Name: visualization of vascular and parenchymal regeneration after 70% partial hepatectomy in normal mice
Uploaded: 2016-09-13T13:00:00.000+00:00
Duration: 11 min 10 s
Description: Watch this Scientific Journal Video about Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice at JoVE.com

The authors acknowledge funding by the German Ministry of Education and Research (BMBF) via the systems biology network "Virtual Liver", grant numbers 0315743 (ExMI), 0315765 (UK Jena), 0315769 (MEVIS).The authors also thank Frank Schubert for technical support.

涉及受试动物的程序已通过图林根Landesamt献给Verbraucherschutz营连Tiergesundheit UND Tierschutz，德国。由于门静脉系统是由肝静脉系统分开可视化，还需要对不同的血管树单独的动物。 1.试剂准备<ol><li>肝素盐溶液<ol><li>加入0.1 ml肝素到10ml盐水（5单位/毫升）。 </li></ol></li><li>有机硅化合物的混合物<ol><li>加2ml MV-120在1个5毫升的试管。通过添加造成的40％溶液3毫升MV稀释剂稀释的MV-120。 </li></ol></li></ol> 2.门静脉系统硅胶射出<ol><li>剖腹<ol><li>鼠标放置在麻醉诱导室，用2％异氟烷和0.3升/分钟的氧气麻醉它。 </li><li>修正了在使用磁带连续吸入手术台麻醉小鼠的2％oflurane和0.3升/分钟的氧气。检查鼠标的脚趾捏撤回反射，如果反射是没有开始运作。 </li><li>就用剪刀对皮肤层和电凝的肌肉层的腹部的横切口。搬出用棉提示肠内到左侧和盖用盐水浸泡的纱布肠子。 </li></ol></li><li>导管插入<ol><li>解剖采用微型镊子在显微镜下门静脉。放置一个6-0丝线缝合肝外门静脉下方，在其分叉约为1毫米的距离和松散的领带以备后用。 </li><li>注入用于全身肝素化通过阴茎静脉（男性）制备的肝素盐溶液或下腔静脉（女）5分钟。 </li><li>将26号（26 G）与导管针进入门静脉，并用夹子固定它</li></ol></li><li>肝素盐溶液和有机硅化合物灌注<ol><li>负载肝素盐水溶胶ution在5ml注射器中，并打开灌注设备。与肝素盐水完全填充导管，以避免气泡。 </li><li>延长管连接到导管，紧紧固定。为0.4毫升/分钟的灌注速率开始肝素盐水灌注。 </li><li>结扎预置6-0丝线缝合双固定导管和脾静脉和肠系膜静脉阻断血流。通过灌注Euthanatize放血鼠标麻醉下。 </li><li>冲洗整个灌注过程期间使用生理盐水肝脏以保持湿润。 </li><li>添加0.1毫升固化剂成的MV-120管。更改肝素盐水的注射器来注射硅胶。 </li><li>为0.2毫升/分钟的灌注速率为约1分钟到预填导管系统和填满门静脉系统启动硅氧烷灌注。停止硅氧烷灌注时的表面上的船只变为蓝色。 </li></ol></li><li>采样<ol><li>保持原位 UNT肝IL硅氧烷完全后大约15至30分钟聚合。解剖连接肝脏和邻近器官小心保持完好肝韧带。植肝，放入福尔马林固定。 </li></ol></li></ol> 3.肝静脉系统硅胶射出<ol><li>按照步骤执行2.1剖腹探查执行并充分暴露手术视野。 </li><li>导管插入<ol><li>解剖采用微型镊子在显微镜下门静脉。放置一个6-0丝线缝合肝外门静脉下方，在其分叉约为1毫米的距离和松散的领带以备后用。 </li><li>注入用于全身肝素化通过阴茎静脉（男性）制备的肝素盐溶液或下腔静脉（女）5分钟。 </li><li>插入一个＆G导管（导管1）用针进入门静脉，并用夹子固定。插入另一＆G导管（导管2）针刺入下腔静脉，并用夹子固定它。 </li><li>结扎下腔静脉（包括左和右的肾静脉）和其远端的使用6-0合成的，单丝，非吸收性聚丙烯缝线的分支。 </li></ol></li><li>肝素盐溶液和有机硅化合物灌注<ol><li>加载肝素盐溶液的5ml注射器中，并打开灌注设备。与肝素盐水填充导管1完全避免气泡产生。延长管连接到导管1，紧紧地修复它。以0.4毫升/分钟的速率开始肝素盐水灌注。 </li><li>结扎预置6-0丝线缝合双固定导管和脾静脉和肠系膜静脉阻断血流。通过灌注Euthanatize放血鼠标麻醉下。 </li><li>冲洗整个灌注过程期间使用生理盐水肝脏以保持湿润。添加0.1毫升固化剂成的MV-120管。交易所肝素盐水的注射器针筒硅胶。 </li><li>将一个夹在suprahepatIC下腔静脉，阻碍肝脏的流出。 </li><li>连接延伸管到导管2，并开始硅氧烷灌注0.2毫升/分钟的灌注速率为约2分钟以达到一个目标肝血管体积为报道。停止硅氧烷灌注时的表面上的船只变为蓝色。 </li></ol></li><li>采样<ol><li>解剖肝韧带避免任何损害肝脏。植肝，放入福尔马林固定。 </li></ol></li></ol> 4.微CT（μCT）扫描使用μCT扫描植肝脏样品，需要下列步骤。 <ol><li>取肝脏样品从固定的解决方案。将肝到μCT床。把μCT床与肝样品放入μCT。 </li><li>在开始扫描之前获得topogram。用于小肝癌实验一子扫描和两个副扫描的​​大样本。 </li><li>选择一个81; CT协议具有高分辨率（ 例如 ，HQD-6565-390-90）。该协议每副扫描90秒的扫描时间一整圈时获得720预测与1032点¯x1012像素。启动μCT扫描。 </li></ol> 5.组织学连续切片<ol><li>嵌入石蜡的肝标本整体μCT扫描之后。切整个石蜡样品放入4μm的切片，从而产生一系列的2000到2500的部分。 </li><li>用适当的染色技术染色切片形象化感兴趣分子事件如Ki-67的作为增殖标志物和HMGB1的局部缺血性损伤的标记物。确定在尊重科学问题染色序列。 </li><li>使用整个幻灯片扫描仪的数字化染色切片。 </li><li>执行血管树（多个）（已经可行）的3D重建和可视化在相对于血管树（研究正在进行中）的分子事件的三维分布。 </li></ol>

