本研究获得中国国家自然科学基金 (81501382 号赠款) 和浙江省自然科学基金 (批准号:) 的支持。LQ13H100003 和 LQ16H100002)。

所有的程序都遵循了温州医科大学动物政策和福利委员会批准的《啮齿动物外科指南》 15。
1. 动物
<ol><li>用雄性 Lewis 老鼠称250-350 克作为捐献者和受体。</li>
</ol>2. 操作环境
<ol><li>在手术显微镜下执行所有显微外科手术, 放大率从7X 到20X 不等。手术前要消毒所有外科器械。 注意: 操作室和操作表清洁, 但不消毒。</li>
</ol>3. 基本显微手术机动
<ol><li>自由, 解剖, 并动员船只。<ol>
<li>首先, 使用直的微钳来解除血管周围的组织。</li>
		<li>第二, 反复轻轻地打开和关闭弯曲微钳的尖端, 这样细微的提示就可以切开并剥开血管前壁上的周围组织。</li>
		<li>第三, 使用直式微钳钳夹并提起血管以暴露后间隙。然后, 使用弯曲微钳的尖端剥离血管后壁上的周围组织。最后, 一个没有任何周围组织连接的秃头血管应该被提出。</li>
	</ol>
</li>
	<li>分裂或双结扎, 并划分血管或组织。<ol>
<li>首先, 使用两个微钳16在容器或组织上系两个方结。第二, 用微剪刀切断两个方结之间的血管或组织。</li>
	</ol>
</li>
</ol>4. 袖套和胆道支架的制备
<ol><li>用12口径和14口径静脉导管分别制作门静脉和 infrahepatic 腔静脉的袖口, 而胆道支架则采用22口径静脉导管。袖口由身体部分和手柄部分组成。使用身体部位的沉, 将容器壁固定在袖口上 (图 1)。</li>
</ol>5. 大鼠的麻醉和固定
<ol><li>在5% 异氟醚与0.5 升/敏氧混合的感应箱中轻轻放置路易斯大鼠, 诱导吸入麻醉。监测大鼠的呼吸频率, 以防止过量异氟醚引起的意外死亡。减少异氟醚的浓度为 2%, 以保持麻醉时, 大鼠不响应疼痛刺激。</li>
	<li>将老鼠移动到一个30厘米 x 30 厘米的橡木板上, 用消毒的手术垫毛巾盖住, 然后把老鼠放在仰卧位。用内侧胶带绑住大鼠的四只四肢, 随后用四图钉固定胶带。一次性用70% 酒精消毒皮肤, 碘伏三次。</li>
</ol>6. 捐助者的行动
<ol><li>暴露手术现场<ol>
<li>在上腹部切开一小横切口, 用电动凝固将两侧下腹血管横断面。</li>
		<li>用手术剪刀将横切口伸展至上腹部的肋缘。</li>
		<li>反向和固定上腹壁和剑突的过程, 分别对颅侧。</li>
		<li>用两根湿棉布把肠子从腹腔移出。用一块湿纱布盖住肠道。</li>
	</ol></li>
	<li>切断韧带和修剪 suprahepatic 腔静脉 (SHVC)<ol>
<li>falciformal 韧带、冠状动脉韧带和三角韧带依次切除。</li>
		<li>用微钳从隔膜中释放左膈静脉。然后, 双结扎使用6-0 丝缝线。 注意: 不要在这个步骤中划分这个静脉。</li>
		<li>用微剪刀切断 gastrohepatic 韧带, 释放下尾叶和 hepatoesophageal 韧带和动脉。</li>
	</ol></li>
	<li>在切除前阻断微光和 ML 的血液供应 (图 2)<ol>
<li>首先, 使用弯曲钳仔细解剖的公共树干的后壁, 提供微光和左中位叶 (爱我的生活) 从实质。第二, 双结扎, 并将其与相应的胆管和肝动脉分开。</li>
		<li>轻轻地解剖和分割 RML 的 PV, 因为这种静脉紧贴肝脏实质。 注: 建议使用14X 放大倍数。不要伤害右上叶的 pv, 这是接近 RML 的 pv。</li>
		<li>鉴别肝动脉和胆管的 RML 位于 PVs 之间的 RML 和爱我的生活。双结扎, 用6-0 丝缝线将 RML 的肝动脉和胆管分开。 注意: 从红色到深红色的微光和 ML 颜色的变化是成功阻断血液供应的指标。深入了解血管解剖是识别和解剖相应血管的必要条件。以前的研究在大鼠清楚地说明了肝脏解剖由腐蚀铸造10。</li>
	</ol></li>
	<li>切除微光<ol>
<li>结扎的椎弓根使用3-0 周丝缝合咬合左外侧肝静脉的血流。使用微剪刀, 切断肝脏立即结扎以上切除肝脏肿块。</li>
	</ol></li>
	<li>用我的生命去切除爱<ol>
<li>用蚊子止血钳夹住中位裂 (图 3A) 中的左中位肝静脉。用微剪刀把爱我生命中的所有肝实质都清除掉。</li>
		<li>在左中位肝静脉周围的镊子下, 放置一条穿透性缝合线, 立即渗入肝实质。然后, 在松开镊子之前, 系上一个方形结结扎。</li>
		<li>在镊子下再放两个穿刺缝线, 环绕着我生命中敞开的伤口。然后, 用两个方结来缝合切口的其余部分。释放蚊子止血钳, 检查是否有出血。</li>
	</ol></li>
	<li>正在切除 RML (图 4)<ol>
<li>放置蚊子止血钳, 将 RML 的底座环绕在0.5 厘米至 SHVC 的距离 (图 4B)。然后, 用蚊子止血钳交叉夹住底座, 咬合右中肝静脉。</li>
