这项工作得到了 2022 年 JST 基础研究（日本移植学会）的支持。

该实验方案得到了京都大学动物实验委员会的批准。该研究使用了从商业来源获得的 C57BL/6 小鼠，年龄超过 10 周，体重在 25 g 至 30 g 之间（参见 材料表）。所有动物均用2.5%异氟烷麻醉（遵循机构批准的方案），在特定的无病原体条件下维持，所有实验程序均按照京都大学的《动物实验条例》进行。用于研究的适当仪器列在 材料表 中，如图 1 和 图 2 所示。
1. 动物选择
<ol><li>使用体重为25-30克的小鼠。 注意：虽然小鼠肝脏同种异体移植物2 在主要组织相容性复合物屏障中被普遍接受，但我们在这项研究中选择了同基因 MOLT 模型，以关注冷缺血再灌注损伤10 的详细机制。不建议使用体重小于 25 g 的小鼠，因为不可能将内部支架插入细胆管。同样，由于血管周围存在大量腹内脂肪，因此不建议体重超过 30 克的小鼠。</li>
</ol>2. 袖口制作
<ol><li>分别为 PV 和 IHIVC 准备 20 G 和 16 G 静脉导管。</li>
	<li>使用手术刀切开导管以形成袖带。袖带的主体长度应为2 mm，延伸手柄为1 mm（图3A）。 注意：如果手柄太大，插入袖带可能会变得困难。</li>
	<li>在袖带的表面，使用手术刀刀片的背面创建一个浅凹槽，用于螺纹固定。 注意： 将手术刀刀片的背面贴在袖带上时，请确保刀片的尖端呈大约 60 度角（如图 3B 所示）。制作第一个凹槽后，固定手术刀刀片并旋转袖带。理想情况下，建议使用两个凹槽，但单个凹槽就足够了，没有问题。</li>
</ol>3. 袖带附件
<ol><li>在一个塑料矩形容器中，放置小冰块，并在顶部放置一个小金属杯（直径 6 厘米）。用器官保存液填充杯子（参见 材料表），并将同基因移植物放入其中。</li>
	<li>用棉签轻轻滚动同基因肝移植物（从另一只供体小鼠中提取），确保肝门朝上。用器官保存液彻底清除储存在 IVC 腔中的任何血液。</li>
	<li>使用例如连接到脾静脉的螺纹将 PV 穿过袖带（见 图 4A）。</li>
	<li>将袖带手柄置于 12 点钟位置，用斗牛犬夹固定手柄和袖带amps（参见 材料表），将其放置在距离 PV 边缘 2 至 3 毫米的位置。</li>
	<li>使用大血管镊子和固定模具进一步固定斗牛犬夹以建立手术区域（见 图4B）。</li>
	<li>以大约 15 至 20 倍的放大倍率仔细检查 PV 流明。如有必要，滴水可以提高管腔的可见度。</li>
	<li>轻轻抓住 PV 流腔并通过袖带将其外部化。</li>
	<li>一旦完全外化，使用 8-0 通过双重连接将其固定丝线沿着袖口的凹槽（图4C，D）。 注意： 确保结扎线没有松动。另外，注意结扎点不要太大，因为它可能会干扰袖带插入。结扎点可以定位在任何方向上。</li>