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The authors declare that they have no competing financial interests.

通过对比硅胶注射和μCT扫描血管树已在肿瘤模型和神经系统疾病模型中引入经常研究进展血管5,7,8,10。在硅氧烷喷射的方法的改进，在本研究中提出的可视化和小鼠肝部分切除后定量血管生长。 有许多的关键步骤注意以达到良好的灌注质量。首先，全身肝素高度用肝素或盐水冲洗肝脏，以避免肝脏内凝血前推荐的。从管路气泡消除也是重要的实现硅氧烷灌注的优良品质。灌注速率和压力的确定应首先基于生理血液动力学参数17。灌注时间反映总的注射量，估计走的是预期的血管内体积和导管系统的预填量考虑在内。 硅氧烷是从血液或盐水的不同的高粘性化合物。因此，需要用于调节灌注速率和压力的几个试验。保持恒定的喷射流速和压力是必要的，以防止严重的扩张或甚小血管的破裂。灌注时间根据所估计的所需注入量设定。然而，灌注应该立即停止时的蓝色化合物变为在肝脏的表面上的船只可见。否则，硅能排入血窦和成另一种血管系统将与后重建和干扰分析。 典型地，硅氧烷是通过在最发表的报告1,3,15左心室系统灌注。左心室很容易暴露，使注射部位自由出入。然而，缺点在于，此路线是岭疗法间接因为​​造影剂具有到达目标部位之前进行循环。因此，硅氧烷注射技术的外科手术已经在本研究中进行修改。在对比由他人进行的应用程序的频繁选择间接路线，硅氧烷化合物直接注射到感兴趣血管系统，代替对比全身。以这种方式，灌注的速度和体积可更好地控制。门静脉系统和肝静脉系统可灌注和购买个别分析分别重构，如图视频。 这个修改​​过程的限制是技术难度。它是具有挑战性的成功执行使用在小鼠中高粘性化合物的静脉内注射，因为血管结构是如此精致。这是相当容易的过程期间破坏血管。因此，门静脉解剖和导管樱雪rtion应轻轻地进行。 所述外植肝脏的血管树和随后的μCT成像的对比是用于可视化和部分切除术后量化血管再生的有用工具。定量血管参数可以被用于更好地理解血管生长的肝再生的进展动力学的。此外，基于连续切片都肝血管系统的三维重建在技术上是可行的，虽然代表一项繁重的工作负荷。 这种技术可以在多个模型被应用，其中可视化和血管生长的定量是重要的。此外，在相对于下层的血管树的分子事件的三维可视化是在覆盖面。感兴趣的分子事件的例子可以是增殖标志物的检测，例如Ki-67的或缺血性损伤的标记物，如HMGB1。评估空间分辨分子E​​V经济需求测试在再生肝实质内再生血管树的附近为先进的多尺度系统生物学建模的前提。这种硅胶注射技术是朝着实现此目标的一种试验性的一步。