		<li>使用微剪刀, 去除所有的肝实质的 RML 沿上表面的镊子。一定要非常接近钳面, 留下一个普通的和薄残端的 ML。</li>
		<li>将第一条穿刺缝线穿透在镊子下的肝实质包围中位肝静脉。然后, 用方结结扎中位肝静脉。用同一方法将第二穿刺缝合结扎右中肝静脉。在6.5.2 的描述之后, 再放置两条穿孔缝合线, 靠近切口的其余部分。</li>
	</ol></li>
	<li>infrahepatic 腔静脉的修整 (IHVC)<ol>
<li>切开打开的腹腔, 露出 IHVC。用弯曲的微钳从周围组织中释放 IHVC 和右肾静脉。解剖和结扎右肾上腺静脉, 从后方排泄到 IHVC。</li>
		<li>在结扎右肾静脉和右肾动脉后, 进行正确的肾切除术, 以节省足够的血管长度, 以便在以后的步骤中将袖口安装到 IHVC 上。</li>
	</ol></li>
	<li>安装胆道支架<ol>
<li>用微钳从第一个肝门动员胆总管 (CBD)。结扎在幽门静脉的交叉点和 cbd 使用6-0 丝缝合。</li>
		<li>切开前壁的一个横切口在 CBD 的近侧结扎。插入一个由22口径静脉注射导管组成的胆道支架, 通过切口进入 CBD 的腔内。用圆周6-0 丝缝线固定胆道支架。在6-0 丝缝线之间进行样条。</li>
	</ol></li>
	<li>修整光伏<ol>
<li>结扎的幽门静脉使用6-0 丝缝合远端的 PV。接下来, 结扎的幽门静脉使用7-0 聚丙烯缝合接近 PV。在这两个结扎之间的幽门静脉样条。双结扎, 并以同样的方式划分脾静脉。 注: 用7-0 聚丙烯缝线结扎血管, 使其与6-0 丝缝线相比, 更小的结扎结。一个大的结可能会干扰袖口安装在最后一步。幽门静脉和脾静脉的血管壁薄, 因此, 在解剖过程中, 温和的操作是必不可少的。在17手术前, 应阅读一篇以前发表的文章, 其中有更多关于解剖和划分幽门静脉和脾静脉的技术说明和插图。</li>
	</ol></li>
	<li>普通肝动脉的修整 (茶)<ol>
<li>用两个微钳从周围组织中仔细地释放茶及其两个分岔、GDA 和胃十二指肠动脉。双结扎的 GDA 使用6-0 丝缝线在岔 GDA。</li>
	</ol></li>
	<li>Heparinizing 与灌注肝移植术<ol>
<li>在1毫升生理盐水中通过小弟弟静脉注入50的肝素。等待5分钟, 实现系统性肝素化。</li>
		<li>用三弯曲微 serrefines 对 IHVC 和茶进行交叉钳咬合肝血流。在 PV 前壁上做一个小切口, 插入22口径导管。灌注在 20 cmH2O 的压力下, 通过导管在4摄氏度的部分肝移植。</li>
		<li>用手术剪刀快速切开胸腔和胸腔静脉, 排出血液和灌流液。不要停止灌注, 直到整个部分肝移植的颜色均匀变黄。</li>
	</ol></li>
	<li>移植部分肝移植术<ol>
<li>用微剪刀对两个结扎 (6.2.2 中描述的) 左膈静脉进行样条。</li>
		<li>接下来, 横断面 SHVC 靠近隔膜, 以保持血管长度尽可能长。用微剪刀在脾脏静脉的水平进行样带。将两个结扎之间的 GDA, 并与腹腔主干接近的横断面。</li>
		<li>将6-0 丝结扎术远端的肾上腺血管横断面放置在6.7.2。用手术剪刀将 IHVC 在左肾静脉的水平。</li>
		<li>将部分肝脏转移到后桌盘 (30 毫米培养皿), 用生理盐水填充4摄氏度。</li>
	</ol></li>
</ol>7. 后表操作 (接枝准备)
<ol><li>安装血管袖口 (图 5)<ol>
<li>用12口径的静脉注射导管用微钳将 PV 拉到袖口的腔内。用容器钳暂时固定袖口手柄和 PV。埃弗特将光伏容器壁覆盖到袖口的外表面。用圆周6-0 丝缝合固定容器壁和袖口位置。 注意: 不要拧下容器。袖口手柄应保持在12点的方向。</li>
		<li>按照7.1.1 中的说明安装 IHVC 袖口。</li>
	</ol>
</li>
	<li>将两条停留缝线预置到 SHVC 上<ol>
<li>在3点和9点方向分别使用两7-0 聚丙烯缝合线从外到内穿透 SHVC 的容器壁。 注意: 不要打结。</li>
	</ol>
</li>
	<li>保存嫁接<ol>
<li>将部分肝移植在生理盐水中浸泡4摄氏度, 以保存后表操作。</li>
	</ol>
</li>
</ol>8. 收件人操作
<ol><li>重复在 6.1-6.2 和6.7 的程序, 除了右肾切除, 在接受者暴露的手术现场, 横断面韧带和修剪血管。</li>
	<li>IHVC、胆管、肝动脉和门静脉在接受者中的制备<ol>
<li>首先, 将 IHVC 的部分调动到右肾静脉。第二, 用6-0 丝缝线结扎右肾上腺血管。第三, 利用微钳从第一肝门动员 CBD。 注意: 不要在 CBD 周围修剪脂肪组织。</li>
		<li>用6-0 丝缝线缝合双结扎术后, 在肝门的第一个分岔处横断面。然后, 用6-0 丝缝合法在双结扎术后用微剪刀对临时医管局进行横断面。将从肝门到幽门静脉的 PV 部分动员起来。</li>
	</ol></li>
	<li>调动肝脏的后方<ol>
<li>用两根棉花棒将接受者肝脏退到左侧。然后, 从 SHVC 到右肾上腺静脉切除肝脏后方的韧带, 如前一篇可视化文章18所述。在此之后, 通过 SHVC 的后空间引入橡皮筋。</li>