	<li>使用类似于PV的程序将袖带连接到IHIVC（图4E）。 注意：用袖带夹住 IHIVC 时，请避免咬入肝实质。由于颈部比 PV 短，因此请仔细确定夹紧位置。</li>
	<li>通过将线绕在 IHIVC 的底部以暂时连接 IHIVC 来创建一个滑结（图 5）。再灌注后将移除该线。</li>
</ol>4. 门静脉重建
<ol><li>通过用2.5%的异氟烷诱导麻醉来麻醉受体小鼠，并将其降低至0.6%，然后再提取已发表的文献2中所述的天然肝脏。在剖腹手术前，用交替的碘伏和 70% 乙醇对手术区域进行至少 3 次 dinsini 感染。</li>
	<li>确保肝叶位置正确，并且套囊 PV 没有扭结和扭转。</li>
	<li>使用 Pean 镊子轻轻抓住接受者的 PV 边缘，并用钳子将其固定。 注意：在本文中，钳子是指用于将豌豆镊子固定在桌子上的机械装置。有关虎钳的更多信息，请参阅 材料清单。</li>
	<li>将血管钳夹应用于接受者的 PV 并通过 8-0丝线缠绕在它周围。</li>
	<li>创建一个 1/4 圆周截面，距离 PV 边缘约 0.5 mm（图 6A）。使用专用仪器（一根 27 G 针头，尖端切断并弯曲成 L 形）将盐水溶液通过管腔时扩大孔。</li>
	<li>使用直镊子握住袖带手柄并将其插入管腔。</li>
	<li>完全插入后，用 8-0 固定袖带丝线。单次结扎就足以进行固定。</li>
	<li>取下血管钳夹并重新灌注肝脏。</li>
	<li>小心地释放豌豆镊子并完成门静脉重建。整个光伏重建过程大约需要 5 分钟。</li>
</ol>5.肝下腔静脉重建
<ol><li>适当放置肝叶，并将连接到受体 IHIVC 背侧的 Pean 镊子移动到移植物的右下叶，用虎钳固定。</li>
	<li>用棉签轻轻擦拭，去除接受者 IVIHC 周围任何残留的肝组织。</li>
	<li>以8-0的比分丝线缠绕在接受者的 IVIHC 周围。</li>
	<li>在距受体的 IVIHC 边缘约 0.5 毫米处创建一个 1/4 圆周截面（图 6B）。</li>
	<li>将生理盐水溶液引入管腔后，将袖带插入管腔。 注意：由于颈部较短，袖带通常比 PV 中的插入得更浅。</li>
	<li>小心地用 8-0 固定袖带丝线，然后取下血管钳和豌豆钳。</li>
</ol>6.术后护理
<ol><li>利用加热垫和加热灯来促进术后恢复。</li>
	<li>持续监测血压、心率、呼吸频率、体温，以及食物和水的消耗。</li>
	<li>给药者皮下注射卡洛芬（5 mg/kg，每 6 小时或直到需要）以减轻术后疼痛。遵循当地的机构准则。 注意：如果小鼠的状况恶化，实验将立即停止，并通过吸入二氧化碳对小鼠实施安乐死。</li>
	<li>为了收集组织样本，重新麻醉小鼠并通过IACUC批准的方法对它们实施安乐死。</li>
</ol>

