这项工作得到了美国国立卫生研究院 T32AI 106704-01A1 和俄亥俄州立大学器官移植、灌注、工程和再生的 t 鲁道夫·弗莱切基金的支持。

所有程序都是按照《机构动物保育指南》和国家研究理事会《人道护理和使用实验动物手册》 (IACUC) 进行的, 并经俄亥俄州立大学 IACUC 委员会批准。
1. 初步设置
<ol><li>
结合以下方法制备灌注液:86 毫升25% 白蛋白, 184 毫升的威廉姆斯的培养基, 30 毫升青霉素/链霉素 (10 的 u/毫升青霉素和0.01 毫克/毫升链霉素), 胰岛素 (50 u/升), 肝素 (0.01 u/毫升), l-谷氨酰胺 (0.292 克/升), 和氢化可松 (0.010 克/升), 总体积为300毫升。对于基灌流液和 peg 猫组, 添加 625 U/毫升的 peg 猫。
	<ol>
<li>用三 (羟甲基) 氨基甲烷 (THAM) 将灌流液溶液缓冲到 pH 值7.4。使用动脉血气机确认灌流液 pH 值。</li>
	</ol>
</li>
	<li>
设置电路 (图 1)。
	<ol>
<li>打开水浴温暖和设置它到37°c。让风琴室暖和起来。</li>
		<li>将混合和缓冲的灌流液倒入水库, 开始循环。 注: 本步骤中提到的灌流液是在步骤1.1.1 中编写的。</li>
		<li>通过在线肺打开氧气气体 (95% 氧气和5% 二氧化碳) 到流量计数器。</li>
		<li>打开数据收集软件, 然后单击 "开始" 以记录实验的持续时间。</li>
	</ol>
</li>
	<li>
设置手术显微镜和手术室 (图 2)。
	<ol>
<li>打开所有设备, 包括暖板, 烙, 麻醉和生命体征 (心率和氧气饱和度) 监测设备。 注: 显微镜的设置会因使用的显微镜而异, 并可根据用户的舒适度进行调整。</li>
		<li>填充10毫升麻醉注射器与10毫升的液体异氟醚吸入 (分子量184.5 克/摩尔) 和放置在麻醉单位。</li>
		<li>适当放置0.5 毫升肝素注射器 (50 U), 手术器械, 4-0 和7-0 丝缝合, 无菌棉签, 4 厘米 x 4 厘米无纺布纱布海绵 (图 2)。</li>
	</ol>
</li>
	<li>准备异氟醚室。</li>
</ol>2. 麻醉的诱导
<ol><li>佩戴以下个人防护设备 (PPE): 手术面罩、手术手套、一次性长袍。</li>
	<li>称一下老鼠。 注意: 我们在 250–350 g 之间使用大大鼠。</li>
	<li>打开氧气压缩机和异氟醚。在被称量后, 将老鼠放入异氟醚腔内, 并确保盖子的安全。用2升/分氧的6% 异氟醚诱导麻醉。 注: 确切使用异氟醚剂量将取决于所使用的特定麻醉系统。</li>
	<li>用电子剪刀夹住动物的腹部毛发.
</li>
	<li>替换异氟醚腔内的动物。</li>
	<li>打开手术室里的麻醉装置。</li>
	<li>当麻醉完全诱发时, 将大鼠从异氟醚腔中取出。用脚趾捏确认麻醉深度。</li>
</ol>3. 采购程序
<ol><li>
准备16克的入口袖口 (图 3)。
	<ol>
<li>从16克 angiocatheter 开始。切割7毫米的油管。确定7毫米段的中点, 测量3.5 毫米切割在中点和移除前半部分油管。</li>
		<li>用止血粉碎这个现在平坦的部分。用打火机融化 angiocatheter 的另一端, 形成唇。不要把笔尖直接放到火焰中, 否则它会着火。</li>
	</ol>
</li>
	<li>
准备胆管插管。
	<ol>
<li>取27克 angiocath, 切断注射口, 只留下导管。将此连接至27克 cannular 油管的10厘米段。</li>
	</ol>
</li>
	<li>将大鼠的鼻子放在麻醉鼻锥中, 并将其四四肢固定。通过将显示器连接到左后肢来监测生命体征。执行脚趾捏, 以确认适当的麻醉深度。继续麻醉在4% 异氟醚 (对体重 > 250 克的动物)。</li>
	<li>用70% 异丙醇喷洒动物的腹部。允许干燥。把不育的褶皱放在动物身上。</li>
	<li>用锋利的剪刀和伸展皮肤 (图 4) 从剑突到耻骨进行中线切口。轻轻地进入腹膜, 切开肌肉。注意避免损害这个切口的下一面和肝脏在这个切口的上级方面的膀胱。</li>
	<li>将切口侧向左侧和右侧, 在肝脏下边界的水平处形成十字。</li>
	<li>将麻醉降低到 2% (为动物称重 > 250 克)。</li>
	<li>用弯曲的蚊子钳和肋骨拉钩 (图 5) 收回剑突过程。</li>
	<li>用锋利的剪刀切开镰、膈和 gastrohepatic 韧带。</li>
	<li>在膈静脉上定位并栓上7-0 条缝合线, 尽可能靠近其原点以防止渗漏。</li>
	<li>阉割用一种不育的湿棉尖涂抹器, 用0.9% 生理盐水蘸纱布包扎肠道。注意不要伸展小肠的血管。</li>
	<li>解剖下腔静脉以去除多余的组织。解剖后的静脉刚好优于分岔和通过一个循环4-0 丝缝合为以后使用 (图 6)。</li>
	<li>收回右肾以提供暴露在右肾上腺静脉。用纱布将肝脏的右叶缩回。用7-0 丝缝线将右肾上腺静脉系好, 尽可能接近烧灼, 并将其穿过远端的领带 (图 7)。</li>
	<li>仔细解剖脾静脉, 用两条7-0 丝缝线将其绑起来, 在两条缝线之间切开。</li>
