Name: assessment of plasma coagulation on liver tissue in a large animal model in vivo
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遵循德国法律的动物研究法规以及实验动物保育原则 (国立卫生研究院出版版 8, 2011)。官方许可来自政府动物护理办公室 (Landesamt Natur, Umwelt 和 Verbraucherschutz Nordrhein, Westfalen, 德国)。
1. 动物
<ol><li>使用雌性德国长白猪 (重25-30 公斤) 存放在开放的笼子里。</li>
	<li>每组使用5只动物 (氩和氦)。</li>
	<li>允许动物在实验前至少一周适应环境。快速动物为 24 h 在手术之前以自由地获得水。</li>
</ol>2. 麻醉
<ol><li>Premedicate 的动物肌肉注射氯胺酮 (15 毫克/千克体重 [体重]), 甲苯噻嗪 (10 毫克/千克体重), 和阿托品 (0.1 毫克/千克体重) 10 分钟前诱导麻醉。</li>
	<li>通过将22口径套管放置到耳静脉中, 可建立外周静脉通路。</li>
	<li>静脉注射丙泊酚2毫克/千克体重诱发全麻。</li>
	<li>将动物置于仰卧位, 在颈静脉沟内进行纵向皮肤切口, 长度为2厘米. 通过钝性的皮下组织的制备找到静脉。插入套管, 然后 Seldinger 导线。</li>
	<li>缩回套管并插入14法郎导管在导丝上。退刀导丝。将导管连接到延伸处, 用皮带或缝合固定导管。</li>
	<li>拔掉舌头, 插入直喉镜。使用喉镜的尖端拉下会厌。用声带插入管子。把袖口放在声门下, 充气。</li>
	<li>通风与40% 氧气在20-26 呼吸/分钟和潮汐容量10毫升/公斤保持末端潮汐部分二氧化碳紧张在36和42毫米汞之间。</li>
	<li>用异氟醚保持麻醉, 浓度为 1-1. 5%, 芬太尼浓度为3-4 µg/千克/小时。</li>
	<li>以4毫升/千克/小时的初始速率供应响铃的乳酸溶液, 在剖腹手术后增加8毫升/公斤/小时的恒定输液率。</li>
</ol>3. 手术和血浆凝固
<ol><li>将动物置于仰卧位, 放置在标准手术台上。</li>
	<li>使用标准的外科消毒剂 (2 丙醇45克/100 克, 1 丙醇10克/100g, Biphenyl-2-ol 0.2 克/100 克) 消毒皮肤, 用手术拭子治疗3次。</li>
	<li>用手术刀进行宽中线剖腹手术, 从剑突过程到耻骨, 并安装手术拉钩。</li>
	<li>开关在等离子凝固装置, 打开氩或氦气瓶, 取决于使用的载体气体。将气体流量调整到3升/分. 选择混凝设备输出功率根据需要。 注: 惰性气体、氩气或氦均可用于等离子凝固。凝固作用是可比较的。有关详细信息, 请参阅参考资料7 。</li>
	<li>在左肝叶上进行血浆凝固, 如前所述7。使用钛模 (方形孔径 1 x 1 厘米2) 来规范凝固区。凝聚为 5 s 与探针距离 1 cm。不同电源设置的 coagulations 可以并排执行, coagulations 之间的距离为5毫米 (图 1)。</li>
	<li>为了收割肝脏, 将所有韧带的连接与肝脏分开。分离和分割上十二指肠弯曲以上的肝蒂, 留下门静脉和胆总管的长部分。将静脉的静脉分在肝脏的上方和下方, 然后取出器官。</li>
	<li>在收获肝脏之后, 猪通过静脉注射管理被安乐死0.16 克/千克戊巴比妥。</li>
	<li>对于爆裂压力实验, 切除一半左内侧肝叶与锋利的剪刀。等离子凝固的切割表面 (100W 输出功率) 或密封的切割表面与纤维蛋白密封剂 (图 2)。</li>
</ol>4. 微循环测量
