我们要感谢罗马维乔雷克和彼得 Kurdybacha 的出色编辑协助, 以及里昂. 德克尔和乌利 Heuter 的出色技术援助。

涉及动物的程序由 RWTH 亚琛大学的机构动物护理和使用委员会 (IACUC) 批准, AZ 84-02.04.2012. A234. 
 注意: 使用 CD1 背景的成年雄性野鼠演示了该过程。在手术前后将小鼠放在一个专门的实验室里, 确保适当地获得食物、专门的兽医控制和治疗。如果这些动物是从外面买来的, 在做手术前允许一周的驯化. 
 1. 捐赠者的准备 <ol> <li> 使用无菌材料和器械在手术期间保持无菌状态以避免感染. </li>
	<li> 麻醉每只老鼠用1.5% 的混合异氟醚和100% 氧气通过口罩。将鼠标放在仰卧位的平台上, 并将其所有的腿粘在手术台上。通过捏后腿来检查其反射, 以确保鼠标足够麻醉。将眼部药膏放在眼部, 以防在手术过程中干燥. </li>
	<li> 用脱毛凝胶或剃须刀从腹部取出所有毛发。在无菌条件下进行操作。用洗必泰和无菌水交替擦洗腹部消毒. </li>
	<li> 通过带剪刀或手术刀的中线腹切口移除供体主动脉。将肠道手动缩回右侧。用无粉手套轻轻地将肠道反射到一侧. 
	<ol> <li> 将肠道放在一块用盐水浸湿的纱布上, 以保持其湿润. </li>
		<li> 用镊子将腹主动脉从周围组织中仔细解剖. </li>
		<li> 用镊子将大动脉之间的动脉段与腔静脉分叉分开. </li>
		<li> 使用11-0 单丝单线缝合, 确保此段的所有小分支都非常小心. </li>
		<li> 在移除主动脉之前, 将含 50 U 肝素的盐水溶液注入0.5 毫升 (毫升) 到下腔静脉. </li>
		<li> 让供体动物血在主动脉段后被移除. </li>
		<li> 将移植物完全用盐水冲洗干净, 然后立即将其转移到 ice-cold 盐水的容器中. </li>
	</ol> </li> </ol> 2。准备收件人 <ol> <li> 麻醉1.5% 的混合体, 由异氟醚和100% 氧气通过面罩, 然后删除头发和消毒 (1 节) 的受体动物。用手术刀将中线切开, 从剑到骨盆, 并缩回腹壁。将眼部药膏放在眼部上, 防止在手术过程中出现干燥. </li>
	<li> 在盐水溶液中用湿纱布包裹肠道, 并轻轻地移到动物和 #39; 右. </li>
	<li> 解剖下主动脉游离于肾动脉下部与镊子远端分岔之间. </li>
	<li> 使用11-0 单丝单线缝合, 确保此段的所有小分支都非常小心. </li>
	<li> 用6-0 单丝缝合钳夹住主动脉的近端和远端部分. </li>
	<li> 在夹钳中间划分主动脉, 用素盐水冲洗切口末端, 使管腔张开. </li>
	<li> 将移植物放在原位, 将喂食容器端插入接收容器, 然后用11-0 单丝进行缝合, 以避免主动脉的任何扭转, 适当地对准施主和接受者 ( 图 1 ) 10 . </li> <li> 在对吻合进行检查后, 仔细地松开绷带。先松开远端钳。这导致在释放近端高压侧之前, 将墙壁压在一起的低压. </li>
	<li> 灌注立即进行移植, 并检查是否有可见的脉搏。轻轻地除去丝绸的残余。供体和受体主动脉的最佳重叠长度为 1-2 mm. </li>
	<li> 将腹内内容返回腹腔, 并用3-0 聚酸缝合闭合整个伤口. </li>
	<li> 在终止麻醉前给小鼠丁丙诺啡 (0.05 毫克/千克体重皮下 (SC)). </li>
	<li> 在完全清醒之前, 不要把动物留在无人看管的状态。管理疼痛治疗与 buprenophine 0.05 毫克/千克体重管理 SC 三次, 每天三天的行动后, 经机构监督机构批准. </li>
	<li> 用于组织捕捞, 麻醉上述的受体小鼠, 用磷酸盐缓冲盐水 (pbs) 冲洗血管, 然后用4% 甲醛/pbs (pH = 7.4) 通过心脏穿刺。轻轻取出移植物。经过隔夜固定4% 甲醛/PBS, 过程标本进一步和嵌入石蜡. </li>
</ol>