	<li>如果需要的话, 在门静脉上用7-0 丝缝线缝合并结扎额外的静脉。 注: infrahepatic 在右肾上腺静脉与下肝之间有时有小的分支。</li>
	<li>解剖周围的胃十二指肠动脉, 用7-0 丝缝线栓住胃十二指肠动脉, 结扎胃十二指肠动脉。</li>
	<li>解剖周围的肝动脉, 然后放置7-0 丝缝合领带围绕它 (图 8)。</li>
	<li>
解剖出胆管。
	<ol>
<li>检查胆管的长度。用7-0 丝缝线将远端的胆管系上。在胆管周围放置一圈7-0 丝缝线, 尽可能下部。</li>
		<li>用小剪刀把管子直径的一半切成一个洞, 然后将27克导管放进胆管下部。使用罗马凉鞋领结缝合导管 (图 9)。</li>
	</ol>
</li>
	<li>用27克针将0.5 毫升肝素 (50 U) 注入到动物的小弟弟静脉或静脉内。 注: 还可以使用27克胰岛素注射器代替。</li>
	<li>用先前放置的4-0 丝缝合线钳住并系上静脉。</li>
	<li>用先前放置的7-0 丝缝线栓住肝动脉。</li>
	<li>用显微外科夹子夹住门静脉。用22克 angiocatheter Cannulate 门静脉。冲洗门静脉与60毫升冷0.9% 生理盐水与1毫升肝素 (100 U), 直到肝脏 blanches (图 10)。 注: 如果肝脏不立即漂白, 它可以用无菌的棉尖喷头按摩。</li>
	<li>暴露 suprahepatic 的静脉, 并在胸部尽可能高的切割。</li>
	<li>执行肝切除术如下。切开隔膜, 切开肝动脉, 切开静脉, 切开门静脉, 切开其他韧带, 取出肝脏。将肝脏置于冰冷的0.9% 生理盐水中 (图 11)。</li>
	<li>在门静脉放置16克的血管袖口 (图 12)。将肝脏放在前体常温肝脏灌注回路中。</li>
</ol>4.体内常温肝灌注
注: 此处使用的灌流液是在协议步骤1.1.1 中编写的。
<ol><li>将门静脉插管置于袖口的门静脉内 (图 13)。</li>
	<li>在门静脉套管放置时, 保持灌流液通过电路2毫升/分钟的流量开始。观察在门静脉压力下的任何尖峰的显示器;这可能表明该船只已被堵塞, 并重新定位套管是必要的。</li>
	<li>用7-0 丝缝线缝合灌流液返流的静脉插管。</li>
	<li>一旦两个套管到位, 开始转动的流量在1毫升/分钟, 直到生理压力的范围内的 10–16 cmH2O 达到。</li>
	<li>
在0、30、60、90、120、150、180、210和240分钟的灌注中, 从前、后端口中抽取1毫升样品。将1毫升样品分成两个0.5 毫升的样品。 注: 0.5 毫升的这将用于协议步骤 4.5.1, 0.5 毫升将用于协议步骤4.5.2。<ol>
<li>在液氮中低温管中对此样品进行0.5 毫升冻结。</li>
		<li>使用剩余的0.5 毫升灌流液进行动脉血气分析。</li>
		<li>在每个时间点 (0、30、60、90、120、150、180、210和240分钟) 运行血气分析后, 检查 ph 值, 并根据需要将灌流液缓冲以返回到 pH 值7.4。</li>
	</ol>
</li>
	<li>在4小时灌注的结论中, 将肝脏与灌注回路断开。把肝脏分成 0.5 g 段。将低温管内的肝组织冷冻在液氮中。</li>
</ol>5. 实验后分析
<ol><li>
使用商业色度测定试剂盒测定灌流液中丙氨酸转氨酶 (ALT) 水平0、30、60、90、120、150、180、210和240分钟。
	<ol>
<li>简而言之, 用37摄氏度的反应混合试剂孵化灌流液60分钟. 使用微板块读取器测量 570 nm 的光学密度值。</li>
	</ol>
</li>
	<li>
融汇0.5 克肝组织, 100 µL 裂解缓冲液, 分析三磷酸腺苷 (ATP)、谷胱甘肽 (GSH) 和丙二醛 (MDA) 的组织裂解。
	<ol>
<li>简而言之, 使用商业化验试剂盒测量肝脏组织样品的 ATP 水平。将样品与反应缓冲剂混合, 室温孵育30分钟. 使用微板块读取器测量 570 nm 的光学密度。</li>
		<li>使用商业化验试剂盒测量肝组织样品的 GSH 含量。将组织样品与化验鸡尾酒混合。测量405–414纳米的光学密度值。</li>
		<li>使用商业化验试剂盒测量肝脏组织样品的 MDA 水平。将样品与 TBA 和热量混合到95摄氏度, 60 分钟离心反应物, 将上清液转移到96井板上。测量光学密度在532毫微米。</li>
	</ol>
</li>
	<li>
融汇0.5 克的肝组织与100µL 的裂解缓冲, 并分析组织裂解剂的相对 caspase-3/7 活动使用商业化验试剂盒。
	<ol>
<li>将组织裂解剂与 caspase-3-7 试剂检测缓冲器混合, 室温孵育30分钟。</li>
		<li>使用微板块读取器测量每个井中的荧光水平。</li>
	</ol>
</li>
	<li>
使用商业原位死亡检测试剂盒确定肝脏组织样本中凋亡细胞的水平。
	<ol>
<li>用 10 U/毫升蛋白酶 K 的0.5 克组织切片预处理10分钟, 然后用反应混合物在37°c 上孵育60分钟. 用荧光显微镜进行分析。</li>
	</ol>
</li>
</ol>