注: 激光多普勒光谱可以通过测量红细胞运动引起的多普勒移位来确定组织中的血流。激光信号与运动红细胞数目有关。激光多普勒技术是临床应用 (如移植医学), 并已验证了多次15。
<ol><li>开关激光多普勒流量计和分光光度计。使用平面探头。</li>
	<li>对流速和流速进行基线测量。保存或记下值。</li>
	<li>按照3.5 的描述进行凝固。</li>
	<li>将平探头放在凝固部位, 测量流速和流速。再次保存或记下值。</li>
	<li>对凝固设备的所有电源设置重复。</li>
</ol>5. 测温
<ol><li>切换系统 (红外相机、笔记本和红外线温度计), 并让它在执行测量之前至少运行1小时。</li>
	<li>调整焦点和视图框架在红外相机上凝固地点。红外序列可以检测到的空间分辨率为 1024年 x 768 像素, 温度分辨率大于 20 mK。考虑到, 凝血区和周围组织--受传热影响--位于视图中间。 注意: 它应该包含尽可能多的帧像素, 以实现最佳的空间分辨率。</li>
	<li>用红外相机在2分钟的时间内用等离子凝固在肝脏表面记录凝固过程。</li>
	<li>用热像分析软件分析图像序列: 定义感兴趣的区域。 注: 软件计算随时间推移的相应平均温度的过程。</li>
</ol>6. 混凝深度测量
<ol><li>用锋利的剪刀收割左内侧肝叶。</li>
	<li>用1厘米厚的凝固部位进行消费。切成3毫米厚的纵段进行进一步加工。</li>
	<li>用中性10% 缓冲福尔马林固定4摄氏度的组织样本。热石蜡2°c 在熔点和嵌入切片。进程过夜。</li>
	<li>执行苏木精/伊红染色。<ol>
<li>Deparaffinize 和水合组织随后浸泡在2x 二甲苯, 100% 乙醇 (乙醇), 95% 乙醇, 70% 乙醇, 去离子 H2O 为2分钟每。</li>
		<li>用迈耶的苏木精溶液染色组织样品3分钟。</li>
		<li>现在用自来水冲洗5分钟。</li>
		<li>用3分钟的伊红溶液染色组织。</li>
		<li>冲洗2x 乙醇 95%, 然后二甲苯3分钟。安装标准安装介质。</li>
	</ol>
</li>
	<li>切换系统 (显微镜连接的摄像头, 成像软件)。查看所有具有40X 放大倍数的部分。</li>
	<li>在放大40X 的情况下, 取一个物体千分尺的图像。在 "目标" 窗口中按 "重新校准" 按钮。选择手动校准。在100µm 的千分尺图像上画一条线. 在对话框中输入01毫米, 然后按 "确定"。</li>
	<li>在 "视图" > "分析控件" > "注释和测量" 窗口中选择 "长度"。测量从肝脏表面到凝固边缘与老鼠。导出或记下结果。对同一张幻灯片上的另一个位置重复测量。 注: 凝血深度可以很容易地与正常肝组织的区别, 由正常肝脏线和坏死区之间的明显缩小细胞质, 固缩核, 出血区。</li>
	<li>计算两个测量值的平均值。</li>
</ol>7. 爆破压力测量
<ol><li>切换系统 (自动泵, 压力表)。根据步骤3.7 准备肝脏标本。 注: 使用两个并联泵通过3路旋塞阀连接。单泵不能获得1500毫米汞的最大压力。</li>
	<li>用剪刀将门静脉、普通肝动脉和胆管隔离在触觉蒂内。钳门静脉与 overholt 钳和结扎 monofil 缝合4-0。钳共肝动脉 overholt 钳和结扎 monofil 缝合4-0。</li>
	<li>将 Ch-16 导管插入胆总管, 结扎2-0 丝缝合。将导管连接到自动泵, 安装3路旋塞阀与压力表 (图 3)。</li>
	<li>用生理盐水填满灌注注射器。</li>
	<li>启动自动泵, 交货率为99毫升/小时。</li>
	<li>监测肝切口表面和压力表的渗漏和记录爆裂压力。 注: 为便于识别泄漏, 专利蓝可以添加到生理盐水 (2 毫升专利蓝 + 18 毫升生理盐水)。通过注意压力表压力损失时间, 可以更容易地观察爆破压力。</li>
</ol>