小鼠肝移植中有效血管吻合的策略

<ol>
	<li>Qian, S. G., Fung, J. J., Demetris, A. V., Ildstad, S. T., Starzl, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+liver+transplantation+in+the+mouse.">Orthotopic liver transplantation in the mouse.</a> Transplantation. 52 (3), 562-564 (1991).</li><li>Yokota, 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=Orthotopic+mouse+liver+transplantation+to+study+liver+biology+and+allograft+tolerance.">Orthotopic mouse liver transplantation to study liver biology and allograft tolerance.</a> Nat Protoc. 11 (7), 1163-1174 (2016).</li><li>Shen, X. 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=Inflammatory+responses+in+a+new+mouse+model+of+prolonged+hepatic+cold+ischemia+followed+by+arterialized+orthotopic+liver+transplantation.">Inflammatory responses in a new mouse model of prolonged hepatic cold ischemia followed by arterialized orthotopic liver transplantation.</a> Liver Transpl. 11 (10), 1273-1281 (2005).</li><li>Kageyama, 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=Ischemia-reperfusion+injury+in+allogeneic+liver+transplantation:+A+role+of+CD4+T+cells+in+early+allograft+injury.">Ischemia-reperfusion injury in allogeneic liver transplantation: A role of CD4 T cells in early allograft injury.</a> Transplantation. 105 (9), 1989-1997 (2021).</li><li>Li, T., Hu, Z., Wang, L., Lv, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Details+determining+the+success+in+establishing+a+mouse+orthotopic+liver+transplantation+model.">Details determining the success in establishing a mouse orthotopic liver transplantation model.</a> World J Gastroenterol. 26 (27), 3889-3898 (2020).</li><li>Tian, Y., R&#252;diger, H. A., Jochum, W., Clavien, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+arterialized+and+nonarterialized+orthotopic+liver+transplantation+in+mice:+Prowess+or+relevant+model.">Comparison of arterialized and nonarterialized orthotopic liver transplantation in mice: Prowess or relevant model.</a> Transplantation. 74 (9), 1242-1246 (2002).</li><li>Steger, U., Sawitzki, B., Gassel, A. M., Gassel, H. J., Wood, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+hepatic+rearterialization+on+reperfusion+injury+and+outcome+after+mouse+liver+transplantation.">Impact of hepatic rearterialization on reperfusion injury and outcome after mouse liver transplantation.</a> Transplantation. 76 (2), 327-332 (2003).</li><li>Chen, J., 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+review+of+various+techniques+of+mouse+liver+transplantation.">A review of various techniques of mouse liver transplantation.</a> Transplant Proc. 45 (6), 2517-2521 (2013).</li><li>Kojima, 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=T+cell+carcinoembryonic+antigen-related+cell+adhesion+molecule+1-T+cell+immunoglobulin+domain+and+mucin+domain-containing+protein+3+cross+talk+alleviates+liver+transplant+injury+in+mice+and+humans.">T cell carcinoembryonic antigen-related cell adhesion molecule 1-T cell immunoglobulin domain and mucin domain-containing protein 3 cross talk alleviates liver transplant injury in mice and humans.</a> Gastroenterology. 165 (5), 1233-1248 (2023).</li><li>Yokota, S., Yoshida, O., Ono, Y., Geller, D. A., Thomson, 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=Liver+transplantation+in+the+mouse:+Insights+into+liver+immunobiology,+tissue+injury,+and+allograft+tolerance.">Liver transplantation in the mouse: Insights into liver immunobiology, tissue injury, and allograft tolerance.</a> Liver Transpl. 22 (4), 536-546 (2016).</li></ol>

任何作者都没有任何利益冲突需要披露。

学习血管重建是成功实现 MOLT 最具挑战性的方面。鉴于小鼠5 的体型较小，袖带的质量显着影响了重建的难度。本文提供了袖带准备、连接和重建的详细方案。
虽然与以前的报告在袖带准备和连接方面没有重大差异 2,5，但应考虑一些小问题。首先，袖带手柄的厚度应限制在总周长的 1/6 左右。如果太薄，手柄很容?...