<ol>
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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reperfusion+liver+injury-induced+superoxide+dismutase+and+catalase+expressions+and+the+protective+effects+of+N-acetyl+cysteine.">Reperfusion liver injury-induced superoxide dismutase and catalase expressions and the protective effects of N-acetyl cysteine.</a> Transplant Proc. 39 (4), 858-860 (2007).</li><li>Chen, B., Tang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effects+of+catalase+on+retinal+ischemia/reperfusion+injury+in+rats.">Protective effects of catalase on retinal ischemia/reperfusion injury in rats.</a> Exp Eye Res. 93 (5), 599-606 (2011).</li><li>He, Y. Y., Hsu, C. Y., Ezrin, A. M., Miller, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethylene+glycol-conjugated+superoxide+dismutase+in+focal+cerebral+ischemia-reperfusion.">Polyethylene glycol-conjugated superoxide dismutase in focal cerebral ischemia-reperfusion.</a> Am J Physiol. 265 (1 Pt 2), H252-H256 (1993).</li><li>I&#351;lekel, S., I&#351;lekel, H., G&#252;ner, G., Ozdamar, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+superoxide+dismutase,+glutathione+peroxidase+and+catalase+activities+in+experimental+cerebral+ischemia-reperfusion.">Alterations in superoxide dismutase, glutathione peroxidase and catalase activities in experimental cerebral ischemia-reperfusion.</a> Res Exp Med (Berl). 199 (3), 167-176 (1999).</li><li>Li, G., Chen, Y., Saari, J. T., Kang, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalase-overexpressing+transgenic+mouse+heart+is+resistant+to+ischemia-reperfusion+injury.">Catalase-overexpressing transgenic mouse heart is resistant to ischemia-reperfusion injury.</a> Am J Physiol. 273 (3 Pt 2), H1090-H1095 (1997).</li><li>Nowak, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunotargeting+of+catalase+to+lung+endothelium+via+anti-angiotensin-converting+enzyme+antibodies+attenuates+ischemia-reperfusion+injury+of+the+lung+in+vivo.">Immunotargeting of catalase to lung endothelium via anti-angiotensin-converting enzyme antibodies attenuates ischemia-reperfusion injury of the lung in vivo.</a> Am J Physiol Lung Cell Mol Physiol. 293 (1), L162-L169 (2007).</li><li>Beckman, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+dismutase+and+catalase+conjugated+to+polyethylene+glycol+increases+endothelial+enzyme+activity+and+oxidant+resistance.">Superoxide dismutase and catalase conjugated to polyethylene glycol increases endothelial enzyme activity and oxidant resistance.</a> J Biol Chem. 263 (14), 6884-6892 (1988).</li><li>Yabe, Y., Nishikawa, M., Tamada, A., Takakura, Y., Hashida, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+delivery+and+improved+therapeutic+potential+of+catalase+by+chemical+modification:+combination+with+superoxide+dismutase+derivatives.">Targeted delivery and improved therapeutic potential of catalase by chemical modification: combination with superoxide dismutase derivatives.</a> J Pharmacol Exp Ther. 289 (2), 1176-1184 (1999).</li><li>Yabe, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+neutrophil-mediated+hepatic+ischemia/reperfusion+injury+by+superoxide+dismutase+and+catalase+derivatives.">Prevention of neutrophil-mediated hepatic ischemia/reperfusion injury by superoxide dismutase and catalase derivatives.</a> J Pharmacol Exp Ther. 298 (3), 894-899 (2001).</li><li>Ushitora, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+hepatic+ischemia-reperfusion+injury+by+pre-administration+of+catalase-expressing+adenovirus+vectors.">Prevention of hepatic ischemia-reperfusion injury by pre-administration of catalase-expressing adenovirus vectors.</a> J Control Release. 142 (3), 431-437 (2010).