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作者没有什么可透露的。

肝脏手术的啮齿动物模型建立了很长时间16。然而, 大型动物模型提供了一定的优势: 不需要显微外科设备作为人体标准手术设备, 手术技术可与临床使用相媲美, 标准的临床评价方法可以转移到实验。例如, 标准的临床血液测试可以进行, 而不需要特殊的实验室测试方法 (图 10)。
猪是适当的实验动物为心肺研究作为他们的生理相似人<sup c...

氩离子凝固 (APC) 是腹部手术中广泛使用的仪器, 三年来1、2。通过封堵肝切口表面, 防止术后出血3, 是大鼠肝切除术后继发止血的标准技术。等离子凝固是射频烙的一种特殊形式, 通过电离气体电弧传递电能。提供单电热止血, 这种非接触技术具有防止电极黏附在组织4的优势。电离气体光束被自动定向到最低电阻的区域, 当电阻上升时, 由于干燥到其他尚未干燥的区域, 就会被转走。这产生一个统一的有限深度的凝固5,6。影响凝血效果的因素有活化时间、凝固装置的功率设置以及探针与组织的距离。氦是另一种载体气体, 可用于等离子凝固7。最近的临床研究集中在临床结果, 而不是组织学和功能发现3,8,9, 而实验研究集中在体外调查10或实验在分离的被灌注的器官11。
基础协议允许研究在接近于临床应用的大型动物模型中的血浆凝固作用在猪附近使用标准人的设备: 微循环由激光多普勒流量计无创性评估和分光光度计, 这是一个标准的临床工具为这个征兆12,13。凝固过程中的温度变化用红外线温度计和红外相机进行监测。在采集组织样品后, 测定组织学上的苏木素/伊红染色切片的凝固深度。为与其他二次止血方法进行比较, 进行了爆破压力实验。与以前描述的技术14相比, 这些都是在说明器官上进行的, 以排除血压相关的影响。除了所述的对血浆凝固局部效应的研究外, 还可以在猪模型中进行标准血液检测。