肝再生通常通过测量肝脏重量和体积的增加和通过评估肝细胞增殖率16确定。然而，肝再生不仅诱导再生实质，而且血管再生6。因此，血管生长应进一步相对于调查其在肝再生的进展的作用。肝血管系统的可视化是推进我们的血管再生的理解是至关重要的。许多间接方法已被开发，研究肝血管再生的分子机制。传统上，检测的细胞因子（血管内皮生长因子，血管内皮生长因子）14，趋化因子和它们的受体（CXCR4 / CXCR7 / CXCL12）4已经用于研究血管再生的支柱。然而，共同的3D模型与血管的定量分析将增加关键解剖信息更好地了解肝实质和血管再生的重要关系。 为了显现肝血管系统，这需要对比血管树，小鼠用不透射线的聚硅氧烷橡胶的造影剂直接注射到门户或肝静脉血管树。硅氧烷和器官的外植的聚合后，将肝脏样品用CT扫描仪进行μCT扫描。该扫描导致体素​​图像表示 硅氧烷喷射的试样9。 在质量控制方面，对血管系统最早是在使用3D软件的临床前可视化。分割通过设定所述软组织的强度和容器强度之间的阈值执行的。由此产生的容器面膜使用面绘制可视化。该软件还允许手工测定vascul两个参数AR增长:最大船舶长度和半径。 那么临床前软件进行三维重建血管树木和供给或引流血管分布区13后续计量。此外，该软件自动确定血管生长的某些参数，所有可见的血管结构也被称为总边缘长度或总容器体积的总长度，如。 在幼稚小鼠和在该行的70％部分肝切除（PH）的小鼠进行硅酮灌注过程。肝脏在不同的观察时间点切除使用上述可视化和定量技术分析血管和实质肝再生后收集。 该薄膜的主要目标是:（1）表明，以达到最佳反衬和（2）示出的潜在利益所得FR所需的微妙的注射技术嗡使用μCT和组织学连续切片所得样品的详细分析。看完这部片子后，读者应该有一个更好的了解如何注入聚硅氧烷化合物为特定的血管系统和技术的实用性和适用性。