	</ol></li>
	<li>在无肝期阶段准备仪器和材料<ol>
<li>重新检查无肝期阶段所需的所有仪器和材料的可用性, 例如用弯曲针、微剪刀、微针支架、微钳、两个微止血钳、两7-0 聚丙烯缝合线和6-0 的5毫升注射器。真丝缝合材料。 注: 搜索材料或仪器可能会延长无肝期阶段。</li>
	</ol></li>
	<li>在接受者中切除肝脏<ol>
<li>用血管钳和直接在幽门静脉上方的 PV 交叉钳住肾脏静脉上方的 IHVC。从这个时刻记录无肝期阶段的持续时间, 直到 PV 上的钳位被释放。</li>
		<li>立即将异氟醚的浓度降低到0.5%。</li>
		<li>用预设的橡皮筋拉 SHVC 和隔膜。用斗牛犬钳夹住隔膜的部分和胸腔静脉, 保持0.5 厘米到 SHVC 的距离。 注意: 不要夹住老鼠的肺。</li>
		<li>使用微剪刀, 沿肝实质的上边缘仔细切开 SHVC 的血管壁, 尽可能长时间保持血管壁。</li>
		<li>用微剪刀对肝门第一分岔处的 PV 进行样带。然后, 对右下叶实质的下腔静脉进行横断面, 而不是在血管壁上, 将周向肝实质留在腔静脉周围。保持在右下叶的切割边缘在4毫米的距离 IHVC。transecting 血管后, 将整个肝脏从腹腔移出。</li>
	</ol></li>
	<li>重建 SHVC<ol>
<li>将部分肝移植到腹腔原位。使用两个预设的停留缝线刺穿 SHVC 的容器壁, 从内到外沿3点和9点方向。把两个留缝线拉到右边, 然后在他们被绑成一个结与自己的两端。</li>
		<li>刺穿 SHVC 血管壁从外到内接近结在3点的方向, 引入针的权利留缝线到血管腔内。</li>
		<li>吻合从3点方向到9点方向, 在血管腔内的一处连续缝合后的 SHVC 后壁。</li>
		<li>在9点方向的拐角处, 刺穿血管壁, 从内到外靠近结, 将针从血管腔中引入。</li>
		<li>使用此缝合吻合前壁使用一个正在进行的缝合线外的血管腔从9点方向到3的 o ' 时钟方向。最后, 将缝合线与它自己的末端绑3次 (图 6)。 注: 在重建的 SHVC 腔内注入3-5 毫升生理盐水, 以在最后2-3 针前强制发出气泡。SHVC 吻合的更详细的描述可以在文章中找到 Delriviere et 等。19。</li>
	</ol></li>
	<li>重建光伏发电<ol>
<li>将两个7-0 聚丙烯停留缝合在接收端的 PV 结束后, 说明在7.2。在相反的方向拉两个停留缝合, 以延长容器壁。使用两个微蚊子止血钳固定两个停留缝线。</li>
		<li>将大约1毫升的盐水注入到接收方的 PV 的血管腔内, 以强制发出气泡。快速将门静脉袖套插入到接收器中的 PV 的血管腔内, 用微钳打开。用圆周6-0 丝缝线固定袖口吻合。交叉钳。</li>
	</ol></li>
	<li>肝移植再灌注<ol>
<li>按顺序松开 SHVC 和 PV 上的夹具, reperfuse 肝移植。记录无肝期阶段的时间。检查吻合口出血。根据大鼠对疼痛刺激的反应, 将异氟醚的浓度提高到1-2%。</li>
	</ol></li>
	<li>重建 IHVC<ol>
<li>将1毫升生理盐水注入接收方 IHVC 的血管腔内, 以迫使气泡。将袖套插入 IHVC 的圆柱形血管腔内, 由圆周肝实质支撑 (图 7)。在 IHVC 的后空间引入6-0 丝缝线。用6-0 丝缝线快速固定袖口吻合。</li>
		<li>释放 IHVC 中的两个容器夹, 重新启动灌注。用微剪刀将周围的肝实质仔细修剪在圆周丝缝线上。</li>
	</ol></li>
	<li>肝动脉重建术<ol>
<li>在3点和9点方向分别使用两个11-0 聚丙烯缝合线从外到内穿透在肝移植中的茶的血管壁。咬合使用两个弯曲的微 serrefines, 在接受者中 GDA 的血流量。</li>
		<li>用微剪刀将接受者在其根部的临时医用小断面暴露在血管腔内。纵向切开部分血管壁, 以适应肝移植中的茶径, 扩大接受方临时医管局的血管腔。</li>
		<li>采用 SHVC 吻合术 (8.6. 2-8. 6.7) 所述的运行缝合技术, 将肝移植到扩大的吻合的端到端方式。 注: 详细插图可以在文章中找到黄 et al14。</li>
	</ol></li>
	<li>重建 CBD<ol>
<li>将肠道移回腹腔, 以减少胆管两端之间的距离。在接受者的胆管前壁进行横切口。</li>
		<li>将胆道支架拉入部分肝移植物中, 通过横切口将其插入到接受者胆管的腔内。用6-0 周丝缝线固定支架。</li>
		<li>将两个6-0 周围丝缝线缝合在胆管之间, 以减少吻合的张力。</li>
	</ol></li>
	<li>关闭腹部<ol>
<li>在连续缝合模式下用3-0 丝缝线缝合两层腹部。</li>
	</ol></li>
</ol>9. 术后治疗 (镇痛和抗生素)
<ol><li>治疗所有接受者0.1 毫克/千克丁丙诺啡皮下。治疗头孢呋辛 (16 毫克/千克) 除了止痛药。向老鼠提供自来水和标准实验动物周向ad 随意。</li>
</ol>

