感谢朱棣文的 & 和组织学的表现;还有麦卡蒂的讨论。研究得到了弗吉尼亚和交流的癌症研究基金会的资助;国家心脏、肺和血液研究所 (R01HL058770 和 U01HL099999);加州再生医学研究所 (RC1 00354) 资助 i.w.y. R. 得到了人类前沿科学项目职业发展奖 (CDA00017)、德国研究基金会 (RI 2787/1)、Siebel 干细胞研究所和托马斯和史黛西 Siebel 基金会 (1119368-104-GHBJI)。J.M.T. 得到了美国国立卫生研究院 (T32GM007365)、国家研究服务奖 (1F30DK108561) 和保罗和黛西. 索罗斯为新美国人提供的奖学金的支持。

所有动物实验都严格按照国际实验动物保育协会 (AAALAC) 和斯坦福大学实验室动物管理小组所规定的指导方针进行。护理 (亚太), (议定书号 #10266) 和在美国, 或《欧洲动物福利法》, 指令 2010/63/欧盟。该议定书获得了德国巴伐利亚政府动物实验伦理学委员会的批准, 并获得了许可号: 55.2-1-54-2532-150-2015。
1. 动物制剂
<ol><li>在加热垫上准备一个有适当床上用品的空笼子。</li>
	<li>在用手套抚摸动物之前, 把母亲的被褥揉到手套上。</li>
	<li>把所有幼崽从母亲身上移开, 放入空笼子里。删除一些母亲的床上用品, 并把它放在空笼与幼崽。</li>
	<li>把母亲放在一个独立的、干净的、干燥的笼子里, 远离手术场。</li>
	<li>新的, 干净的无菌手套在外科领域准备之前。</li>
</ol>2. 手术现场准备
<ol><li>使用10毫升吸管, 添加10毫升磷酸盐缓冲盐水 (PBS) 到10厘米培养皿。</li>
	<li>吸管2毫升的 betadine 或等效的抗脓毒溶液在10厘米培养皿。</li>
	<li>在手术领域放置解剖范围。打开解剖范围灯, 并调整光的水平, 以外科医生的舒适。小狗可以放置在解剖范围灯下面的手术领域。足够的光线可以通过小狗腹面上没有阴影来证实。</li>
	<li>用鼻锥准备异氟醚麻醉室 (参见材料表)。该分庭应在没有尿液或粪便证据的情况下进行清洗。将鼻锥及相关导管置于手术领域的解剖范围内。将氧气和异氟醚的流动完全转移到鼻锥上。</li>
	<li>准备手术后恢复区与加热垫设置在大约37°c。手术后的恢复应包括4片纱布放在加热垫上。如果可能的话, 擦纱布垫在母亲的床上用品, 粪便, 和尿液, 以保存母亲的气味。 注意: 使用热灯是不明智的, 因为这使得控制温度变得困难。高温会导致新生鼠死亡。</li>
	<li>用高压釜准备和消毒所有外科器械。所需的工具包括: 显微解剖剪刀, 显微解剖钳, 纱布, 止血, 和任何6-0 单丝不可吸收缝合。手术器械可以 resterilzed 任何玻璃珠消毒器。</li>
</ol>3. 部分小叶切除术
<ol><li>麻醉把小狗放在鼻子锥上, 然后轻轻地把脚和手贴在原处。小狗应该在氧气中接收5% 异氟醚。<ol>
<li>允许幼犬坐5分钟或直到充分麻醉, 这可以通过脚趾捏测试验证。</li>
		<li>在切口前注射5毫克/千克卡洛芬。 注: 整个手术应不超过30分钟. 在全身麻醉下的新生儿中, 可能会发现较贫穷的结局30分钟以上。采取预防措施, 最大限度地减少手术的长度, 通过彻底的现场准备和润湿皮肤之前关闭, 以尽量减少缝合引起的皮肤撕裂。</li>
	</ol>
</li>
	<li>用 betadine 湿的小纱布轻轻擦拭后腹壁。允许 betadine 干燥1分钟。</li>
	<li>用微解剖剪刀和镊子将左中锁骨0.5 厘米切口立即放在肋骨笼下面。用镊子轻轻地将皮肤分开, 然后将第二个更深的切口插入腹膜腔 (参见图 1C, 左和中心)</li>