<table><tbody><tr><td>Xylazine 20 mg/mL</td><td>Vetoquinol GmbH</td><td>Xylapan</td><td></td></tr><tr><td>Ketamine 100 mg/mL</td><td>Ceva GmbH</td><td>Ceva Ketamine Injection</td><td></td></tr><tr><td>Atropine 100 mg / 10 mL</td><td>Dr. Franz K&amp;ouml;hler Chemie GmbH</td><td>Atropinsulfat K&amp;ouml;hler 100mg Amp.</td><td></td></tr><tr><td>Propofol</td><td>Fresenius Kabi GmbH</td><td>Propofol 1% MCT Fresenius</td><td></td></tr><tr><td>Fentanyl</td><td>KG Rotexmedica GmbH</td><td>Fentanyl 0,5mg Rotexmedica</td><td></td></tr><tr><td>Isoflurane</td><td>Abbot GmbH</td><td>Forene 100% (V/V) 250 mL</td><td></td></tr><tr><td>Ringer&#039;s lactate solution</td><td>Baxter Deutschland GmbH</td><td></td><td>sodium 131mmol/l, potassium 5 mmol/l, calcium 2 mmol/l, cloride 111 mmol/l, lactate 29 mmol/l</td></tr><tr><td>Surgical disinfactant</td><td>Sch&amp;uuml;lke &amp;amp; Mayr GmbH</td><td>Kodan Tinktur forte gef&amp;auml;rbt 1l 104804</td><td></td></tr><tr><td>Motorized microscope</td><td>Nikon Instruments Europe</td><td>Eclipse TE2000-E</td><td></td></tr><tr><td>Microscope camera</td><td>Nikon Instruments Europe</td><td>Digitalsight DS-Qi1Mc</td><td></td></tr><tr><td>Imaging software</td><td>Nikon Instruments Europe</td><td>NIS elements Vers. 4.40</td><td></td></tr><tr><td>Plasma coagulator</td><td>S&amp;ouml;ring GmbH</td><td>CPC-1000</td><td></td></tr><tr><td>Argon gas</td><td>Linde AG</td><td>Argon 4.8&amp;nbsp;</td><td></td></tr><tr><td>Helium gas</td><td>Linde AG</td><td>Helium 4.8</td><td></td></tr><tr><td>O2C</td><td>LEA Medizintechnik GmbH</td><td>O2C Version 1212</td><td>with LF-2 or LF-3 probe</td></tr><tr><td>Infrared thermometer</td><td>Voltcraft</td><td>VOLTCRAFT IR 260-8S</td><td></td></tr><tr><td>Thermographic camera</td><td>InfraTec GmbH</td><td>VarioCAM HD head 820</td><td></td></tr><tr><td>Thermographic analysis software</td><td>InfraTec GmbH</td><td>IRBIS 3</td><td></td></tr><tr><td>Mayer&#039;s Hematoxylin solution</td><td></td><td>Merck 1.09249</td><td></td></tr><tr><td>Eosin solution</td><td>VWR International GmbH</td><td>Merck 1.09844</td><td></td></tr><tr><td>Rollerpump Masterflex L/S easy Load</td><td>Cole-Parmer Instrument Company</td><td>model 7518-10</td><td></td></tr><tr><td>Perfusorpump</td><td>B. Braun Melsungen AG</td><td>Perfusor secura FT</td><td></td></tr><tr><td>Digital pressure meter</td><td>Greisinger electronic</td><td>GMH 3161</td><td></td></tr><tr><td>Perfusorsyringe, 50 mL</td><td>B. Braun Melsungen AG</td><td>REF 8728810 F</td><td></td></tr><tr><td>Perfusor line, Type IV Standard, PVC Luer lock</td><td>B. Braun Melsungen AG</td><td>REF 8722960</td><td></td></tr><tr><td>3-Way stopcock, Dicofix C35C</td><td>B. Braun Melsungen AG</td><td>REF 16494 C</td><td></td></tr><tr><td>Silk 2-0. 3 metric</td><td>Resorba</td><td>REF H5F</td><td></td></tr><tr><td>Vicryl 4-0 Sutupak</td><td>Ethicon</td><td>V1224H</td><td></td></tr><tr><td>NaCl 0.9 %</td><td>B. Braun Melsungen AG</td><td></td><td></td></tr></tbody></table>

assessment of plasma coagulation on liver tissue in a large animal model in vivo

血浆凝固作为一种烙的形式, 用于肝脏手术数十年, 以封闭大肝切除术后, 主要切除后, 以防止出血的后期阶段。血浆凝固对肝脏组织的确切的作用只被检查的很少。在我们的猪模型中, 凝血作用可以接近临床应用。联合激光多普勒流量计和分光光度计文件在8毫米组织深度无创性凝血过程中微循环的变化, 提供了超出主观临床印象的可量化的止血信息。在凝固过程中, 用红外线温度计对凝血部位的温度进行评估, 在凝固期间用红外相机测量气体束温度, 因为器件的上阈值是不可能的。用目标测微仪标定后的苏木精/红/红染色切片, 对凝固深度进行了显微测量, 并给出了功率设定-凝固深度关系的准确信息。在胆管上检查密封效果, 因为血浆凝固无法封堵较大的血管。对说明器官进行了爆裂压力实验, 排除了血压的相关影响。

微循环:通过对血浆凝固后止血的诊断装置, 可以通过微循环的改变来证明。毛细管血流 (显示为任意单位 (AU)) 从基线值减少 142.7, 76.08 au 到 57.78, 49.57 au 在 25 w 设备输出功率, 48.5 @ 7.26 au 在 50 w 和 5.04 @ 1.31 au 在 100 w (图 4)。
温度:用红外相机测量凝固部位的温度 (图 ...