<ol>
	<li>Rowinska, Z., 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=Non-+invasive+in+vivo+analysis+of+a+murine+aortic+graft+using+high+resolution+ultrasound+microimaging.">Non- invasive in vivo analysis of a murine aortic graft using high resolution ultrasound microimaging.</a> Eur J Radiol. 81 (2), 244-249 (2012).</li><li>Daugherty, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+atherosclerosis.">Mouse models of atherosclerosis.</a> Am J Med Sci. 323 (1), 3-10 (2002).</li><li>Xu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+arteriosclerosis:+from+arterial+injuries+to+vascular+grafts.">Mouse models of arteriosclerosis: from arterial injuries to vascular grafts.</a> Am J Pathol. 165 (1), 1-10 (2004).</li><li>Daugherty, A., Cassis, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+abdominal+aortic+aneurysms.">Mouse models of abdominal aortic aneurysms.</a> Arterioscler Thromb Vasc .Biol. 24 (3), 429-434 (2004).</li><li>Zernecke, A., Shagdarsuren, E., Weber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+in+atherosclerosis:+an+update.">Chemokines in atherosclerosis: an update.</a> Arterioscler Thromb Vasc Biol. 28 (11), 1897-1908 (2008).</li><li>Chereshnev, I., 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=Mouse+model+of+heterotopic+aortic+arch+transplantation.">Mouse model of heterotopic aortic arch transplantation.</a> J Surg Res. 111 (2), 171-176 (2003).</li><li>Koulack, J., McAlister, V. C., Giacomantonio, C. A., Bitter-Suermann, H., MacDonald, A. S., Lee, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+mouse+aortic+transplant+model+of+chronic+rejection.">Development of a mouse aortic transplant model of chronic rejection.</a> Microsurgery. 16 (2), 110-113 (1995).</li><li>Engelbrecht, G., Kahn, D., Duminy, F., Hickman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+rapid+technique+for+renal+transplantation+in+the+rat.">New rapid technique for renal transplantation in the rat.</a> Microsurgery. 13 (6), 340-344 (1992).</li><li>Baxter, K., Hao, P. M., Howden, B. O., Saunder, A., Jablonski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+technique+of+abdominal+heart+transplantation+in+the+rat.">Modified technique of abdominal heart transplantation in the rat.</a> J Heart Lung Transplant. 17 (11), 1057-1064 (1998).</li><li>Dambrin, C., Calise, D., Pieraggi, M. T., Thiers, J. C., Thomsen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+aortic+transplantation+in+mice:+a+new+model+of+allograft+arteriosclerosis.">Orthotopic aortic transplantation in mice: a new model of allograft arteriosclerosis.</a> J Heart Lung Transplant. 18 (10), 946-951 (1999).</li><li>Siemionow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathology+of+microarterial+anastomoses:+end-to-end+versus+end-in-end+(sleeve)+technique.">Histopathology of microarterial anastomoses: end-to-end versus end-in-end (sleeve) technique.</a> J Hand Surg Am. 15, 619-625 (1990).</li><li>Charo, I. F., Ransohoff, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+roles+of+chemokines+and+chemokine+receptors+in+inflammation.">The many roles of chemokines and chemokine receptors in inflammation.</a> N Engl J Med. 354 (6), 610-621 (2006).</li><li>Sun, 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=Improved+surgical+technique+for+the+establishment+of+a+murine+model+of+aortic+transplantation.">Improved surgical technique for the establishment of a murine model of aortic transplantation.</a> Microsurgery. 18 (6), 368-371 (1998).</li><li>Guo, L., Agarwal, A., George, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+aortic+transplantation+in+mice+for+the+study+of+vascular+disease.">Orthotopic aortic transplantation in mice for the study of vascular disease.</a> J Vis Exp. (69), e4338 (2012).</li></ol>

作者声明他们没有竞争的金融利益。

在本研究之前, 对小鼠其他各种移植模型进行了全面分析3,6,7,10,13,14。采用 Dambrin et al.修饰的袖状技术进行主动脉移植的模型选择, 因为它符合我们的标准, 并显示了与传统的端部缝合方法相比, 套管吻合的高可靠性1,10。
这种技术在许多方面都是有利的, 阻断时间大大减少了手术期间对主动脉损伤的最小化。此外, 还观察到血栓形成率低的情况, 以避免捐赠者和受赠人之间的血管口径可能不匹配。上述观察使该技术非常适合于研究小鼠主动脉移植中的血管疾病。
在一项随访研究中, 超声在移植后8周内进行, 未发现明显的变化。这证实了在手术中对主动脉的任何损伤都是最小的1。
本文提出的嫁接过程保证了移植物完整性和功能的不损害。因此, 这一实验性移植模型可以作为对基因工程小鼠退行性血管病的分子和药理学研究的重要工具。
我们认为, 视频指南可以作为教学材料, 说明使用这个简单的动动脉模型, 它将有助于进一步丰富的辩论中的许多重要问题的血管病理。这种快速吻合的方法可用于研究基因工程小鼠的血管疾病。它也可作为一种改良的动脉瘤模型结合移植。
在过程中有关键点。缝合线本身的放置是最关键的一步。外科医生必须小心, 以避免任何扭转的主动脉通过适当的协调捐助者和受体。在检查吻合口后, 要小心地取下夹具。远端钳位应始终首先释放导致低压保持墙壁在一起之前, 在近端高压侧释放。不正确地跟随释放序列的后果将是流血。