<table><tbody><tr><td>PERFUSOR&amp;reg; VI</td><td>B.BRAUN</td><td>87 222/0</td><td></td></tr><tr><td>Pipetus&amp;reg;-akku</td><td>Hirschmann</td><td>9907200</td><td></td></tr><tr><td>Pipets</td><td>Greiner</td><td>606180</td><td></td></tr><tr><td>micro scissors</td><td>Fine Science Tools (F&amp;middot;S&amp;middot;L)</td><td>No. 14058-09</td><td></td></tr><tr><td>micro serrefine</td><td>Fine Science Tools (F&amp;middot;S&amp;middot;L)</td><td>No.18055-05</td><td></td></tr><tr><td>Micro clamps applicator</td><td>Fine Science Tools (F&amp;middot;S&amp;middot;L)</td><td>No. 18057-14</td><td></td></tr><tr><td>Straight micro forceps</td><td>Fine Science Tools (F&amp;middot;S&amp;middot;L)</td><td>No. 00632-11</td><td></td></tr><tr><td>Curved micro forceps</td><td>Fine Science Tools (F&amp;middot;S&amp;middot;L)</td><td>No. 00649-11</td><td></td></tr><tr><td>needle-holder</td><td>Fine Science Tools (F&amp;middot;S&amp;middot;L)</td><td>No. 12061-01</td><td></td></tr><tr><td>1 ml syringe</td><td>B.Braun</td><td>9161406V</td><td></td></tr><tr><td>5 ml syringe</td><td>B.Braun</td><td>4606051V</td><td></td></tr><tr><td>extension and connection lines</td><td>B.Braun</td><td>4256000</td><td>30 cm, inner &amp;oslash; 1.2 mm</td></tr><tr><td>6-0 silk (Perma-Hand Seide)</td><td>Ethicon</td><td>639H</td><td></td></tr><tr><td>6-0 prolene</td><td>Ethicon</td><td>8711H</td><td></td></tr><tr><td>Microfil&amp;reg; MV diluent</td><td>FLOW TECH, INC</td><td></td><td></td></tr><tr><td>Microfil&amp;reg; MV - 120</td><td>FLOW TECH, INC</td><td>MV - 120 (blue)</td><td></td></tr><tr><td>MV curing agent</td><td>FLOW TECH, INC</td><td></td><td></td></tr><tr><td>Heparin 2500 I.E./5 ml</td><td>Rotexmedica</td><td>ETI3L318-15</td><td></td></tr><tr><td>Saline</td><td>Fresenius Kabi Deutschland GmbH</td><td>E15117/D DE</td><td></td></tr><tr><td>Imalytics Preclinical software</td><td>Experimental Molecular Imaging, RWTH Aachen University, Germany</td><td></td><td></td></tr><tr><td>HepaVision</td><td>Fraunhofer MEVIS, Bremen, Germany</td><td></td><td></td></tr><tr><td>NanoZoomer 2.0-HT&amp;nbsp;Digital slide scanner</td><td>Hamamatsu Electronic Press, Japan&amp;nbsp;</td><td>C9600</td><td></td></tr><tr><td>Tomoscope Duo CT&amp;nbsp;</td><td>CT Imaging GmbH, Erlangen, Germany</td><td>TomoScope&amp;reg;&amp;nbsp;Synergy</td><td></td></tr></tbody></table>

visualization of vascular and parenchymal regeneration after 70% partial hepatectomy in normal mice

一种改进硅氧烷注射步骤，用于肝血管树的可视化。此过程包括在体内注射硅氧烷化合物组成，经由＆G导管，进入门或肝静脉。硅胶注射后，器官植及离体微CT（μCT）扫描准备。硅胶注射过程在技术上具有挑战性。取得成功的，需要从医生丰富的经验显微。之一的这个过程的挑战涉及确定为聚硅氧烷化合物的适当的灌注速率。需要基于感兴趣血管系统的血流动力学被定义为聚硅氧烷化合物的灌注速率。不适当的灌注率可导致一个不完整的灌注，人工扩张和破裂的血管树。 血管系统的三维重建是基于CT扫描和使用达到临床前的软件如HepaVision。重构的血管树的质量直接与硅氧烷灌注的质量。表示血管生长的随后计算血管的参数，如总血管体积，基于所述血管重建计算。对比用硅酮血管树允许试样μCT扫描后后续的组织学后处理。试样可以进行连续切片，组织学分析和整个幻灯片扫描，并在其后的三维重建基于组织学图像中的血管树。这是用于检测分子事件和它们相对于分配给所述血管树的前提。此改性硅氧烷注入步骤也可以用于可视化和重建其它器官的血管系统。该技术具有的潜力可广泛应用于研究关于在各种动物血管解剖学和生长第二疾病模型。