Watch this Scientific Journal Video about Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo at JoVE.com

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

在这里, 我们提出了一个实验性的评估血浆凝固在体内的肝脏组织的协议。在猪模型中, 通过激光多普勒检查微循环, 用红外测温仪和红外相机对凝血深度进行组织学测量, 凝血部位温度, 并通过爆裂压力记录管道密封效果。实验。

大动物模型肝组织血浆凝固的评价

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent ...

assessment-plasma-coagulation-on-liver-tissue-large-animal-model

Research

JoVE Journal

Medicine

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

8.8K 次观看.  University of Bonn. 在这里, 我们提出了一个实验性的评估血浆凝固在体内的肝脏组织的协议。在猪模型中, 通过激光多普勒检查微循环, 用红外测温仪和红外相机对凝血深度进行组织学测量, 凝血部位温度, 并通过爆裂压力记录管道密封效果。实验。

视频: Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

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 ...

科研

生物工程

Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring

Uncontrolled hemorrhage is an important cause of preventable deaths among trauma patients. We have developed a murine model of uncontrolled hemorrhage via a liver laceration that results in consistent blood loss, hemodynamic alterations, and survival.
Mice undergo a standardized resection of the left-middle lobe of the liver. They are allowed to bleed without mechanical intervention. Hemostatic agents can be administered as pre-treatment or rescue therapy depending on the interest of the investigator. During the time of hemorrhage, real-time hemodynamic monitoring via a left femoral arterial line is performed. Mice are then sacrificed, blood loss is quantified, blood is collected for further analysis, and organs are harvested for analysis of injury. Experimental design is described to allow for simultaneous testing of multiple animals.
Liver hemorrhage as a model of uncontrolled hemorrhage exists in the literature, primarily in rat and porcine models. Some of these models utilize hemodynamic monitoring or quantify blood loss but lack consistency. The present model incorporates quantification of blood loss, real-time hemodynamic monitoring in a murine model that offers the advantage of using transgenic lines and a high-throughput mechanism to further investigate the pathophysiologic mechanisms in uncontrolled hemorrhage.

Uncontrolled hemorrhage, an important cause of mortality among trauma patients, can be modeled using a standard liver laceration in a murine model. This model results in consistent blood loss, survival, and allows for testing hemostatic agents. This article provides the step-by-step process to perform this valuable model.

通过实时血液动力学监测在小鼠中通过肝脏浸润建立的不受控制的出血性休克

Uncontrolled hemorrhage is an important cause of preventable deaths among trauma patients. We have developed a murine model of uncontrolled hemorrhage via ...

医学

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 ...

Invasive Hemodynamic Characterization of the Portal-hypertensive Syndrome in Cirrhotic Rats

This is a detailed protocol describing invasive hemodynamic measurements in cirrhotic rats for the characterization of portal hypertensive syndrome. Portal hypertension (PHT) due to cirrhosis is responsible for the most severe complications in patients with liver disease. The full picture of the portal hypertensive syndrome is characterized by increased portal pressure (PP) due to the increased intrahepatic vascular resistance (IHVR), hyperdynamic circulation, and increased splanchnic blood flow. Progressive splanchnic arterial vasodilation and increased cardiac output with elevated heart rate (HR) but low arterial pressure characterizes the portal hypertensive syndrome.
Novel therapies are currently being developed that aim to decrease PP by either targeting IHVR or increased splanchnic blood flow &#8212; but side effects on systemic hemodynamics may occur. Thus, a detailed characterization of portal venous, splanchnic, and systemic hemodynamic parameters, including measurement of PP, portal venous blood flow (PVBF), mesenteric arterial blood flow, mean arterial pressure (MAP), and HR is needed for preclinical evaluation of the efficacy of novel treatments for PHT. Our video article provides the reader with a structured protocol for performing invasive hemodynamic measurements in cirrhotic rats. In particular, we describe the catheterization of the femoral artery and the portal vein via an ileocolic vein and the measurement of portal venous and splanchnic blood flow via perivascular Doppler-ultrasound flow probes. Representative results of different rat models of PHT are shown.