</li><li>Kakizaki, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effects+of+Short-Term+Subnormothermic+Perfusion+after+Cold+Preservation+on+Liver+Grafts+from+Donors+after+Cardiac+Death:+An+Ex+Vivo+Rat+Model.">The Effects of Short-Term Subnormothermic Perfusion after Cold Preservation on Liver Grafts from Donors after Cardiac Death: An Ex Vivo Rat Model.</a> Transplantation. , (2018).</li><li>Kumar, R., Chung, W. Y., Dennison, A. R., Garcea, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+Vivo+Porcine+Organ+Perfusion+Models+as+a+Suitable+Platform+for+Translational+Transplant+Research.">Ex Vivo Porcine Organ Perfusion Models as a Suitable Platform for Translational Transplant Research.</a> Artif Organs. , (2017).</li><li>Nativ, N. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+defatting:+an+alternative+approach+to+enable+steatotic+liver+transplantation.">Liver defatting: an alternative approach to enable steatotic liver transplantation.</a> Am J Transplant. 12 (12), 3176-3183 (2012).</li><li>Yeung, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+adenoviral+vector+gene+delivery+results+in+decreased+vector-associated+inflammation+pre-+and+post-lung+transplantation+in+the+pig.">Ex vivo adenoviral vector gene delivery results in decreased vector-associated inflammation pre- and post-lung transplantation in the pig.</a> Mol Ther. 20 (6), 1204-1211 (2012).</li><li>Goldaracena, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducing+Hepatitis+C+Virus+Resistance+After+Pig+Liver+Transplantation-A+Proof+of+Concept+of+Liver+Graft+Modification+Using+Warm+Ex+Vivo+Perfusion.">Inducing Hepatitis C Virus Resistance After Pig Liver Transplantation-A Proof of Concept of Liver Graft Modification Using Warm Ex Vivo Perfusion.</a> Am J Transplant. 17 (4), 970-978 (2017).</li><li>Van Raemdonck, D., Neyrinck, A., Rega, F., Devos, T., Pirenne, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Machine+perfusion+in+organ+transplantation:+a+tool+for+ex+vivo+graft+conditioning+with+mesenchymal+stem+cells?.">Machine perfusion in organ transplantation: a tool for ex vivo graft conditioning with mesenchymal stem cells?.</a> Curr Opin Organ Transplant. 18 (1), 24-33 (2013).</li><li>Pratschke, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Results+of+the+TOP+Study:+Prospectively+Randomized+Multicenter+Trial+of+an+Ex+Vivo+Tacrolimus+Rinse+Before+Transplantation+in+EDC+Livers.">Results of the TOP Study: Prospectively Randomized Multicenter Trial of an Ex Vivo Tacrolimus Rinse Before Transplantation in EDC Livers.</a> Transplant Direct. 2 (6), e76 (2016).</li><li>Pratschke, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+TOP-Study+(tacrolimus+organ+perfusion):+a+prospective+randomized+multicenter+trial+to+reduce+ischemia+reperfusion+injury+in+transplantation+of+marginal+liver+grafts+with+an+ex+vivo+tacrolimus+perfusion.">Protocol TOP-Study (tacrolimus organ perfusion): a prospective randomized multicenter trial to reduce ischemia reperfusion injury in transplantation of marginal liver grafts with an ex vivo tacrolimus perfusion.</a> Transplant Res. 2 (1), 3 (2013).</li><li>Nativ, N. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+sensitivity+of+macrosteatotic+hepatocytes+to+hypoxia/reoxygenation+stress+is+reversed+by+a+novel+defatting+protocol.">Elevated sensitivity of macrosteatotic hepatocytes to hypoxia/reoxygenation stress is reversed by a novel defatting protocol.</a> Liver Transpl. 20 (8), 1000-1011 (2014).</li><li>Lonze, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+ex+vivo+delivery+of+short+hairpin+RNAs+for+control+of+hepatitis+C+viral+transcript+expression.">In vitro and ex vivo delivery of short hairpin RNAs for control of hepatitis C viral transcript expression.</a> Arch Surg. 147 (4), 384-387 (2012).</li></ol>