<ol>
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所有作者都没有相互冲突的利益来披露。

大鼠 PLTx 是一种复杂的显微外科手术, 其培训计划成本高且持续时间为20。大鼠 PLTx 协议的复杂性使得研究人员无法使用这种动物模型。与全尺寸大鼠 LTx 相比, 大鼠 PLTx 的 microsurgeon 不仅与移植过程的挑战有关, 而且与小动物肝脏切除的挑战有关。因此, 详细描述整个过程的可视化显微外科协议是建立这一复杂模型的必要条件。Nagai et所报告的先前可视化的文章提出了一项50% 种局部原位 LTx 与肝动脉重建的协议 ( 表 2)。该协议使用了一个前体肝切除术, 临床上类似于分裂 LTx 的过程。
与分裂 LTx 相比, 活体捐献者 LTx 切除掉活体捐献者的部分肝移植, 而不是在后表手术中分裂肝脏。体内肝切除可能导致更多的手术操作, 更长的门静脉高压, 甚至更温暖的缺血性损伤的部分肝移植。因此, 使用体内肝切除的几种鼠 PLTx 模型已在相关研究中建立6,8,21,22。许多协议涉及切除肝叶后, 简单结扎在椎弓根或基部2,3,6,8,21,23。然而, 不同的肝叶有不同的形状。大鼠的 ML 有一个广泛的基础, 半包围腔静脉10,24。简单的结扎在 ML 的底部容易收缩下腔。为了避免腔静脉的收缩, Madrahimov et . 开发了一种以保肢为导向的肝切除术, 在2个步骤中切除了四条穿刺缝线, 使 ml 的残端扁平10. 然而, 穿刺缝线结扎的肝叶 PVs 在软组织中隐藏。由于缺少或伤害了 PV 的小分支而造成的残端出血无法完全避免11。在我们的协议中, 首先进一步解剖相应的 PVs 和肝动脉。术前切除肝叶的血供结扎单独闭塞。因此, 使用我们的协议 (图 10) 降低残端出血率。在 ML 的残端放置四穿刺缝线的技术也被用于结扎肝静脉, 而不是 PV 和肝动脉。这些结扎使用多穿孔缝合和一步明智的切除, 保留残肝残肢的扁平形状, 避免了腔静脉的收缩。
对于 IHVC 重建, 对卡玛达et 等描述的袖套吻合进行了轻微的修改。25. 接受者的下腔静脉在右下叶的实质中被切断, 而不是在 IHVC 血管上, 在腔静脉周围留下一个圆周肝实质。在腔静脉周围的肝实质有助于维持受体 IHVC 的血管末端的圆柱形, 而不是平坦的形状。因此, IHVC 的流明是自然打开的, 没有留下缝合和镊子的帮助。这一修改有助于节省放置缝合线的步骤, 并促进插入 IHVC 袖口的血管腔的接受者。此外, 这种修改延长了 IHVC 的血管长度, 从而减少了袖套吻合期间两端的张力。
以快速血管吻合为优势, 在大鼠原位 LTx 中经常使用双袖技术进行网状和 IHVC。宫田et . 甚至提出了一个协议, 涉及三袖技术吻合光伏, IHVC 和 SHVC。然而, 袖口技术也有它的缺点。首先, 固定尺寸的袖口不能完全符合相应容器的大小。此外, 袖套技术干扰血液流动, 导致血栓形成的发病率和异物反应的袖口26。SHVC 是大鼠的主要血管, 呈高血流。血管内的任何收缩或血栓都可能导致严重的术后并发症。因此, 在我们的 PLTx 协议中, 我们选择使用手缝技术重建 SHVC, 使吻合口的调整尽可能接近物理设置。然而, 采用手工缝合技术完成高质量的 SHVC 吻合术, 需要很高的显微外科技术。对于初学者, 书桌的三角化技术建议用于网状血管27。通过使用这项技术, 三保持缝合线定期放置在周围的 SHVC。SHVC 的3D 形状是通过在三个方向上缩回三的缝合线来保持的, 这可以防止 SHVC 在吻合过程中出现狭窄。
重新动脉肝移植是一个有争议的话题。我们认为, 重新动脉不是确保接受者生存率高的关键步骤, 但它可能是确保高质量肝移植的关键步骤。在传统的大鼠 LTx 的双袖技术协议中, 肝动脉没有重建, 因为肝动脉灌注不足不会影响大鼠 LTx12的结果。此外, 以前对小鼠 LTx 的研究是为了研究再动脉对长期移植存活、组织学改变、缺血性肝损伤和早期免疫活化途径的影响13。研究人员得出结论, 动脉肝移植术对患者的存活率和免疫活化程度没有重大影响。但是, 黄et最近的一项研究表明, 重新动脉对肝切除术后肝脏实质的恢复非常重要, 特别是在流出的阻塞28的情况下。部分肝移植的非动脉加重肝损伤, 延缓局灶性坏死的恢复。因此, 推荐部分肝移植的动脉。如图所示, 采用端对端吻合技术重建肝动脉, 这是我们的一个作者14先前描述的。这项技术的优点是选择吻合两个厚动脉, 茶和 GDA, 而不是薄的 PHA, 减少了重建肝动脉的难度, 保持了高的吻合率。
卡玛达et. 提出的经典鼠 LTx 协议建议控制无肝期阶段, 使其小于26分钟, 因为当无肝期阶段持续时间长于26分钟25时, 没有动物幸存。我们的经验与这一建议是一致的, 虽然我们以前观察过几个存活病例, 无肝期相时间长于26分钟。我们认为, 稳定的无肝期阶段优先于较短的无肝期阶段的外科研究。在我们组中, 无肝期阶段精确地控制在特定时间内。在达到预设的无肝期时间之前, 无肝期阶段不会停止, 尽管 SHVC 和 PV 的吻合可以更早完成。然而, 在培训项目中, 鼓励更短的无肝期阶段。
虽然已经进行了许多改进, 以缩短训练阶段和消除不必要的手术, 鼠 LTx 和 PLTx 仍然是复杂的显微外科手术20。因此, 除了可视化的协议外, 基于计划-检查-操作周期的步进训练协议对于实现高生存率和高运行质量具有重要意义。我们在使用这项训练协议的初步经验表明, 需要40-73 手术来掌握大鼠原位 LTx。还需要另外9-13 个手术来掌握大鼠重新静脉 PLTx。Czigány et . 提出的培训协议声称, 50-60 LTxs 足以掌握原位 LTx 和 PLTx29。
在我们的计划-检查-行动培训协议中, 必须强调每个培训步骤和每个个体操作的质量控制, 尤其是在这样一个复杂的模型中。高存活率并不等同于高质量的程序。在代表性的结果, 列出了评价显微外科的质量的几个标准, 包括组织学, 肝酶, 体重恢复, 黄疸 (血清胆红素和黄尿), 无肝期阶段的持续时间, 和通畅肝动脉。只有满足所有标准, 才能实现高质量。