正如先前的一项研究指出的, 对小鼠主动脉移植模型给予了极大的关注, 这种模式允许在移植物本身引起的特定血管反应和与 arteriogenic 环境有关的某些系统因素之间进行区分。1,2,3. 在这里扮演关键角色的主要因素是淘汰赛和转基因小鼠的可用性。他们参与这种模式提供了确定和确定新的病理生理学途径的可能性与发展的退行性血管疾病, 如动脉粥样硬化和动脉瘤形成4,5。
值得注意的是, 在移植过程中, 血管内的缺血/再灌注损伤可能会出现。因此, 在术后期间发生的具体问题与移植完整性或意想不到的炎症反应不能排除可能解除退行性血管疾病的病理生理改变3 ,4,5,6,7。套管吻合是一种替代的 end-in-end 方法, 用于动脉吻合直径小于一毫米的血管, 并已成功应用于大鼠的肾和心脏移植, 随后适应主动脉小鼠移植的 Dambrin et al.8,9,10,11。
主动脉损伤使用袖状移植技术是最小的, 技术故障率非常低, 由于它只持续20分钟平均。我们以前的研究结果表明, 在移植使用套筒技术1后, isograft在体内具有优异的功能和结构特性。Dambrin et al.描述在短学习曲线之后, 成功率超过 78%10。并发症, 如血栓形成是罕见的, 例如 Engelbrecht et al.没有观察到血栓形成使用套筒技术在肾移植在大鼠8。
小鼠主动脉移植模型与套筒吻合是一种快速、简便的研究同种异体移植血管反应的工具。这段视频说明了在我们实验室进行的主动脉移植手术。这种移植模型可能有助于确定血管退行性疾病的潜在病理机制, 并可能有助于进一步评价分子和药理干预措施12。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Halsey Needle Holder</td>
			<td>Fine Science Tools</td>
			<td>12001-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5-45 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-35&#8232;</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps -</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Lexer-Baby Scissors</td>
			<td>Fine Science Tools</td>
			<td>14079-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Castroviejo Micro Needle Holder</td>
			<td>Fine Science Tools</td>
			<td>12060-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Scissors</td>
			<td>Aesculap, Germany</td>
			<td>Typ OC498R</td>
			<td></td>
		</tr>
		<tr>
			<td>Castroviejo Suture Forceps</td>
			<td>Geuder, Germany</td>
			<td>19015</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 silk black (Silk)</td>
			<td>Deknatel, Research Triangle Park NC, USA</td>
			<td>18020-60- FST</td>
			<td></td>
		</tr>
		<tr>
			<td>11-0 monofilament (Ethilon)</td>
			<td>Ethicon, Norderstedt, Germany</td>
			<td>EH7438G</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 polyglycolic acid suture (Serafit)</td>
			<td>Serag-Wiessner, Naila, Germany</td>
			<td>60203214</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluorane</td>
			<td>Any genericon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Any genericon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.9% saline</td>
			<td>Any genericon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Any genericon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bepanthene eye and nose cream</td>
			<td>Bayer, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscop</td>
			<td>Zeiss</td>
			<td>Opmi MDO/S5</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaporiser</td>
			<td>Eickemeyer</td>
			<td>TEC3</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound</td>
			<td>Vevo, Canada</td>
			<td>770,2100</td>
			<td></td>
		</tr>
	</tbody>
</table>

using the sleeve technique in a mouse model of aortic transplantation - an instructional video

采用袖套技术的原位主动脉移植可减少10-20% 的失败率, 降低主动脉的损伤。使用套筒法吻合小鼠主动脉的时间较短, 易于平均20分钟, 允许对同种异体移植进行研究。下面的文章介绍了主动脉移植术在我们的实验室中的应用。小鼠用1.5% 容积异氟醚和100% 氧气的混合物在面罩中麻醉。此时, 肾动脉与其分支之间的动脉段与腔静脉分离, 自由制备, clampedat 近端和远端段, 单丝缝合。在主动脉切除之前, 将含肝素的盐水溶液注入下腔静脉。然后在钳夹之间切断主动脉, 用生理盐水肝素溶液冲洗腔内。采用单丝缝合袖套技术, 在原位移植腹主动脉。