	<li>轻轻地施加腹部两侧的侧压, 使用背部, 钝端的微解剖剪刀和镊子, 以迫使左叶顶端的腹膜腔。左叶的左尖应该很容易被可视化 (图 1C, 右)。</li>
	<li>
从先端, 截肢, 并权衡要去除的组织数量。
	<ol>
<li>使用微解剖剪刀, 从左叶的顶端轻轻地切除所需的组织数量。</li>
		<li>将被截去的组织放进一个1.5 毫升的试管中, 用 PBS 填充。使用分析天平对被截断的组织进行称量 (请参阅材料表)。 注: 在天平上放一块纱布或纸, 并将其包装。然后将被截断的组织放在纱布或纸上, 并测量其质量。</li>
		<li>将2% 多聚甲醛的截面积固定在室温下1小时, 并将最佳切削温度 (OCT) 在干冰中放置, 用于冷冻切片分析。通过使用任何标准恒温器切割7-10 µm 切片来分析冰冻切片。</li>
	</ol>
</li>
	<li>使用卷纱布, 轻轻地将左叶替换成腹腔。把纱布留在洞里直到出血停止。</li>
	<li>取出纱布, 并用 PBS 浸泡的纱布将手术部位和周围区域弄湿。</li>
	<li>关闭手术部位与6-0 单丝, 不可吸收缝线与运行针。腹膜和皮肤应分别闭合。 注: 用 PBS 浸泡纱布轻轻润湿皮肤, 可减少缝合引起的泪。</li>
	<li>轻轻地, 但彻底清洁小狗用纱布浸泡与 PBS, 并确保没有血液或 betadine 遗骸。卷上浸泡的纱布垫, 轻轻擦洗伤口标志, 清除任何血液或 betadine。 注意: 这是特别重要的, 因为母亲可能蚕食幼崽, 如果他们被清理得不充分。</li>
	<li>从鼻锥中取出幼犬, 将其放在恢复区, 包括暴露在母亲粪便和尿液中的纱布垫。一旦幼崽恢复, 用母亲的被褥替换空笼子里的小狗。 注: 请勿使用散热灯。使用热灯会导致新生儿过热。</li>
	<li>对所需的幼崽数重复该过程。虽然所有的幼崽都可以使用 , 但一般是可取的 , 让一些未操作的幼崽与作的幼崽一起替换。</li>
	<li>在母亲的笼子里同时用母亲的被褥替换所有幼崽。</li>
</ol>4. 恢复和分析
<ol><li>
每天跟踪老鼠。
	<ol>
<li>检查伤口是否保持闭合, 并清除任何死幼崽, 如果存在。</li>
		<li>如果伤口重新打开, 准备手术部位和恢复区如前所述, 并重复步骤3.8 至3.12。</li>
		<li>注射5毫克/千克的 carpofen 皮下24和48小时后的程序。</li>
		<li>跟踪幼崽56天或更多。</li>
	</ol>
</li>
	<li>通过二氧化碳 (CO2) 暴露和宫颈脱位, 弄死动物在手术后所需的天数。<ol>
<li>在诱导/安乐死室放置老鼠, 并打开 CO2直到老鼠停止呼吸。</li>
		<li>通过颈椎脱位确保安乐死。使用前指和拇指向下推鼠标的背颈, 然后用另一只手向下拉下尾部。</li>
	</ol>
</li>
	<li>
删除整个肝脏en 集团和权衡它。
	<ol>
<li>仔细地分开每个裂片并且单独称重。 注: 仔细解剖膈肌和肝门血管, 取出肝脏。通过仔细解剖每一个裂片在其近端的附件, 分离出不同的叶。</li>
	</ol>
</li>
	<li>通过将被截离的左叶质量与整个肝脏的质量进行比较, 确定再生的程度。没有受伤的左叶大约是整个肝脏的30%。</li>
</ol>

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作者没有披露。

急性肝损伤传统上是使用化学 (扑热息痛, 乙醇, 四氯化碳), 或外科模型 (70% 部分肝切除)。70% 部分肝切除后再生反应的特点是涉及全球肝细胞肥大和多轮细胞分裂4,5。然而, 为了停止出血, 这个模型是有限的, 因为主要的血管必须结扎离开一个异常的环境, 以再生。因此, 许多研究都采用了其他不那么侵入性的急性损伤模型, 通过化学损伤, 使总的结构发生?...