Here we describe a detailed protocol for invasive measurements of hemodynamic parameters including portal pressure, splanchnic blood flow, and systemic hemodynamics in order to characterize the portal hypertensive syndrome in rats.

肝硬化大鼠门脉高压综合征的侵袭性血流动力学特征

This is a detailed protocol describing invasive hemodynamic measurements in cirrhotic rats for the characterization of portal hypertensive syndrome. ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications &#8212; the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

体外常温肝灌注的小动物模型

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, we have translated a vascular injury model from a small animal into a large animal (canine), with slight modifications that can be used for pre-clinical screening of prophylactic and thrombolytic agents. In addition to the surgical methods, the modified protocol describes the step-by-step methods to assess carotid artery canalization by angiography, detailed instructions to process both the brain and carotid artery for histological analysis to verify carotid canalization and cerebral hemorrhage, and specific parameters to complete an assessment of downstream thromboembolic events by utilizing magnetic resonance imaging (MRI). In addition, specific procedural changes from the previously well-established small animal model necessary to translate into a large animal (canine) vascular injury are discussed.

Here, we present the modifications necessary to a well characterized and commonly used small animal ferric chloride-induced (FeCl3) carotid artery injury model for use in a large animal vascular injury model. The resulting model can be utilized for pre-clinical trial assessment of both prophylactic and thrombolytic pharmacological and mechanical interventions.

氯化铁诱导的犬颈动脉血栓形成: 一种大的血管损伤动物模型

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, ...

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 ...

Porcine Liver Transplantation Without Veno-Venous Bypass As an Extended Criteria Donor Model

Liver transplantation is regarded as the gold standard for the treatment of a variety of fatal hepatic diseases. However, unsolved issues of chronic graft failure, ongoing organ donor shortages, and the increased use of marginal grafts call for the improvement of current concepts, such as the implementation of organ machine perfusion. In order to evaluate new methods of graft reconditioning and modulation, translational models are required. With respect to anatomical and physiological similarities to humans and recent progress in the field of xenotransplantation, pigs have become the main large animal species used in transplantation models. After the initial introduction of a porcine orthotopic liver transplant model by Garnier et al. in 1965, several modifications have been published over the past 60 years.
Due to specifies-specific anatomical traits, a veno-venous bypass during the anhepatic phase is regarded as a necessity to reduce intestinal congestion and ischemia resulting in hemodynamic instability and perioperative mortality. However, the implementation of a bypass increases the technical and logistical complexity of the procedure. Furthermore, associated complications such as air embolism, hemorrhage, and the need for a simultaneous splenectomy have been reported previously.
In this protocol, we describe a model of porcine orthotopic liver transplantation without the use of a veno-venous bypass. The engraftment of donor livers after static cold storage of 20 h - simulating extended criteria donor conditions - demonstrates that this simplified approach can be performed without significant hemodynamic alterations or intraoperative mortality and with regular uptake of liver function (as defined by bile production and liver-specific CYP1A2 metabolism). The success of this approach is ensured by an optimized surgical technique and a sophisticated anesthesiologic volume and vasopressor management.
This model should be of special interest for workgroups focusing on the immediate postoperative course, ischemia-reperfusion injury, associated immunological mechanisms, and the reconditioning of extended criteria donor organs.