所有作者都报告说, 他们没有相关的披露。

移植的同种异体肝有很大的不足, 在反应中, 捐献者的标准已经扩大了1、2、3、4、5。由于捐助者短缺, NEVLP 已被引入作为评估和修改器官功能6,7的方法。我们设计了一种 NEVLP 的大鼠模型。此外, 我们使用这个模型来证明它的一个重?...

在肝移植的等待名单上有14578名患者, 每年约有7000人进行移植手术,1,2。针对这一严重的捐助者短缺问题, 已扩大了肝捐献者的标准;这些通常被称为边际器官或延长的标准捐赠者, 并预期在移植后的表现不如同种异体标准标准, 具有较高的原发性移植功能障碍和延迟移植功能3, 4,5,6。结果, NEVLP 作为评价和修改器官功能6,7的方法被引入。我们设计了一个大鼠 NEVLP 模型, 并用该模型证明了它的一个重要的潜在应用--对肝脏灌流液的新型分子添加剂的检测。
NEVLP 已经被评估在鼠 (鼠) 和猪模型, 以及在被丢弃的人体器官6,8,9。NEVLP 第一次人体试验的结果最近也发表在10。虽然低温机灌注已明显成为肾脏保存的标准, 但肝机灌注的温度仍有争议。与低温和 subnormothermic 灌注相比, NEVLP 有许多建议的优点。这包括减少保存伤害, 恢复正常的器官功能在生理条件下, 能力评估器官的性能, 并作为一个平台的器官修复, 重塑和修改7,11, 12,13,14,15,16,17。
大量的研究已经完成使用猪 NEVLP 模型。尽管这些模型在考虑使用废弃人体器官或人体临床试验模型时比较便宜, 但与我们的小动物 NEVLP 模型相比, 它们是非常昂贵的。每个实验成本的一个重要组成部分是灌流液。我们能够完成4小时的灌注与300毫升的灌流液在相对较低的成本。此外, 小动物的成本, 包括大鼠是非常低的成本相比, 猪。
与其他模型的 NEVLP 在大鼠, 这里提出的模型是相对简单的实施和具有广泛的应用范围。在图 1中可以看到灌注电路。灌流液开始在灌流液水库 (1), 这是一个水套容器。灌流液是由一个滚子泵 (2) 从水库中拉出, 并推入 windkessel (3), 然后是肺 (4)。肺为逆流气体和灌流液流提供最大气体交换。灌流液然后进入加热线圈 (5) 内的灌注室, 以确保它是在生理温度, 和气泡陷阱 (6), 以防止空气气泡灌注有前器官 (7) 和后器官 (8) 样本端口, 这使得灌流液采样。灌流液然后通过门静脉插管进入肝脏。门静脉套管附在压力监测器上, 用于图表数据收集软件上的值。灌流液然后通过静脉插管退出肝脏, 并流入压力均衡器块 (9)。最后, 将灌流液从压力块中拉回, 通过滚子泵, 倒入储层中。该模型包括对门静脉进行连续灌注, 并将脉动流留到肝动脉和透析用在其他一些模型中, 每一个都需要一个单独的和额外的电路, 但以前已经证明不需要9,13。
为了探索新的治疗分子在灌流液中的加入, 我们选择了酶过氧化氢。过氧化氢酶是一种内源性 ROS 清除剂, 是细胞内部防御机制的一部分, 以减轻 ROS18的影响。过氧化氢酶在肝缺血再灌注损伤中的表达增加19。实验性地添加过氧化氢酶已被证明可以减少缺血再灌注眼, 脑, 肺20,21,22,23,24。聚乙二醇化已被证明的目标过氧化氢酶的内皮和帮助过氧化氢酶吸收到内皮细胞25。在减少肝缺血再灌注损伤方面, 对 PEG 进行系统的管理, 疗效有限;然而, 我们假设, 添加 PEG 猫到一个孤立的器官灌注电路将导致改善结果26,27,28。在这里, 我们添加 PEG 猫到我们的基地灌流液, 并表明其能力减轻肝脏保护伤害。