目前, 捐赠肝脏的数量和等待捐献肝脏的病人数量之间存在很大的差异。肝脏器官的短缺是一个全球性的问题。为了扩大捐献者的水池, 分裂肝移植 (LTx) 和活体捐献者 LTx 被开发用于使用部分肝脏作为移植物1。
为进一步研究部分肝移植 (PLTx) 的机制, 建立了相关动物模型2,3,4,5。在大鼠 PLTx 中, 肝叶切除体内和前体, 以模拟活体捐献者 LTx 的条件, 并分别在人类体内分裂 LTx。在可视化实验杂志上发表的一篇论文提出了一个详细的协议, 涉及50% 只老鼠 PLTx 使用前体肝切除5。然而, 在可视化文献中还没有报道一只老鼠 PLTx 与体内肝切除术有关。
除了体内和前体hepatectomies 之间的差异外, 执行肝切除术本身也在确定 PLTx 的结果方面起着重要作用. 目前, 在许多外科研究中使用大鼠 PTLx模型, 肝叶切除后, 放置一个简单的结扎在肝瓣的椎弓根2,3,6,7,8,9。然而, 在切除前放置一个简单的结扎不适合所有肝叶, 因为不同的肝叶有不同的形状和大小。在正中裂片的基础上进行简单结扎, 有很高的风险导致腔静脉收缩, 这可能最终影响部分肝移植的流出10,11。因此, 在大鼠 PTLx 的研究中, 需要以大鼠肝脏解剖知识为基础的更新肝切除技术。
在本研究中所述的协议中, 将最新的以血管为导向的70% 肝切除术纳入大鼠 PLTx 的程序中。在肝实质切除前分别对左外侧叶 (微光) 和正中叶 (ML) 的门静脉和肝动脉进行解剖和分切。然后用穿刺缝线结扎了微光和 ML 的肝静脉。通过使用单独的结扎和多重穿刺缝合, 而不是简单的结扎, 毫升的残余树桩能够扩散到腔静脉。因此, 避免了手术结扎引起的 infrahepatic 腔静脉的收缩。另外, 在切除肝实质之前, 单独结扎对微光和 ML 血供的阻断减少了残肝残端的出血率, 从而最大限度地减少了血液损失对实验的影响11。
对于 microsurgeons, 由于该血管直径极小, 重建肝移植肝动脉 (PHA) 是一项重大的挑战。虽然在 LTx 中重新动脉是否真的是必要的问题仍在辩论中12,13,14, 已经提出了许多重建肝动脉的显微外科技术14.本文介绍了一种新的肝移植动脉技术, 它以端到端的方式将普通肝动脉 (CHA) 与扩大后的医用血管吻合。使用这种端对端技术, 两个肝动脉连接到一个更大的直径吻合。吻合口直径越大, 缝合越容易, 吻合通畅的改善越大。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Surgical microscope</td>
			<td>M&#246;ller-Wedel, Germany</td>
			<td>654359</td>
			<td></td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>OSRAM (China) Lighting Co., Ltd</td>
			<td>64627</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane Vaporizer</td>
			<td>MIDMARK Corporation</td>
			<td>VIP3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter Healthcare Corporation</td>
			<td>40032609</td>
			<td>For Inhalation anesthesia</td>
		</tr>
		<tr>
			<td>Normal Saline</td>
			<td>Zhejiang Tianrui Pharmaceutical Co., Ltd.</td>
			<td>716092103</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone Iodine Solution</td>
			<td>HANGZHOU MINSHENG PHARMACEUTICAL</td>
			<td>H33021567</td>
			<td>For disinfection</td>
		</tr>
		<tr>
			<td>Hemostatic forceps</td>
			<td>Shanghai Medical Instruments(Group) Ltd</td>
			<td>J31020</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Shanghai Medical Instruments(Group) Ltd</td>
			<td>JC2116</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle-holder</td>
			<td>Shanghai Medical Instruments(Group) Ltd</td>
			<td>J32030</td>
			<td></td>
		</tr>
		<tr>
			<td>Dressing Forceps</td>
			<td>Shanghai Medical Instruments(Group) Ltd</td>
			<td>J42020</td>
			<td></td>
		</tr>
		<tr>
			<td>Mosquito hemostatic forceps</td>
			<td>Shanghai Medical Instruments(Group) Ltd</td>
			<td>W40340</td>
			<td>For liver resection; For retracting the stay sutures.&#160;</td>
		</tr>
		<tr>
			<td>Micro-scissors</td>
			<td>Shanghai Medical Instruments(Group) Ltd</td>
			<td>WA1040</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro needle-holder</td>
			<td>Shanghai Medical Instruments(Group) Ltd</td>
			<td>WA2060</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved micro typing forceps</td>
			<td>Shanghai Medical Instruments(Group) Ltd</td>
			<td>WA3061</td>
			<td></td>
		</tr>
		<tr>
			<td>straight micro typing forceps</td>
			<td>Shanghai Medical Instruments(Group) Ltd</td>
			<td>WA3060</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro hemostatic forceps</td>
			<td>Shanghai Medical Instruments(Group) Ltd</td>
			<td>WA3020</td>
			<td></td>
		</tr>
		<tr>
			<td>Bulldog clamp</td>
			<td>Shanghai Medical Instruments(Group) Ltd</td>
			<td>XEC350</td>
			<td></td>
		</tr>
		<tr>
			<td>Vessel clamps</td>
			<td>Shanghai Medical Instruments(Group) Ltd</td>
			<td>W40160</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Serrefine-curved</td>
			<td>&#160;Fine Science Tools, Inc</td>
			<td>18055-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Dacryosyrinx</td>
			<td>Shi Zhuo medical instruments Co., Ltd.</td>
			<td>5# curve</td>
			<td>Use as the curved needle for forcing out the air bubbles in the vascular lumen</td>
		</tr>
		<tr>
			<td>Sterilized gauze swabs</td>
			<td>Fuqing Health &#38; Integral Medical</td>
			<td>20230R</td>
			<td></td>
		</tr>
		<tr>
			<td>5mL syringe</td>
			<td>Zhejiang Yusheng Medical Instrument Co.,Ltd</td>
			<td>811932416</td>
			<td></td>
		</tr>
		<tr>
			<td>18G needles</td>
			<td>Shanghai Kindly Enterprise Development Group Co., Ltd</td>
			<td>01-014</td>
			<td>For fixing the upper abdomen wall</td>
		</tr>
		<tr>
			<td>Intima II 22G intraveous catheter</td>
			<td>Becton Dickinson Medical Devices (Shanghai) Co Ltd.</td>
			<td>383207y</td>
			<td>For making biliary stent; For infusing perfusate</td>
		</tr>
		<tr>
			<td>7-0 polypropylene sutures</td>
			<td>Ningbo Medical Needle Co., Ltd</td>
			<td>151026</td>
			<td>For SHVC anastomsis</td>
		</tr>
		<tr>
			<td>6-0 silk suture</td>
			<td>Shanghai Pudong Jinhuan Medical Products Co., Ltd</td>
			<td>2650182</td>
			<td>For ligation</td>
		</tr>
		<tr>
			<td>Culture dish (100mm)</td>
			<td>Corning Incorporated</td>
			<td>430165</td>
			<td>used in back table for storage ice. The back table dish was placed on it.&#160;</td>
		</tr>
		<tr>
			<td>Culture dish (30 mm)</td>
			<td>Corning Incorporated</td>
			<td>3296</td>
			<td>As a vessel for organ preservation</td>
		</tr>
		<tr>
			<td>Angiocath 12G intravenous catheter</td>
			<td>Becton Dickinson Medical Devices (Shanghai) Co Ltd.</td>
			<td>382277</td>
			<td>For making a IHVC cuff</td>
		</tr>
		<tr>
			<td>Angiocath 14G intravenous catheter</td>
			<td>Becton Dickinson Medical Devices (Shanghai) Co Ltd.</td>
			<td>382269</td>
			<td>For making a PV cuff</td>
		</tr>
		<tr>
			<td>Buprenorphine (hydrochloride)</td>
			<td>Tianjin Institute of Pharmaceutical Research Pharmaceutical Co., Ltd</td>
			<td>H12020275</td>
			<td>For analgesic</td>
		</tr>
		<tr>
			<td>Cefuroxime sodium for injection</td>
			<td>Esseti FarmaceuticiS.r.l</td>
			<td>H20160013</td>
			<td>As an antibiotics</td>
		</tr>
	</tbody>
</table>