小鼠原位肝移植（MOLT）是一种有效的实验方法，于1991年首次报道1。这种利用转基因小鼠和各种研究试剂的实验模型在研究冷暖缺血和再灌注损伤方面发挥了关键作用。然而，该模型的高技术复杂性阻碍了肝移植基础医学的发展2。MOLT 涉及三个主要步骤：（1） 从供体小鼠中取出肝脏，（2） 背台手术，以及 （3） 将肝脏植入受体。在这些受体手术中，血管吻合术构成了最大的挑战。虽然肝上下腔静脉吻合术通常通过手工缝合完成2，但肝下下腔静脉 （IHIVC） 和门静脉 （PV） 可以使用塑料袖带代替手工缝合线更有效地重建。
肝期表示切除受体的天然肝脏和移植物植入之间的间隔。为确保一致的结果，必须将肝脏分离时间限制在20分钟以内。因此，采用这一模式的研究仅限于特定机构3,4,5,6,7,8,9。在 MOLT 的各个阶段中，实现平稳的 PV 和 IHIVC 重建对于最大限度地减少肝功能时间并确保移植成功至关重要。
PV 和 IVC 重建通常使用血管袖带进行，因为与手工缝合线相比，袖带技术简化了血管吻合术 2,5,8。血管袖带准备和袖带安全连接所涉及的技术显着影响受体血管重建的复杂性。我们的目标是为与袖带方法相关的众多技术技巧提供详细的视觉指导，从而减少学习曲线。这些视频剪辑将清楚地了解如何将袖带连接到血管上，并在接受者手术期间重建 PV 和 IHIVC。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16 G intravenous catheter</td>
			<td>TERUMO</td>
			<td>SR-FF2032</td>
			<td>IHIVC cuff</td>
		</tr>
		<tr>
			<td>20 G intravenous catheter</td>
			<td>TERUMO</td>
			<td>SR-FF1651</td>
			<td>PV cuff</td>
		</tr>
		<tr>
			<td>8-0 braid silk</td>
			<td>Natsume Seisakusho</td>
			<td>CR9-80B2</td>
			<td>8-0 silk</td>
		</tr>
		<tr>
			<td>Belzer UW Cold Storage Solution&#160;</td>
			<td>Astellas</td>
			<td></td>
			<td>Organ preservation fluid</td>
		</tr>
		<tr>
			<td>Bulldog clamp</td>
			<td>B BRAUN</td>
			<td>FB329R</td>
			<td>Bulldog clamp</td>
		</tr>
		<tr>
			<td>C57BL/6 mice&#160;&#160;</td>
			<td>Oriental Bio Service</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane inhalation solution</td>
			<td>Viatris</td>
			<td></td>
			<td>Anesthesic</td>
		</tr>
		<tr>
			<td>Micro Blunted Tips 0.1 mm x 0.06 mm&#160;</td>
			<td>F.S.T</td>
			<td>11253-20</td>
			<td>Straight microforceps</td>
		</tr>
		<tr>
			<td>Micro Serrefine Clamp Applicator with Lock</td>
			<td>F.S.T</td>
			<td>18056-14</td>
			<td>Vessel clip applicator</td>
		</tr>
		<tr>
			<td>Micro Serrefines&#160;</td>
			<td>F.S.T</td>
			<td>18055-4</td>
			<td>Vessel clip</td>
		</tr>
		<tr>
			<td>No.11 Spare Blades</td>
			<td>FEATHER Safety Razor</td>
			<td>11</td>
			<td>Blades</td>
		</tr>
		<tr>
			<td>Ophthalmic scissor, round handle</td>
			<td>B BRAUN</td>
			<td>FD103R</td>
			<td>Microscissor</td>
		</tr>
		<tr>
			<td>Plastic rectangular-shaped container&#160;&#160;</td>
			<td>Daiso</td>
			<td></td>
			<td>10 cm long, 15 cm wide and 6 cm high</td>
		</tr>
		<tr>
			<td>SuperGrip Tips</td>
			<td>F.S.T</td>
			<td>00649-11&#160;</td>
			<td>Curved microforceps</td>
		</tr>
		<tr>
			<td>SZX7</td>
			<td>Olympus</td>
			<td>SZX7</td>
			<td>Microscope</td>
		</tr>
	</tbody>
</table>

vascular reconstruction with the cuff technique in mouse orthotopic liver transplantation

小鼠原位肝移植是研究肝缺血和再灌注损伤潜在机制的有效方法。然而，技术挑战对利用这种宝贵的实验模型并将这些技能传授给下一代构成了障碍。该手术最具挑战性的方面是血管重建，包括门静脉 （PV）、肝下下腔静脉 （IHIVC） 和肝上下腔静脉。使用塑料袖带而不是缝合线，可以更顺利地进行 PV 和 IHIVC 重建。通过将由静脉导管制成的袖带连接到移植物血管的尖端并将袖带插入受体血管来重建血管。两个最关键的方面是正确地观察血管的内腔和避免使用过度的力。我们的目标是提供在接受者手术中使用袖带技术进行血管重建的技术概述。这些袖带技术的技术技巧有望帮助显微外科医生促进血管重建并推进他们的研究。