<table>
	<tbody>
		<tr>
			<td>Perfusate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>8% Albumin</td>
			<td>CLS Behring, King of Prussia, PA</td>
			<td>0053-7680-32</td>
			<td></td>
		</tr>
		<tr>
			<td>Williams Media</td>
			<td>Sigma Aldrich, St. Louis, MO</td>
			<td>W1878</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma Aldrich, St. Louis, MO</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Eli Lilly, Indianapolis, IL</td>
			<td>0002-8215-91</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Fresnius Lab, Lake Zurich, IL</td>
			<td>C504701</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma Aldrich, St. Louis, MO</td>
			<td>G3126</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma Aldrich, St. Louis, MO</td>
			<td>H0888</td>
			<td></td>
		</tr>
		<tr>
			<td>THAM</td>
			<td>Hospira, Inc,</td>
			<td>0409-1593-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene Glycol - Catalase</td>
			<td>Sigma Aldrich</td>
			<td>S9549 SIGMA</td>
			<td></td>
		</tr>
		<tr>
			<td>Personal Protective Equipment </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Mask</td>
			<td>Generic</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Protective Gown</td>
			<td>Generic</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Gloves</td>
			<td>Generic</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Liver Procurement</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague-Dawley Rat</td>
			<td>Harlan Sprague Dawley Inc.</td>
			<td>250 -350 grams</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Microscope</td>
			<td>Leica</td>
			<td>M500-N w/ OHS</td>
			<td></td>
		</tr>
		<tr>
			<td>Charcoal Canisters</td>
			<td>Kent Scientific</td>
			<td>SOMNO-2001-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal Healthcare</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure-Lok Precision Analytical Syringe&#160;</td>
			<td>Valco Instruments Co, Inc.</td>
			<td>SOMNO-10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrosurgical Unit</td>
			<td>Macan</td>
			<td>MV-7A</td>
			<td></td>
		</tr>
		<tr>
			<td>Warming Pad</td>
			<td>Braintree Scientific</td>
			<td>HHP2</td>
			<td></td>
		</tr>
		<tr>
			<td>SomnoSuite Small Animal Anesthesia System</td>
			<td>Kent Scientific</td>
			<td>SS-MVG-Module</td>
			<td></td>
		</tr>
		<tr>
			<td>PhysioSuite</td>
			<td>Kent Scientific</td>
			<td>PS-MSTAT-RT</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane chamber</td>
			<td>Kent Scientific</td>
			<td>SOMNO-0530LG</td>
			<td></td>
		</tr>
		<tr>
			<td>SurgiVet</td>
			<td>Isotec</td>
			<td>CDS 9000 Tabletop</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen</td>
			<td>Praxair</td>
			<td>98015</td>
			<td></td>
		</tr>
		<tr>
			<td>Rib retractors</td>
			<td>Kent Scientific</td>
			<td>INS600240</td>
			<td></td>
		</tr>
		<tr>
			<td>GenieTouch</td>
			<td>Kent Scientific</td>
			<td>GenieTouch</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Saline</td>
			<td>Baxter</td>
			<td>NDC 0338-0048-04</td>
			<td></td>
		</tr>
		<tr>
			<td>4x4 Non-Woven Sponges</td>
			<td>Criterion</td>
			<td>104-2411</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Q-Tips</td>
			<td>Henry Schein Animal Health</td>
			<td>1009175</td>
			<td></td>
		</tr>
		<tr>
			<td>U-100 27 Gauge Insulin Syringe</td>
			<td>Terumo</td>
			<td>22-272328</td>
			<td></td>
		</tr>
		<tr>
			<td>5mL Syringe</td>
			<td>BD</td>
			<td>REF 309603</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 Braided Silk Suture</td>
			<td>Deknatel, Inc.</td>
			<td>198737LP</td>
			<td></td>
		</tr>
		<tr>
			<td>7-0 Braided Silk Suture</td>
			<td>Teleflex Medical</td>
			<td>REF 103-S</td>
			<td></td>
		</tr>
		<tr>
			<td>16 gauge Catheters</td>
			<td>BBraun Introcan Safety</td>
			<td>4252586-02</td>
			<td></td>
		</tr>
		<tr>
			<td>14 gauge Catheters</td>
			<td>BBraun Introcan Safety</td>
			<td>4251717-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Bile Duct Cannular Tubing</td>
			<td>Altec</td>
			<td>01-96-1727&#160;&#160;&#160;&#160;&#160; &#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Liver Perfusion Circuit Components </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water Bath Warmer</td>
			<td>Lauda Ecoline Staredition</td>
			<td>E103</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Collection Software</td>
			<td>ADInstruments&#160;</td>
			<td>Labchart 7</td>
			<td></td>
		</tr>
		<tr>
			<td>Liver Perfusion Circuit</td>
			<td>Harvard Apparatus</td>
			<td>73-2901</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane Oxygenator</td>
			<td>Mediac SPA</td>
			<td>M03069</td>
			<td></td>
		</tr>
		<tr>
			<td>Roller Pump</td>
			<td>Ismatec</td>
			<td>ISM827B</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas (95% oxygen and 5% carbon dioxide)</td>
			<td>Praxair</td>
			<td>98015</td>
			<td></td>
		</tr>
		<tr>
			<td>Organ Chamber</td>
			<td>Harvard Apparatus</td>
			<td>ILP-2</td>
			<td></td>
		</tr>
		<tr>
			<td>1.8 mL Arcticle Cryogenic Tube</td>
			<td>USA Scientific</td>
			<td>1418-7410</td>
			<td></td>
		</tr>
		<tr>
			<td>Mucasol</td>
			<td>Sigma-Aldrich</td>
			<td>Z637181</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsurgical Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small Scissors</td>
			<td>Roboz</td>
			<td>RS-5610</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Scissors</td>
			<td>S&#38;T</td>
			<td>SAA-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps - Large Angled</td>
			<td>S&#38;T</td>
			<td>JFCL-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps - Small Angled</td>
			<td>S&#38;T</td>
			<td>FRAS-15 RM-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Clip Applier</td>
			<td>ROBOZ</td>
			<td>RS-5440</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors - non micro</td>
			<td>FST 14958-11</td>
			<td>14958-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps - Straight Tip</td>
			<td>S&#38;T</td>
			<td>FRS-15 RM8TC</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Microsurgical Clip</td>
			<td>Fine Scientific Tools</td>
			<td>18055-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Microsurgical Clip</td>
			<td>Fine Scientific Tools</td>
			<td>18055-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Microsurgical Clip</td>
			<td>Fine Scientific Tools</td>
			<td>18055-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Microsurgical Clip</td>
			<td>Fine Scientific Tools</td>
			<td>18055-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Mosquito Clamps</td>
			<td>Generic</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Post-Experiment Analysis </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alanine Aminotransferase (ALT) Activity Colorimetric/Fluorometric Assay Kit</td>
			<td>BioVision</td>
			<td>K752</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine Triphosphate (ATP) Colorimetric/Fluorometric Assay Kit</td>
			<td>BioVision</td>
			<td>K354</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione Assay Kit</td>
			<td>Cayman Chemical</td>
			<td>703002</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipid Peroxidation (MDA) Assay Kit</td>
			<td>Abcam</td>
			<td>ab118970</td>
			<td></td>
		</tr>
		<tr>
			<td>Caspase-Glo 3/7 Assay Systems</td>
			<td>Promega</td>
			<td>G8090</td>
			<td></td>
		</tr>
		<tr>
			<td>POLARstar OMEGA Microplate Reader</td>
			<td>BMG LABTECH</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