re-arterialized rat partial liver transplantation with an in vivo vessel-oriented 70% hepatectomy

在临床上发展了肝移植和活体肝供肝移植, 以更有效地利用肝脏器官。为更好地了解这些手术过程的机制, 建立了大鼠部分肝移植 (PLTx) 模型进行相关的外科研究。由于大鼠 PLTx 模型的复杂性, 需要一个具有详细描述的协议。此前发表的一篇文章报告了一项协议, 其中前体肝切除术用于实现50% 大鼠 PLTx。与此协议相比, 我们引入了一个 PLTx体内70% 肝切除的重新静脉。以血管为导向的肝切除术被纳入大鼠 PLTx, 以改进显微外科手术。肝实质切除前, 左外侧叶和正中叶的门静脉和肝动脉分别进行解剖和结扎, 从而降低残肝残端出血的几率。此外, 还介绍了肝动脉与肝动脉扩张的端到端血管吻合术, 以重新 arterialize 肝脏动脉。采用端到端血管吻合技术, 扩大了吻合口的直径, 降低了手缝的难度, 保持了较高的吻合通畅率。此外, infrahepatic 腔静脉的袖套吻合略有改变。在袖状吻合术期间, 在接受者的腔静脉周围有一节环性肝实质, 以维持血管腔的三维形状。这部分肝实质在完成吻合术后被切除。经此修改, 省略了放置缝合线的步骤, 从而进一步缩短了袖套吻合时间。采用本协议对大鼠 PLTx, 在显微外科手术后实现了低肝酶水平、完整的肝小叶结构和高存活率。