再生器官的能力, 或恢复形式和功能, 一直被认为是在进化时期失去了大部分。在急性化学或物理损伤后, 成年哺乳动物肝脏的再生潜能涉及动员所有剩余的肝细胞, 导致肥大和几轮细胞分裂, 导致功能性但建筑上不同的器官1,2,3,4,5。最近, 研究已经开始描述新生儿哺乳动物器官在生命的第一周内对损伤的再生反应6,7,8。这些研究表明, 当在新生儿发育期间受伤时, 某些哺乳动物器官以形态学再生代替补偿生长或纤维化7,8。
最近的研究表明, 全球结构和功能的再生发生在早期新生儿期6,7,8。建立的肝损伤协议涉及化学伤害或酒精的管理9,10,11, 扑热息痛12,13,14,15, 四氯化碳16,17,18,19, 70% 部分肝切除4,20,21, 或删除左侧和中位裂片。化学管理导致肝细胞死亡, 但经常留下微和宏观结构完好。由于整体的肝脏结构没有被抹杀, 所以在这种背景下不能轻易地研究形态学再生。70% 部分肝切除术包括缝合结扎的主要血管, 这是必要的止血, 但留下一个非生理环境与永久性中断的血管。此外, 该方法仅用于成年啮齿动物, 其在新生儿中的应用在技术上极其困难。考虑到这一点, 我们开发了一种方法, 其中20-30% 的左叶顶点被移除在新生的 P0 鼠标 (图 1A-1B) 中。该方法手术保守, 微创, 不具技术挑战性, 导致形态学毛损, 不结扎血管, 留出再生空间。由此得出的分步协议, 允许任何研究员对新生小鼠进行部分小叶切除, 以便研究哺乳动物在产后早期的新生。这种方法在再生医学和干细胞生物学的比较研究中也有明显的应用, 因为它可以在生命后期的肝脏中使用。
最常见的急性肝损伤研究是化学性损伤, 成人肝脏截肢, 或70% 部分肝切除。化学损伤通常包括静脉注射、腹腔或口服乙酰氨基酚、四氯化碳或乙醇, 是一种相对容易和无创性的损伤模型。如前所述, 化学损伤导致肝细胞死亡, 但往往使基质和软组织结构完好, 使人们难以对形态学再生提出要求。化学损伤往往集中在肝脏血管, 使其成为一个有用的技术来研究现场和细胞特定的伤害, 但也使它难以在整个器官层面上审问其他人群, 可能是进一步从船只, 并可能有助于再生。尽管有这些限制, 化学损伤仍然是一个有用和高度生理相关的伤害模型。
成人70% 部分肝切除术包括切除肝血管结扎后的左侧和正中裂片。对肝切除的反应有很好的特点: 截肢后的肝14天后70% 部分肝切除与原始未受损的小叶形成严重不同的结构, 作为剩余右、尾状裂片的肝细胞经历肥厚和几轮细胞分裂4,5。这弥补了丢失的质量和功能, 但未能再生两个截切的裂片, 因此不取代毛形态。因此, 对70% 例部分肝切除术的损伤反应有助于研究有限再生的代偿生长机制。
在这里, 我们充分描述了一个新生儿部分小叶切除的协议。该程序涉及适当的动物选择和准备, 外科领域的准备, 手术和恢复。对议定书的不同应用可能需要对这些步骤进行优化和调整。
我们对野生型 C57BL/6J 幼崽 (jax-rpc 000664) 的这一协议进行了广泛的执行和优化, 但是, 为了研究不同的细胞种群和再生机制, 我们还使用了各种转基因动物, 包括饲养各种品种的小鼠和CreERT2 转基因和/或敲门 (Axin2CreERT2 jax-rpc 018867,和Sox9CreERT2 jax-rpc 018829) 与荧光记者, 如彩虹和 mTmG 系统 (R26VT2/GK3,R26mT/mG)22,23. 我们发现没有必要改变这种方法的不同的老鼠菌株, 因为没有差异的生存结局或再生潜能被观察到。
除了使用不同的动物菌株, 我们还对小分子治疗的新生小鼠进行了部分小叶 hepatectomies, 如 4-羟基三苯氧胺和 5-乙炔--2′-脱氧尿苷 (教育局)。以二甲基亚砜和乙醇为溶剂, 发现玉米油是引起发病率的重要原因。否则, 我们发现, 腹腔内对小分子的管理不影响生存或再生结果。我们预测, 这个协议将被修改, 以用于其他小分子, 以审问的各个方面的再生。
新生小鼠手术在技术上是有挑战性的, 可能需要专门的动物处理和显微解剖专家。畜牧业专门知识是必要的, 以避免在手术后和在立即恢复期间的孕妇同类相食。