a small animal model of ex vivo normothermic liver perfusion

移植的同种异体肝有很大的不足, 并在反应中扩大了供体的标准。因此, 常温体外肝灌注 (NEVLP) 作为评价和改变器官功能的一种方法。NEVLP 与低温和 subnormothermic 灌注相比有许多优点, 包括减少保存损伤、生理条件下正常脏器功能恢复、器官性能评估, 以及作为器官修复的平台。、改建和改装。对小鼠和猪 NEVLP 模型进行了描述。我们展示了一个大鼠模型的 NEVLP, 并使用该模型显示其重要的应用之一-使用的治疗分子添加到肝灌流液。过氧化氢酶是一种内源活性氧 (ROS) 清除剂, 已被证明可以减少缺血再灌注在眼, 脑, 肺。聚乙二醇化已被证明是以过氧化氢酶为靶向内皮细胞。在这里, 我们增加了 pegylated 过氧化氢酶 (PEG 猫) 的基础灌流液, 并证明了它的能力减轻肝脏保护伤害。我们的啮齿动物 NEVLP 模型的好处是它是便宜的与更大的动物模型比较。这项研究的一个局限是它目前不包括灌注后肝移植。因此, 不能肯定地预测器官移植后的功能。然而, 大鼠肝移植模型的建立是很好的, 可以与该模型结合使用。最后, 我们展示了一个廉价的, 简单的, 易于复制的 NEVLP 模型使用大鼠。该模型的应用包括测试新的 perfusates 和灌流液添加剂, 用于器官评估的测试软件, 以及用于修复器官的实验。

每组三只老鼠的样本大小被使用。ALT 测量了 0, 30, 60, 90, 120, 150, 180, 210, 和240分钟的灌注。我们使用学生的t检验, 以比较的结果之间的基础灌流液和基地灌流液加上 PEG 猫组在每个时间点。在比较基灌流液和基灌流液加上 peg-猫组, 有显着少 (p < 0.05) ALT 在基灌流液加上 PEG 猫组在 150, 180, 210 和240分钟 (图 14A)。
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Watch this Scientific Journal Video about A Small Animal Model of Ex Vivo Normothermic Liver Perfusion at JoVE.com

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

体外常温肝灌注的小动物模型

肝脏捐献者数量严重不足, 肝脏捐献者的标准已经扩大。常温体外肝灌注 (NEVLP) 已发展为评价和修饰器官功能。本研究展示了 NEVLP 的大鼠模型, 并对 pegylated 过氧化氢酶的能力进行了试验, 以减轻肝脏保存损伤。