共完成31例自体静脉大鼠 PLTx 的应用本协议。所有收件人都存活到观察时间结束为止。患者的体重在术后一天 (POD) 4 开始恢复。在 POD 6 (图 8) 之后, 体重的斜率接近正常的 Lewis 鼠, 这间接地暗示了接收方的恢复。组织学上, 接受者观察到胆管的轻微增生, 接受者的肝小叶结构完好。没有发现坏死或明显的正弦扩张 (图 9)。血清丙氨酸转氨酶 (ALT) 在30荚的正常范围内 (图 10)。在这些接受者中, 没有发现由黄疸引起的黄尿, 这是通过每天检查笼垫, 表明胆道支架通畅。正常血清胆红素水平的 POD 30 进一步证实成功重建胆管 (图 11)。在 transecting 的手术中, 对肝动脉的通畅性进行了检查。所有接受者都观察到了临时医管局的出血, 这意味着吻合术的通畅。所有这些结果表明, 肝移植的功能良好。
无肝期相的平均持续时间为 23.03 @ 2.3 分钟。pv 的袖套吻合术 4.68 0.77 分钟, 而 IHVC 袖吻合仅 2.05 @ 0.71 分钟. 与 PV 的袖套吻合相比, IHVC 的袖套吻合保存了放置留缝线的步骤, 从而进一步缩短了阿纳斯tomosis 时间 (表 1)。
在31例血管性肝切除术中, 观察了70% 例肝切除术中残余残端出血的两起病例。在先前的实验中, 8 例中观察到 ML 残余残端的出血, 其中18起是以血管为导向的肝切除术 (图 12)。结果表明, 采用血管导向的70% 肝切除术后, ML 残余肝残端出血率较低。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56392/56392fig1.jpg"> 图 1.袖口和胆道支架。 (A、C)袖口的大小。(B) 袖口上的沉。(D) 身体部分和手柄部分袖口。(E) IHVC 袖口;(b) 光伏袖口。(F) 胆道支架。刻度条 = 5 毫米.<a href="/files/ftp_upload/56392/56392fig1large.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/56392/56392fig2v2.jpg"> 图 2.咬合和 ML 的血液供应. 一个)太阳的常见树干, 肝动脉和胆管的微光和爱我的生活。 b)transecting 的变色和爱我的生活后, 普通的树干。 c)RML 的门静脉。 d)所有的 PVs, 肝动脉和胆管的微光和 ML 被切断。 刻度条 = 5 毫米缩写: 左外侧叶, ML 中位叶, 爱我的生活左中位叶, RML 右正中叶, RIL 右上叶, 右下叶, SCL 优越尾状叶。<a href="/files/ftp_upload/56392/56392fig2v2large.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/56392/56392fig3.jpg"> 图 3.切除左正中叶。 (A) 切除爱的肝脏大量我的生活立刻在钳之上。 (B) 在切口上放置三穿刺缝线。(<img alt="Image 1" src="/files/ftp_upload/56392/56392img1.jpg">) 刻度条 = 5 毫米缩写: RML 右正中叶, RIL 右上叶, 右下叶, SCL 上级尾状叶。<a href="/files/ftp_upload/56392/56392fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56392/56392fig4.jpg"> 图 4.切除右正中叶 (RML). 一个)RML 的基地。 b)在 RML 的底部放置蚊子止血钳。 c)切除钳上 RML 的肝实质。 d)在 SHVC 上留下一层薄薄的残肝组织。 刻度条 = 5 毫米缩写: ML 中位叶, RSL 右上叶。<a href="/files/ftp_upload/56392/56392fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56392/56392fig5.jpg"> 图 5.安装袖口。 (A) 将 PV 从袖口的腔内拉出。 (B) 用容器钳固定袖口手柄和 PV。 (C) 埃弗特光伏血管壁覆盖袖口的外表面。 (D) 确保血管壁和袖口就位。 刻度条 = 5 毫米.<a href="/files/ftp_upload/56392/56392fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56392/56392fig6.jpg"> 图 6.重建 SHVC. (A & B)分别在右侧和左侧拉两条缝线。 (C) 在血管腔内进行缝合, 吻合 SHVC 后壁。 (D) 将 SHVC 的前壁吻合血管腔内。 刻度条 = 5 毫米.<a href="/files/ftp_upload/56392/56392fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56392/56392fig7.jpg"> 图 7.重建 IHVC. (a) 接收方 IHVC 的圆柱形血管腔。 (B) 确保 IHVC 的袖套吻合。 (C) 开始再灌注。 (D) 将周围的肝实质修剪在圆周丝缝合上方。 刻度条 = 5 毫米缩写: PV 门静脉, IHVC 下 infrahepatic 腔静脉, RIL 右下叶。<a href="/files/ftp_upload/56392/56392fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56392/56392fig10v4.jpg"> 图 8.术后体重恢复。 在术后4天后, 接受者的体重开始恢复。术后6天后, 体重的斜率接近正常的 Lewis 鼠, 间接地暗示了受体的恢复。
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56392/56392fig9.jpg"> 图 9.30天后肝移植组织学. 组织学上, 接受者观察到胆管的轻微增生, 接受者的肝小叶结构完好。没有发现坏死或明显的正弦扩张 (400X)。缩放条 = 20 um<a href="/files/ftp_upload/56392/56392fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/56392/56392fig8v4.jpg"> 图 10.血清 ALT 水平 血清 ALT 水平在 POD 30 的正常范围内。(**, P < 0.05)。
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/56392/56392fig11v2.jpg"> 图 11.血清胆红素水平. 30的血清胆红素水平与正常的 Lewis 大鼠 (P > 0.05) 相当。
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/56392/56392fig12v2.jpg"> 图 12.不同切除方法的出血率. 采用血管定向70% 肝切除法观察了 ML 残余肝残端出血率较低的情况。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>程序</td>
			<td>肝切除</td>
			<td>SHVC 吻合术</td>
			<td>光伏吻合</td>
			<td>IHVC 吻合术</td>
			<td>无肝期时间</td>
		</tr>
<tr>
<td>时间 (分钟)</td>
			<td>2.62±0.46</td>
			<td>15.73±2.05</td>
			<td>4.68±0.77</td>
			<td>2.05±0.71</td>
			<td>23.03±2.30</td>
		</tr>
<tr>
<td colspan="6">数据以 Mean±SD 表示。</td>
		</tr>
</tbody></table>表1。无肝期阶段个体手术的时间
<img alt="Table 2" src="/files/ftp_upload/56392/56392tbl2.jpg"> 表2。大鼠 PLTx 两种可视化协议的区别.<a href="/files/ftp_upload/56392/56392tbl2large.jpg" target="_blank">请单击此处查看此表的较大版本.</a>

<img src="/files/ftp_upload/56392/SUPPLEMENTARY_MATERIAL.jpg"> 补充材料

Watch this Scientific Journal Video about Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy at JoVE.com

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

本文提出了一种静脉大鼠部分肝移植的协议。具体地说, 70% 例肝切除术是通过使用一种最新的血管性肝切除技术在体内进行的。肝动脉以端对侧的方式重建。对袖套技术进行了改进, 缩短了 infrahepatic 腔静脉的吻合时间。

静脉大鼠部分肝移植的体内血管定向70% 肝切除术

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

re-arterialized-rat-partial-liver-transplantation-with-an-vivo-vessel

Nanyang Technological University

Research

JoVE Journal

Medicine

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

11.4K 次观看.  The First Affiliated Hospital of Wenzhou Medical University. 本文提出了一种静脉大鼠部分肝移植的协议。具体地说, 70% 例肝切除术是通过使用一种最新的血管性肝切除技术在体内进行的。肝动脉以端对侧的方式重建。对袖套技术进行了改进, 缩短了 infrahepatic 腔静脉的吻合时间。

视频: Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5.
In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat.

大鼠原位肝移植

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

科研

医学

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

部分原位肝移植大鼠模型的肝动脉重建的外科手术

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Heterotopic Auxiliary Rat Liver Transplantation With Flow-regulated Portal Vein Arterialization in Acute Hepatic Failure

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the failing native liver has regenerated.1-3 The APOLT-method, the orthotopic implantation of auxiliary segments- averts most of the technical problems. However this method necessitates extensive resections of both the native liver and the graft.4 In 1998, Erhard developed the heterotopic auxiliary liver transplantation (HALT) utilizing portal vein arterialization (PVA) (Figure 1). This technique showed promising initial clinical results.5-6 We developed a HALT-technique with flow-regulated PVA in the rat to examine the influence of flow-regulated PVA on graft morphology and function (Figure 2).
A liver graft reduced to 30 % of its original size, was heterotopically implanted in the right renal region of the recipient after explantation of the right kidney.&#160; The infra-hepatic caval vein of the graft was anastomosed with the infrahepatic caval vein of the recipient. The arterialization of the donor&#8217;s portal vein was carried out via the recipient&#8217;s right renal artery with the stent technique. The blood-flow regulation of the arterialized portal vein was achieved with the use of a stent with an internal diameter of 0.3 mm. The celiac trunk of the graft was end-to-side anastomosed with the recipient&#8217;s aorta and the bile duct was implanted into the duodenum. A subtotal resection of the native liver was performed to induce acute hepatic failure. 7
In this manner 112 transplantations were performed. The perioperative survival rate was 90% and the 6-week survival rate was 80%. Six weeks after operation, the native liver regenerated, showing an increase in weight from 2.3&#177;0.8 g to 9.8&#177;1 g. At this time, the graft&#8217;s weight decreased from 3.3&#177;0.8 g to 2.3&#177;0.8 g.
We were able to obtain promising long-term results in terms of graft morphology and function. HALT with flow-regulated PVA reliably bridges acute hepatic failure until the native liver regenerates.