质量标准 硅酮注射液的质量可以与过程中的肉眼进行判断。在肝表面的小血管的蓝色复合填写逐渐。如果对肝脏表面观察正常的血管结构，所述硅橡胶注入质量是好的。如果灌注量不足，对肝脏表面小血管没有完全填补。与此相反，在填充引起血管破裂的器官的表面上不规则蓝色斑点所示。两者都导致在3D重建，最终呈现的过程的失败的困难。 对于注射剂质量较好的评价，进行μCT扫描，随后使用临床前软件三维重建血管。注射被确定为成功WH恩分割产生了一个完整的完整的血管树的不可见光外渗表明破裂结构的可视化。如果血管树出现不完整（ 图1A），灌注体积是不够的。如果一个以上的血管树出现或外渗被看见（ 图1B），灌注体积或压力不足。这是一个失败的最可能的原因。灌注压力理论上可以取决于溶液的粘度略有不同。粘度取决于化合物和注入混合到血管系统之间经过聚合时间。由于一些操作所需的混合和注射，时间间隔可以在3分钟之间稍有变化，以5分钟。 如果溶液是低粘度的，灌注压力是低的。在这种情况下，化合物可以通过正弦系统转移到非注入血管树导致稍高的灌注体积。如果溶液是进一步聚合，导致更高的粘度，灌注压将上升，从而引起血管和外渗的破坏。 目标灌注量的测定 由于灌注或灌注不足会导致注射失败，规范灌注量进行了审议。据报道2，肝体积的6％是由血液占据，在肝脏中的血液44％驻留在大血管（ 例如 ，肝动脉，门静脉）。在小鼠肝脏体积约为1.3毫升11。因此，在正常小鼠门静脉系统总血管体积估计0.03毫升和0.04 ml的到的范围内。流动速率为0.2毫升/分钟，总灌注时间设定为导致总注射体积约1分钟的约0.2毫升需要这种看似高容量，因为导管系统必须预先填充，大约需要0.1〜0.15毫升。需要0.05毫升额外体积以填充肝门和导管的结扎之间近端门静脉。流速为肝静脉注射也为0.2毫升/分钟，但总灌注时间延长至2分钟，产生约0.4毫升较高的总体积。肝内总血管体积假定为类似于总门静脉血管体积。然而，体积用于填充肝下结扎和肝上钳用0.2ml估计之间的肝内腔静脉。 成功率 共有49只动物接受硅胶注射:22动物进入光伏系统和27个动物进入日ËHV系统。我们的注射的成功率在PV组中55％（12/22）和HV组中89％（24/27）。考虑到对PV组用于建立在开始硅氧烷注射技术（N = 4），注射的用于光伏系统的成功率应有效地高于55％。然而，从这些注射获得的μCT图象是适于三维重建和定量分析。此外，无论从PV或36只小鼠的高压血管树可以与IMALYTICS临床前软件进行重构。 脑实质和血管再生 一时间分辨μCT一系列小鼠肝脏样本的切除物进行定性分析后。实质再生包括残肝3D的增长。血管再生被定义为在所显示的增加的组织长度和血管茎的直径，其主要的树枝和附加末端分支的生长，同时在门静脉和肝静脉树。 目前，适合于描述血管生长和肝实质再生和血管再生关系中的几个定量参数正在接受调查。这些包括指示血管生长的参数，包括最大血管长度（图2），血管半径和血管密度在总血管长度/肝脏体积或血管体积/肝脏体积而言。 总肝脏体积和选定的肝叶的体积计算。在正常肝，肝脏总体积介于1.2毫升到1.6毫升（N = 6，包括光伏组和高压基）。它降低到0.6到0.7毫升的，由除去左侧纬度执行扩展肝切除后ERAL和中叶。肝生长过程中肝脏体积持续增加。由7术后天（POD 7），肝脏体积增加至其原始体积， 即约88％，由2.6倍。肝脏体积增加肝重量回收率相关。 光伏系统和高压系统的总血管体积计算。高压系统的总血管体积较光伏系统高，因为肝内下腔静脉的一部分被列入。光伏系统的总血管体积0.05〜0.08毫升正常小鼠不等。后70％部分肝切除降低到0.03到0.04毫升通过POD 7，残肝的总血管体积增加至原体积的100％。高压的总血管体积范围从0.14至0.16毫升血管体积切除后降低至0.08至0.09毫升在术后第一周，剩余肝的血管总成交量增加bÿ原始值的94％。 作为结果，增加血管体积和实质体积之间的关系，血管密度算出，更精确的血管体积分数（血管体积由肝脏体积除以）。这表明，血管体积分数在整个再生过程中保持相对稳定。 分子活动的三维可视化 后的CT-扫描选定样品进行连续切片，产生高达2000滑动，这是完全数字化，并用于重建门户以及肝静脉树12。这对于再生期间的分子事件的未来的3D可视化中对于生长血管树的前提。 <i毫克ALT ="图1"SRC ="/文件/ ftp_upload / 53935 / 53935fig1.jpg"/&gt; 图1. 监测硅氧烷喷射的质量。右下门静脉的 。不完全填充（左箭头）和尾门静脉（中右箭头）的临床前IMALYTICS软件进行可视化的血管树的不连续性。这个指示的灌注压或灌注体积不足或存在气泡; B。右下门静脉外渗可视化这说明血管的破坏，由于压力或灌注不足。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53935/53935fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53935/53935fig2.jpg" /> 图 2. 右最大船舶长度的测量劣门静脉。为了确定最大血管长度，一个开始点和一个结束点被放置在根目录和在临床前软件血管掩模（蓝色和品红色球标记）的右下门静脉（RIPV）的远端。获得RIPV（10.95毫米）的血管路径长度为评估"路径曲折"的一部分。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53935/53935fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice at JoVE.com

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

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 ...

visualization-vascular-parenchymal-regeneration-after-70-partial

Universidad San Sebastian

Research

JoVE Journal

Medicine

8.0K 次观看.  Jena University Hospital. 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 µCT and histological serial sections.