<table><tbody><tr><td>&lt;strong&gt;Animals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mother with litter of day 0 neonatal pups (any strain)</td><td></td><td></td><td></td></tr><tr><td>Surrogate mother and surrogate litter (optional)</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Standard Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Phosphate Buffered Serum (PBS)</td><td></td><td></td><td></td></tr><tr><td>Providine-iodine or equivalent antiseptic solution</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Surgical Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dissecting microscope</td><td>Zeiss</td><td>ZEMSDV4L MFR # 435421-9901-000</td><td></td></tr><tr><td>3mm straight spring micro scissors</td><td>Vannas</td><td>72932-01</td><td></td></tr><tr><td>5SF Forceps</td><td>Dumont</td><td>11252-00</td><td></td></tr><tr><td>Straight Kelly forceps</td><td>Grainger</td><td>17-050G</td><td></td></tr><tr><td>Heating pad</td><td>Sunbeam</td><td>000771-810-000</td><td></td></tr><tr><td>Isoflurane</td><td>Abbott Labs</td><td>0044-5260-05</td><td></td></tr><tr><td>Rodent Anesthesia System</td><td>Kent Scientific</td><td>1205S</td><td></td></tr><tr><td>Gauze, 10.16 x 10.16cm</td><td>Fisher Scientific</td><td>13-761-52</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Standard Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1.5ml microcentrifuge tube</td><td>Eppendorf</td><td>22363204</td><td></td></tr><tr><td>6-0 monocryl sutures</td><td>Ethicon</td><td>MCP489G</td><td></td></tr><tr><td>Petri dish</td><td>Fisher Scientific</td><td>S35839</td><td></td></tr><tr><td>Pipet-Aid, Plain, 110V</td><td>Drummond</td><td>4-000-110</td><td></td></tr><tr><td>Mettler Toledo NewClassic ME Analytical Balances</td><td>Fisher Scientific</td><td>01-912-402</td><td></td></tr><tr><td>Low Cost Induction Chamber</td><td>Kent Scientific</td><td>SOMNO-0730</td><td></td></tr></tbody></table>

partial lobular hepatectomy: a surgical model for morphologic liver regeneration

急性组织损伤后的形态学器官再生在下脊椎动物中很常见, 但在哺乳类动物产后生活中很少观察到。70% 部分肝切除后成人肝再生导致肝细胞肥大, 其余裂片复制, 代谢活性恢复, 但损伤叶的形态学和结构永久性丧失。在这里, 我们详细介绍了新生儿的一种新的外科方法, 它留下了一个有利于再生的生理环境。该模型涉及截肢的左叶顶端和随后的保守管理方案, 并缺乏必要的结扎的主要肝血管或化学伤, 留下一个生理环境, 再生可能发生。我们将这项议定书扩展到对幼年 (P7-14) 小鼠的截肢, 在此期间, 受伤的肝脏从器官再生过渡到代偿性生长。提出的, 简短的30分钟协议提供了一个框架, 以研究再生机制, 其年龄相关的下降, 哺乳动物, 和鉴定的肝茎或祖细胞。

图 1A详细介绍新生儿部分小叶切除术的一般时间线 (图 1B中的示意图), 以及预期等待的时间长度, 直到观察到再生。术后7-14 天可以观察到左叶的细微再生。术后56天, 常观察到全再生。小鼠在手术后不应显示生理异常的迹象。
接受部分小叶 hepatectomies 的小鼠可恢复2、7、14、35和56天?...