In this protocol, a model of porcine orthotopic liver transplantation after static cold storage of donor organs for 20 h without the use of a veno-venous bypass during engraftment is described. The approach uses a simplified surgical technique with minimization of the anhepatic phase and sophisticated volume and vasopressor management.

无静脉-静脉旁路术的猪肝移植作为扩展标准供体模型

Liver transplantation is regarded as the gold standard for the treatment of a variety of fatal hepatic diseases. However, unsolved issues of chronic graft ...

Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice

Tail bleeding models are important tools in hemophilia research, specifically for the assessment of procoagulant effects. The tail vein transection (TVT) survival model has been preferred in many settings due to sensitivity to clinically relevant doses of FVIII, whereas other established models, such as the tail clip model, require higher levels of procoagulant compounds. To avoid using survival as an endpoint, we developed a TVT model establishing blood loss and bleeding time as endpoints and full anesthesia during the entire experiment. Briefly, anesthetized mice are positioned with the tail submerged in temperate saline (37&#176;C) and dosed with the test compound in the right lateral tail vein. After 5 min, the left lateral tail vein is transected using a template guide, the tail is returned to the saline, and all bleeding episodes are monitored and recorded for 40 min while collecting the blood. If no bleeding occurs at 10 min, 20 min, or 30 min post-injury, the clot is challenged gently by wiping the cut twice with a wet gauze swab. After 40 min, blood loss is quantified by the amount of hemoglobin bled into the saline. This fast and relatively simple procedure results in consistent and reproducible bleeds. Compared to the TVT survival model, it uses a more humane procedure without compromising sensitivity to pharmacological intervention. Furthermore, it is possible to use both genders, reducing the total number of animals that need to be bred, in adherence with the principles of 3R&#39;s. A potential limitation in bleeding models is the stochastic nature of hemostasis, which can reduce the reproducibility of the model. To counter this, manual clot disruption ensures that the clot is challenged during monitoring, preventing primary (platelet) hemostasis from stopping bleeding. This addition to the catalog of bleeding injury models provides an option to characterize procoagulant effects in a standardized and humane manner.

The refined tail vein transection (TVT) bleeding model in anesthetized mice is a sensitive in vivo method for the assessment of hemophilic bleeding. This optimized TVT bleeding model uses blood loss and bleeding time as endpoints, refining other models and avoiding death as an endpoint.

全麻醉血友病A小鼠尾静脉横断出血模型

Tail bleeding models are important tools in hemophilia research, specifically for the assessment of procoagulant effects. The tail vein transection (TVT) ...

生物学

Intravital Microscopy to Study Platelet-Leukocyte-Endothelial Interactions in the Mouse Liver

Inflammation and thrombosis are complex processes that occur primarily in the microcirculation. Although standard histology may provide insight into the end pathway for both inflammation and thrombosis, it is not capable of showing the temporal changes that occur throughout the time course of these processes. Intravital microscopy (IVM) is the use of live-animal imaging to gain temporal insight into physiologic processes in vivo. This method is particularly powerful when assessing cellular and protein interactions within the circulation due to the rapid and sequential events that are often necessary for these interactions to occur. While IVM is an extremely powerful imaging methodology capable of viewing complex processes in vivo, there are a number of methodological factors that are important to consider when planning an IVM study. This paper outlines the process of conducting intravital imaging of the liver, identifying important considerations and potential pitfalls that may arise. Thus, this paper describes the use of IVM to study platelet-leukocyte-endothelial interactions in liver sinusoids to study the relative contributions of each in different models of acute liver injury.

Intravital microscopy is a powerful tool that provides insight into both the temporal and spatial relationships of rapid and/or sequential processes. Herein, we describe a protocol to assess both protein-protein interactions and platelet-neutrophil-endothelial interactions in liver sinusoids in a murine model of experimental sepsis (endotoxemia).

Inflammation and thrombosis are complex processes that occur primarily in the microcirculation. Although standard histology may provide insight into the ...