Auxiliary liver transplantation provides a temporary support in acute hepatic failure, until regeneration of the failing liver. The heterotopic auxiliary liver transplantation (HALT) with portal vein arterialization (PVA) renders sufficient liver function. We developed an analogous technique in the rat, to examine the influence of the portal vein arterialization on the morphology and function of the graft.

在急性肝衰竭异位辅助性大鼠肝移植流量调节门静脉动脉

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (&#181;CT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.
The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after &#181;CT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections.

之后，正常小鼠70％部分肝切除血管和实质再生的可视化

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) Procedure

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver metastases, the amount of liver tissue left behind after liver resection has become the main limiting factor of resectability of large or multiple liver tumors. A minimal amount of functional tissue is required to avoid the severe complication of post-hepatectomy liver failure, which has high morbidity and mortality. Inducing liver growth of the prospective remnant prior to resection has become more established in liver surgery, either in the form of portal vein embolization by interventional radiologists or in the form of portal vein ligation several weeks prior to resection. Recently, it was shown that liver regeneration is more extensive and rapid, when the parenchymal transection is added to portal vein ligation in a first stage and then, after only one week of waiting, resection performed in a second stage (Associating Liver Partition and Portal vein ligation for Staged hepatectomy = ALPPS). ALPPS has rapidly become popular across the world, but has been criticized for its high perioperative mortality. The mechanism of accelerated and extensive growth induced by this procedure has not been well understood. Animal models have been developed to explore both the physiological and molecular mechanisms of accelerated liver regeneration in ALPPS. This protocol presents a rat model that allows mechanistic exploration of accelerated regeneration.

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy ALPPS Procedure

Inducing rapid liver hypertrophy using Associating Liver Partition and Portal vein ligation for a Staged hepatectomy (ALPPS) has been proposed for resection of borderline resectable liver tumors. This model may elucidate mechanisms involved in rapid hypertrophy and allows testing of drugs that promote or block the acceleration of regeneration.

肝分型与门静脉结扎大鼠分期肝切除 (ALPPS) 手术模型的建立

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver ...

Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological conditions. Partial hepatectomy also facilitates the increased engraftment and proliferation of transplanted cells by accelerating neovascularization and cell migration towards the liver. Here, we describe a simple protocol for performing 30% hepatectomy and transplantation of cells in the spleen of a non-obese diabetic/severe combined immunodeficient NOD.SCID (NOD.CB17-Prkdcscid/J) mouse. 
In this procedure, two small incisions are made. The first incision is to expose and resect the left lobe of the liver, and another small incision is made to expose the spleen for the intrasplenic transplantation of cells. This procedure does not require any specialized surgical skills, and it can be completed in 5-7 minutes with less stress and pain, faster recovery, and better survival. We have demonstrated the transplantation of hepatocytes isolated from a green fluorescent protein (GFP) expressing mouse (Transgenic C57BL/6-Tg (UBC-GFP) 30Scha/J), as well as hepatocyte like cells of human origin (NeoHep) in partially hepatectomized NOD.SCID mice.

We have described a protocol for performing partial hepatectomy (PHx) and cell transplantation via spleen in NOD.SCID (NOD.CB17-Prkdcscid/J) mice. In this protocol, an incision is made to expose and resect the left lobe of the liver followed by another incision for the intrasplenic transplantation of cells.

肝细胞部分肝切除术后脾移植。小鼠

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological ...

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

The preparation and transplantation of adipose tissue derived stem cell (ASC) sheets onto insufficiently sutured colorectal anastomoses in a rat model is presented. This novel application shows that ASC sheets can reduce colorectal anastomosis leakage.

脂肪组织干细胞移植对大鼠部分结肠切除术后渗漏的减少

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...

Generation of a Rat Model of Acute Liver Failure by Combining 70% Partial Hepatectomy and Acetaminophen

Acute liver failure (ALF) is a clinical condition caused by various etiologies resulting in the loss of metabolic, biochemical, synthesizing, and detoxifying functions of the liver. In most irreversible liver damage cases, orthotropic liver transplant (OLT) remains the only available treatment. To study the therapeutic potential of a treatment for ALF, its prior testing in an animal model of ALF is essential. In the current study, an ALF model in rats was developed by combining 70% partial hepatectomy (PHx) and injections of acetaminophen (APAP) that provides a therapeutic window of 48 h. The median and left lateral lobes of the liver were removed to excise 70% of the liver mass and APAP was given 24 h postsurgically for 2 days. Survival in ALF-induced animals was found to be severely decreased. The development of ALF was confirmed by altered serum levels of the enzymes alanine amino transferase (ALT), aspartate amino transferase (AST), alkaline phosphatase (ALP); changes in prothrombin time (PT); and assessment of the international normalized ratio (INR). Study of the gene expression profile by qPCR revealed an increase in expression levels of genes involved in apoptosis, inflammation, and in the progression of liver injury. Diffused degeneration of hepatocytes and infiltration of immune cells was observed by histological evaluation. The reversibility of ALF was confirmed by the restoration of survival and serum levels of ALT, AST, and ALP after intrasplenic transplantation of syngeneic healthy rat hepatocytes. This model presents a reliable alternative to the available ALF animal models to study the pathophysiology of ALF as well as to evaluate the potential of a novel therapy for ALF. The use of two different approaches also makes it possible to study the combined effect of physical and drug-induced liver injury. The reproducibility and feasibility of current procedure is an added benefit of the model.

The acute liver failure animal model developed in the current study presents a feasible alternative for the study of potential therapies. The current model employs the combined effect of physical and drug-induced hepatic injury and provides a suitable time window to study the potential of novel therapies.

通过结合70%部分切除术和对乙酰氨基酚，生成急性肝衰竭大鼠模型

Acute liver failure (ALF) is a clinical condition caused by various etiologies resulting in the loss of metabolic, biochemical, synthesizing, and ...

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver&#39;s common hepatic artery to the recipient liver&#39;s proper hepatic artery.

矫形肝移植大鼠模型中动脉重联后并发症减少

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

毛利精密切肝片作为肝脏生物学的Ex Vivo模型

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...