and prevention of OAD, different animal models of tracheal transplantation in rodents have been developed (1-7). Here, we use two established models of trachea transplantation, the heterotopic and orthotopic model and demonstrate their advantages and limitations.
For the heterotopic model, the donor trachea is wrapped into the greater omentum of the recipient, whereas the donor trachea is anastomosed by end-to-end anastomosis in the orthotopic model.
In both models, the development of obliterative lesions histological similar to clinical OAD has been demonstrated (1-7).
This video shows how to perform both, the heterotopic as well as the orthotopic tracheal transplantation technique in mice, and compares the time course of OAD development in both models using histology.

This video shows and compares two experimental models to study the development of obliterative airway disease (OAD) in mice, the heterotopic and orthotopic tracheal transplantation model.

异位和原位气管移植小鼠模型研究闭塞性气道疾病的发展

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the ...

生物学

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

在小鼠气管插管快速的改进方法

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

小鼠模型中生物合成组织工程气管移植的育苗与植入

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

生物工程

A Mouse Model of Orotracheal Intubation and Ventilated Lung Ischemia Reperfusion Surgery

Ischemia reperfusion (IR) injury frequently results from processes that involve a transient period of interrupted blood flow. In the lung, isolated IR permits the experimental study of this specific process with continued alveolar ventilation, thereby avoiding the compounding injurious processes of hypoxia and atelectasis. In the clinical context, lung ischemia reperfusion injury (also known as lung IRI or LIRI) is caused by numerous processes, including but not limited to pulmonary embolism, resuscitated hemorrhagic trauma, and lung transplantation. There are currently limited effective treatment options for LIRI. Here, we present a reversible surgical model of lung IR involving first orotracheal intubation followed by unilateral left lung ischemia and reperfusion with preserved alveolar ventilation or gas exchange. Mice undergo a left thoracotomy, through which the left pulmonary artery is exposed, visualized, isolated, and compressed using a reversible slipknot. The surgical incision is then closed during the ischemic period, and the animal is awakened and extubated. With the mouse spontaneously breathing, reperfusion is established by releasing the slipknot around the pulmonary artery. This clinically relevant survival model permits the evaluation of lung IR injury, the resolution phase, downstream effects on lung function, as well as two-hit models involving experimental pneumonia. While technically challenging, this model can be mastered over the course of a few weeks to months with an eventual survival or success rate of 80%-90%.

A mouse surgical model to create left lung ischemia reperfusion (IR) injury while maintaining ventilation and avoiding hypoxia.

口腔插管和通气肺缺血再灌注手术小鼠模型

Ischemia reperfusion (IR) injury frequently results from processes that involve a transient period of interrupted blood flow. In the lung, isolated IR ...

免疫与感染

A Heterotopic Mouse Model for Studying Laryngeal Transplantation

Laryngeal heterotopic transplantation, although a technically challenging procedure, offers more scientific analysis and cost benefits compared to other animal models. Although first described by Shipchandler et al. in 2009, this technique is not widely used, possibly due to the difficulties in learning the microsurgical technique and time required to master it. This paper describes the surgical steps in detail, as well as potential pitfalls to avoid, in order to encourage effective use of this technique.
In this model, the bilateral carotid arteries of the donor larynx are anastomosed to the recipient carotid artery and external jugular vein, allowing for blood flow through the graft. Blood flow can be confirmed intraoperatively by the visualization of blood filling in the graft bilateral carotid arteries, reddening of the thyroid glands of the graft, and bleeding from micro vessels in the graft. The crucial elements for success include delicate preservation of the graft vessels, making the correct size arteriotomy and venotomy, and using the appropriate number of sutures on the arterial-arterial and arterial-venous anastomoses to secure vessels without leakage and prevent occlusion. 
Anyone can become proficient in this model with sufficient training and perform the procedure in approximately 3 h. If performed successfully, this model allows for immunologic studies to be performed with ease and at low cost.

The aim of this manuscript is to describe the microsurgical steps required to perform a heterotopic laryngeal transplant in mice. The advantages of this mouse model compared to other animal models of laryngeal transplantation are its cost-effectiveness and the availability of immunologic assays and data.

用于研究喉移植的异位小鼠模型

Laryngeal heterotopic transplantation, although a technically challenging procedure, offers more scientific analysis and cost benefits compared to other ...

A Simple Cuff Technique for Murine Left Lung Transplantation

Over the past decade, our laboratory has made significant progress in developing and refining vascularized mouse lung transplantation models using an efficient and highly reliable "cuff technique" of transplantation. This article describes a sophisticated and comprehensive method for orthotopic lung transplantation in a vascularized orthotopic lung model, representing the most physiologic and clinically relevant model of mouse lung transplantation to date. The transplantation process consists of two distinct stages: donor harvest and subsequent implantation into the recipient. The method has been successfully mastered, and with several months of sufficient training, a skilled practitioner can perform the procedure in approximately 90 min from skin-to-skin. Surprisingly, once individuals overcome the initial learning curve, the survival rate during the perioperative period approaches nearly 100%. The mouse model allows for the use of multiple commercially available transgenic and mutant strains of mice, enabling the study of tolerance and rejection. Additionally, the unique features of this model make it a valuable tool for investigating tumor biology and immunology.

The present protocol describes a cuff technique for a mouse left lung transplantation model. This technique has been developed over several years and has performed well, serving effectively in immunological research.

Over the past decade, our laboratory has made significant progress in developing and refining vascularized mouse lung transplantation models using an ...

<meta charset="utf8"></head><body>小鼠肺移植中细胞动力学的实时监测

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in na&#239;ve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

<meta charset="utf8"></head><body>作者聚焦：通过活体成像推进肺移植免疫学

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

移植小鼠肺的活体双光子显微镜检查

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by ...