小鼠在15-30 分钟内从麻醉中恢复, 没有观察到身体损伤, 尽管血栓形成的风险很高。术后随访采用超声分析。在研究中使用的野生型小鼠, 其腔内的尺寸没有变化。因此, 没有观察到狭窄和动脉瘤形成。移植动物未能表现出血管壁的斑块发育 (图 2)。
常规染色方法如 immunohistology 可用于测定斑块、平滑肌细胞的形态和巨噬细胞的积累。在本研究中, 移植后6周进行组织学和免疫组化染色, 以检验移植物的完整性。组织学染色 (苏木精和曙红 (he) 和免疫组织化学染色 (平滑肌肌动蛋白 (SMA) 和巨噬细胞 (MAC2)) (图 3) 显示我们不变的平滑肌细胞分布模式, 完整的内皮细胞衬里, 没有细胞在内膜中的积累。这些结果表明, 移植血管中没有发现明显的病变或细胞活化 (图 3)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/54915/54915fig1.jpg"> 
图 1: 套筒技术.腹主动脉采用袖套法移植。在这个过程中, 捐献者的主动脉被放置在原位, 表面的叮咬存在于喂食容器中。<a href="//ecsource.jove.com/files/ftp_upload/54915/54915fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/54915/54915fig2.jpg"> 
图 2: 术中和超声图像.例术中 (尸检) 观察移植术后6周 (A), 其三维超声 (B) 和 B 模式视图 (C)。术后随访采用超声进行。图片显示了移植的通畅性, 在流明尺寸上没有变化。此外, 没有观察到狭窄或动脉瘤形成。<a href="//ecsource.jove.com/files/ftp_upload/54915/54915fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/54915/54915fig3.jpg"> 
图 3: 组织学和免疫组织化学图像.移植后6周移植动物的组织学和免疫组化代表图像。在移植脉中未观察到明显的病变 (苏木精和曙红, he, 100X 放大倍率, 刻度棒 = 50 µm), 免疫组织化学染色 (平滑肌肌动蛋白 SMA (红色), 或巨噬细胞 MAC2 (绿色), 200X 放大倍数, 刻度条 =25µm)。细胞核由 DAPI (蓝色) counter-stained。<a href="//ecsource.jove.com/files/ftp_upload/54915/54915fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video at JoVE.com

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

本文采用套筒技术对小鼠原位主动脉移植模型进行了研究。它是一种非常快速的吻合方法, 可用于血管疾病的研究。

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 ...

using-sleeve-technique-mouse-model-aortic-transplantation-an

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

8.9K 次观看.  Ruhr- University Bochum. 本文采用套筒技术对小鼠原位主动脉移植模型进行了研究。它是一种非常快速的吻合方法, 可用于血管疾病的研究。

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

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

主动脉原位移植大鼠模型，研究慢性血管病变的发展

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

科研

医学

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

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 ...

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

原位主动脉瓣移植在小鼠血管疾病的研究

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

采用实时定量PCR检测供体细胞植入一个具有竞争力的小鼠骨髓移植模型

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

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 ...

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 ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

创作和移植的糖尿病伤口愈合模型中脂肪源性干细胞 (ASC) 表

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

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 ...

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 ...

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. However, only minor improvement in the long-term results of transplanted solid organs could be observed over the past decades. In this context, chronic allograft vasculopathy (CAV) still represents the leading cause of late organ failure in cardiac, renal and pulmonary transplantation.
Thus far, the underlying pathogenesis of CAV development remains unclear, explaining why effective treatment strategies are presently missing and emphasizing a need for relevant experimental models in order to study the underlying pathophysiology leading to CAV formation. The following protocol describes a murine heterotopic cervical aortic transplantation model using a modified non-suture cuff technique. In this technique, a segment of the thoracic aorta is interpositioned in the right common carotid artery. With the use of the non-suture cuff technique, an easy to learn and reproducible model can be established, minimizing the possible heterogeneity of sutured vascular micro anastomoses.

Here, we present a protocol of heterotopic aortic transplantation in mice using the non-suture cuff technique in a cervical murine model. This model can be used to study the underlying pathology of chronic allograft vasculopathy (CAV) and can help evaluate new therapeutic agents in order to prevent its formation.

使用改性非缝合铐技术的宫颈主动脉移植模型

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. ...