Mouse orthotopic liver transplantation (MOLT) is an effective experimental method first reported in 19911. This experimental model, which utilizes genetically modified mice and various research reagents, has played a pivotal role in investigating warm and cold ischemia and reperfusion injuries. However, the model&#39;s high technical complexity has hindered the development of basic medicine for liver transplantation2. MOLT involves three major steps: (1) retrieval of the liver from the donor mouse, (2) back table surgery, and (3) implantation of the liver into the recipient. Among these recipient procedures, vascular anastomosis poses the greatest challenge. While the suprahepatic inferior vena cava anastomosis is typically completed by hand stitching2, the infrahepatic inferior vena cava (IHIVC) and portal vein (PV) can be reconstructed more efficiently using plastic cuffs in place of hand-sewn sutures.
The anhepatic period signifies the interval between the removal of the recipient&#39;s native liver and the graft implantation. To ensure consistent results, it is imperative to limit the anhepatic time to less than 20 min. Consequently, studies employing this model have been confined to specific institutions3,4,5,6,7,8,9. Among the various stages of MOLT, achieving smooth PV and IHIVC reconstruction is crucial for minimizing anhepatic time and ensuring successful transplantation.
PV and IVC reconstruction is generally carried out using vascular cuffs since the cuff technique simplifies vascular anastomosis compared to hand-sewn sutures2,5,8. The technique involved in vascular cuff preparation and secure attachment of the cuff significantly impacts the complexity of recipient vascular reconstruction. Our objective is to provide detailed visual guidance for numerous technical tips related to the cuff method, thereby reducing the learning curve. These video clips will provide a clear understanding of how to attach the cuff to the vessels and reconstruct the PV and IHIVC during recipient surgery.

作者聚焦：探索脂肪肝移植中的冷缺血和温再灌注损伤

袖带技术在小鼠原位肝移植中的血管重建

We focus our research on liver transplantation, in particular on cold ischemia and warm reperfusion injury. We use mouse liver transplantation model as a research tool for this purpose. Because the technique is difficult, so it can take a long time to learn and to achieve consistent results.In the future, we'd like to conduct research on fatty liver transplantation. This is still a problem to overcome on clinical liver transplantation.

当松开门静脉时，没有迂曲，肝脏均匀灌注时，PV 重建成功。肝功能时间应小于20分钟，因为肝脏时间超过25分钟会增加小鼠死亡的风险。如果移植物没有血液反流，则认为 IHIVC 重建成功。
使用器官保存溶液在低温下储存移植物1小时，导致再灌注后6小时的血清丙氨酸氨基转移酶水平约为2,000U / L（图7A）。移植后7天的存活率为100%（图7B</s...

Watch this Scientific Journal Video about Author Spotlight: Exploring Cold Ischemia and Warm Reperfusion Injury in Fatty Liver Transplantation at JoVE.com

该协议为在小鼠原位肝移植中使用袖带技术进行血管重建提供了技术信息。

Mouse orthotopic liver transplantation is an effective methodology for investigating the underlying mechanisms of liver ischemia and reperfusion injury. ...

我们的研究重点是肝移植，特别是冷缺血和温再灌注损伤。为此，我们使用小鼠肝移植模型作为研究工具。因为这项技术很困难，所以可能需要很长时间来学习和获得一致的结果。未来，我们想进行脂肪肝移植的研究。这仍然是临床肝移植需要克服的问题。

vascular-reconstruction-with-cuff-technique-mouse-orthotopic-liver

Research

JoVE Journal

Medicine

Vascular Reconstruction with the Cuff Technique in Mouse Orthotopic Liver Transplantation

833 次观看.  Kyoto University. 我们的研究重点是肝移植，特别是冷缺血和温再灌注损伤。为此，我们使用小鼠肝移植模型作为研究工具。因为这项技术很困难，所以可能需要很长时间来学习和获得一致的结果。未来，我们想进行脂肪肝移植的研究。这仍然是临床肝移植需要克服的问题。