Watch this Scientific Journal Video about Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration at JoVE.com

Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration

部分小叶切除术: 形态学肝再生的外科模型

本文提出了新生儿 (0 天) 小鼠左肝叶部分切除的新方法。这一新的协议适用于研究急性肝损伤和损伤反应在新生儿的设置。

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult ...

partial-lobular-hepatectomy-surgical-model-for-morphologic-liver

Research

JoVE Journal

Developmental Biology

12.1K 次观看.  Stanford University School of Medicine. 本文提出了新生儿 (0 天) 小鼠左肝叶部分切除的新方法。这一新的协议适用于研究急性肝损伤和损伤反应在新生儿的设置。

视频: Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration

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 ...

科研

医学

<meta charset="utf8"></head><body>用于肝再生研究的患者来源肝癌细胞类器官

A Hepatocellular Cancer Patient&#45;Derived Organoid Xenograft Model to Investigate Impact of Liver Regeneration on Tumor Growth

Recurrence poses a notable challenge after hepatocellular carcinoma (HCC) treatment, impacting more than 70% of patients who undergo surgical resection. Recurrence stems from undetected micro-metastasis or de novo cancer, potentially triggered by postsurgical liver regeneration. Prior research employed HCC cell lines in orthotopic models to study the impact of liver regeneration, but their limited validity prompted the need for a more representative model. Here, we introduce a novel approach utilizing patient-derived HCC organoids to investigate the influence of liver regeneration on HCC.
Patient tumor tissues are processed to create tumor organoids, embedded in a three-dimensional basement membrane matrix, and cultured in a liver-specific medium. One million organoids are injected into the right superior lobe (RSL) of immunodeficient mice, confirming macroscopic tumor growth through sonography. The intervention group undergoes resection of the left lateral lobe (LLL) (30% of total liver volume) or additionally, the middle lobe (ML) (65% of total liver volume) to induce liver regeneration within the tumor site. The control group experiences re-laparotomy without liver tissue resection. After 2 weeks, both groups undergo tumor and normal tissue explantation.
In conclusion, this patient-derived HCC organoid model offers a robust platform to investigate the impact of liver regeneration post-cancer resection. Its multi-cellular composition, genetic diversity, and prolonged culture capabilities make it an invaluable tool for studying HCC recurrence mechanisms and potential interventions.

<meta charset="utf8"></head><body>肝细胞癌患者来源的类器官异种移植模型，用于研究肝脏再生对肿瘤生长的影响

Organoids generated from patient tumors are orthotopically injected into the mouse liver. The resection of non-tumorous liver tissue leads to a regenerative environment in the liver tissue where the tumor is located.

肝细胞癌患者来源的类器官异种移植模型，用于研究肝脏再生对肿瘤生长的影响

Recurrence poses a notable challenge after hepatocellular carcinoma (HCC) treatment, impacting more than 70% of patients who undergo surgical resection. ...

癌症研究

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

大鼠体内部分肝工程的外科技术基础

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

生物工程

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 ...

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

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

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

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

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. ...

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream single-cell applications is challenging. A marked accumulation of lipids within regenerating hepatocytes is observed during the first 2 days of normal liver regeneration in mice. This so-called transient regeneration-associated steatosis (TRAS) is temporary but partially overlaps the major proliferative phase. Density-gradient purification is the backbone of most existing protocols for the isolation of primary hepatocytes. As gradient purification relies on the density and size of cells, it separates non-steatotic from steatotic hepatocyte populations. Therefore, fatty hepatocytes often are lost, yielding non-representative hepatocyte fractions. 
The presented protocol describes an easy and reliable method for the in vivo isolation of regenerating hepatocytes regardless of their lipid content. Hepatocytes from male C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the remnant, using a retrograde perfusion technique with outflow through the portal vein. Hepatocytes are dissociated by collagenase for their release from the Glisson's capsule. After washing and careful centrifugation, the hepatocytes can be used for any downstream analyses. In conclusion, this paper describes a straightforward and reproducible technique for the isolation of a representative population of regenerating hepatocytes after partial hepatectomy in mice. The method may also aid the study of fatty liver disease.

Lipid-laden hepatocytes are inherent to liver regeneration but are usually lost upon density-gradient centrifugation. Here, we present an optimized cell isolation protocol that retains steatotic hepatocytes, yielding representative populations of regenerating hepatocytes after partial hepatectomy in mice.

小鼠肝部分切除术后再生肝细胞的分离

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream ...

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