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

Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult liver regeneration after 70% partial hepatectomy results in hepatocyte hypertrophy with some replication in remaining lobes with restoration of metabolic activity, but with permanent loss of the injured lobe&#39;s morphology and architecture. Here, we detail a new surgical method in the neonate that leaves a physiologic environment conducive to regeneration. This model involves amputation of the left lobe apex and a subsequent conservative management regimen, and lacks the necessity for ligation of major liver vessels or chemical injury, leaving a physiologic environment where regeneration may occur. We extend this protocol to amputations on juvenile (P7-14) mice, during which the injured liver transitions from organ regeneration to compensatory growth by hypertrophy. The presented, brief 30 min protocol provides a framework to study the mechanisms of regeneration, its age-associated decline in mammals, and the characterization of putative hepatic stem or progenitors.

Here, we present a new method for partial resection of the left hepatic lobe in neonatal (day 0) mice. This new protocol is suitable for studying acute liver injury and injury response in the neonatal setting.

部分小叶切除术: 形态学肝再生的外科模型

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

科研

发育生物学

Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable during renal transplantation. Experimental models that accurately and reproducibly recapitulate renal IRI are crucial in dissecting the pathophysiology of AKI and the development of novel therapeutic agents. Presented here is a mouse model of renal IRI that results in reproducible AKI. This is achieved by a midline laparotomy approach for the surgery with one incision allowing both a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia of the left kidney. By careful monitoring of the clamp position and body temperature during the period of ischaemia this model achieves reproducible functional and structural injury. Mice sacrificed 24 hr&#160;following surgery demonstrate loss of renal function with elevation of the serum or plasma creatinine level as well as structural kidney damage with acute tubular necrosis evident. Renal function improves and the acute tissue injury resolves during the course of 7 days following renal IRI such that this model may be used to study renal regeneration. This model of renal IRI has been utilized to study the molecular and cellular pathophysiology of AKI as well as analysis of the subsequent renal regeneration.

The mouse model of renal ischaemia reperfusion injury described here comprises of a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia that results in acute kidney injury. This model uses a midline laparotomy approach with all steps performed via one incision.

肾缺血再灌注损伤:损伤的小鼠模型和再生

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable ...

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

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 ...

小鼠肝移植和同种异体移植物通畅性分析

Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality images to be obtained of micro-vasculature and are high-fidelity tools in the field of organ transplantation. In this model of orthotopic liver transplantation (OLT) in mice, microCT affords the opportunity to evaluate allograft anastomosis in real time and has the added benefit of not having to sacrifice study animals. The choice of contrast, as well as image acquisition settings, create a high-definition image, which gives researchers invaluable information. This allows for evaluation of the technical aspects of the procedure as well as potentially evaluating different therapeutics over an extended duration of time. In this protocol, we detail an OLT model in mice in a stepwise fashion and finally describe a microCT protocol that can give high-quality images, which aid researchers in in-depth analysis of solid organ transplantation. We provide a step-by-step guide for liver transplantation in a mouse, as well as briefly discuss a protocol for evaluating the patency of the graft through microCT angiography.

作者聚焦:通过小鼠模型中的 MicroCT 血管造影增强移植研究 

In this protocol, we discuss the implementation of a model of successful orthotopic liver transplantation (OLT) in mice. Additionally, adjuvants to further analyze allograft patency after successful OLT in a mouse are discussed as well, specifically utilizing microcomputed tomography (microCT) scans.

利用显微计算机断层扫描血管造影成功对小鼠进行原位肝移植

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality ...

之后，正常小鼠70％部分肝切除血管和实质再生的可视化

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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

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

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

肝细胞部分肝切除术后脾移植。小鼠

静脉大鼠部分肝移植的体内血管定向70% 肝切除术

小鼠肝部分切除术后再生肝细胞的分离

利用显微计算机断层扫描血管造影成功对小鼠进行原位肝移植

利用显微计算机断层扫描血管造影成功对小鼠进行原位肝移植