视频: 作者聚焦：探索脂肪肝移植中的冷缺血和温再灌注损伤

Mouse orthotopic liver transplantation is an effective methodology for investigating the underlying mechanisms of liver ischemia and reperfusion injury. However, the technical challenges pose a barrier to utilizing this valuable experimental model and passing on these skills to the next generation. The most challenging aspect of this procedure is vascular reconstruction, including the portal vein (PV), infrahepatic inferior vena cava (IHIVC), and suprahepatic inferior vena cava. The use of plastic cuffs, rather than sutures, allows for smoother PV and IHIVC reconstruction. Vessels are reconstructed by attaching a cuff made from an intravenous catheter to the tip of the graft vessel and interposing the cuff into the recipient vessel. The two most crucial aspects are properly visualizing the inner lumen of the vessel and avoiding the use of excessive force. Our aim is to provide a technical overview of vascular reconstructions using the cuff technique in recipient surgery. These technical tips for the cuff technique are expected to help microsurgeons facilitate vascular reconstruction and advance their research.

This protocol provides technical information for vessel reconstruction using the cuff technique in mouse orthotopic liver transplantation.

科研

医学

Preparation and Attachment of Vascular Cuffs for Syngeneic Liver Grafting

To begin, prepare 16-gauge and 20-gauge intravenous catheters for the portal vein, or PV, and the infra hepatic inferior vena cava, or IHIVC, respectively. Using a scalpel, cut the catheter to create the cuff. Then with the back of the scalpel, create a shallow groove on the cuff's surface for thread fixation.Place small ice cubes in a rectangular-shaped plastic container, and position a small metal cup on top of the container. Fill the cup with organ preservation fluid and place the syngeneic liver graft in it. Under eight to 10 X magnification, gently roll the liver graft using a cotton swab, ensuring that the porta hepatis faces upwards.Thread the PV through the cuff, using a thread attached to the splenic vein. With the cuff handle at the 12:00 position, secure the handle and cuff with a bulldog clamp, positioning it two to three millimeters from the PV edge. Further secure the bulldog clamp using large vascular forceps and a fixation mold to establish the operative field.Next, increase the magnification to 15 to 20 X and carefully inspect the PV lumen. After grasping the PV lumen, externalize the thin wall of PV through the inner lumen of the cuff. Secure the fully externalized PV lumen with a double ligation using 8-O silk thread along the cuff's groove, and then cut the string.Similarly, attach the cuff to the IHIVC lumen, and fix it using 8-O silk thread. Then, to create a slipknot, pass the thread around the base of the IHIVC to temporarily ligate it.

用于同基因肝移植的血管袖带的制备和连接

Portal Vein and Infrahepatic Inferior Vena Cava Reconstruction in Mouse Liver Transplantation

After extracting the native liver from the anesthetized recipient mouse, gently grasp the recipient's liver portal vein or PV edge using pen forceps and secure it with a vice. Pass an eight oh silk thread around the PV and place a vascular clamp on it. Create a one fourth circumferential section approximately 0.5 millimeters from the PV edge.While passing saline through the hole, use a specialized instrument to enlarge the hole. Grip the cuff handle using straight forceps and insert it into the lumen. Then secure the cuff with an 8.0 silk thread.Remove the vascular clamp from PV to allow reperfusion of the liver. Finally, carefully release the pen forceps to complete the PV reconstruction. After positioning the liver lobe, Move the pen forceps attached to the recipient IHIVC dorsal to the graft's right lower lobe.Then secure the IHIVC with a vice. Rub away any remaining liver tissue around the recipient's IHIVC with a cotton swab. Pass an 8.0 silk thread around the recipient's IHIVC.Create a one fourth circumferential section, approximately 0.5 millimeters from the recipient's IHIVC edge. After flushing the lumen with saline, insert the cuff into the IHIVC lumen. Carefully secure the cuff with an 8.0 silk thread.Finally, remove the vascular clamp and pen forceps. The preservation of the graft at cold temperatures for one hour resulted in serum alanine aminotransferase levels, averaging approximately 2000 units per liter at six hours post reperfusion. A survival rate of 100%was observed at seven days following transplantation.