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

a-small-animal-model-of-ex-vivo-normothermic-liver-perfusion

Research

JoVE Journal

Medicine

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

13.3K 次观看.  Ohio State University Wexner Medical Center. 肝脏捐献者数量严重不足, 肝脏捐献者的标准已经扩大。常温体外肝灌注 (NEVLP) 已发展为评价和修饰器官功能。本研究展示了 NEVLP 的大鼠模型, 并对 pegylated 过氧化氢酶的能力进行了试验, 以减轻肝脏保存损伤。

视频: A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for liver transplantation die without receiving an organ transplant or are delisted for disease progression. One strategy to increase the donor pool is the utilization of marginal grafts, such as fatty livers, grafts from older donors, or donation after cardiac death (DCD). The current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage. In addition, cold static organ storage does not allow graft assessment or repair prior to transplantation.
These shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury, assess liver grafts during preservation, and explore the opportunity to repair marginal livers prior to transplantation. The optimal pressure and flow conditions, perfusion temperature, composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past.
In spite of promising results in several animal studies, the complexity and the costs have prevented a broader clinical application so far. Recently, with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved.
This paper will provide information about liver retrieval, storage techniques, and isolated liver perfusion in pigs. We will illustrate a) the requirements to ensure sufficient oxygen supply to the organ, b) technical considerations about the perfusion machine and the perfusion solution, and c) biochemical aspects of isolated organs.

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

Marginal grafts, such as fatty livers, grafts from older donors, or livers retrieved after cardiac death (DCD) tolerate conventional, cold static storage only poorly. We developed a novel model of subnormothermic ex vivo liver perfusion for preservation, assessment, and repair of marginal liver grafts prior to transplantation.

Subnormothermic的技术<em&gt;前体内</em&gt;肝脏灌注的存储，评估和修复边际移植肝

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for ...

科研

医学

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

的隔离方法<em&gt;离体</em&gt;肺灌注的大鼠模型:教训制定鼠EVLP课程中学

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Ex Situ Normothermic Machine Perfusion of Donor Livers

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous perfusion of organs provides the opportunity to improve organ quality and allows ex situ viability assessment of donor livers prior to transplantation. This video article provides a step by step protocol for ex situ normothermic machine perfusion (37 &#176;C) of human donor livers using a device that provides a pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous perfusion of the portal vein. The perfusion fluid is oxygenated by two hollow fiber membrane oxygenators and the temperature can be regulated between 10 &#176;C and 37 &#176;C. During perfusion, the metabolic activity of the liver as well as the degree of injury can be assessed by biochemical analysis of samples taken from the perfusion fluid. Machine perfusion is a very promising tool to increase the number of livers that are suitable for transplantation.

Ex Situ Normothermic Machine Perfusion of Donor Livers

Here we present a protocol describing oxygenated ex situ machine perfusion of donor liver grafts. This article contains a step by step protocol to procure and prepare the liver graft for machine perfusion, prepare the perfusion fluid, prime the perfusion machine and perform oxygenated normothermic machine perfusion of the liver graft.

供肝易地常温灌注机

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

常温<em&gt;离体</em&gt;肾脏的灌注肾的移植保护移植前

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

直接节段性肝内交付使用大鼠肝门部夹具模型方法

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

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

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 hemorrhages at a later stage. The exact effects of plasma coagulation on liver tissue are only poorly examined. In our porcine model, the coagulation effects can be examined close to the clinical application. A combined laser Doppler flowmeter and spectrophotometer documents microcirculation changes during coagulation at 8 mm tissue depth noninvasively, providing quantifiable information about hemostasis beyond the subjective clinical impression. The temperature at coagulation site is assessed with an infrared thermometer prior and post coagulation and with a thermographic camera during coagulation, a measurement of the gas beam temperature is not possible due to the upper threshold of the devices. The depth of coagulation is measured microscopically on hematoxylin/eosin stained sections after calibration with an object micrometer and gives an exact information about the power setting-coagulation depth-relation. The sealing effect is examined on the bile ducts as it is not possible for a plasma coagulator to seal larger blood vessels. Burst pressure experiments are carried out on explanted organs to rule out blood pressure related effects.

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

Here we present a protocol to experimentally assess plasma coagulation in liver tissue in vivo. In a porcine model, microcirculation is examined by laser Doppler, coagulation depth is measured histologically, temperature at coagulation site by infrared thermometer and thermographic camera, and duct sealing effect is documented by burst pressure experiments.

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 ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation time and increases the risk of adverse post-transplantation outcomes. Moreover, the static nature of CS does not allow for organ evaluation or intervention during the preservation interval. Normothermic ex situ heart perfusion (ESHP) is a novel method for preservation of the donated heart that minimizes cold ischemia by providing oxygenated, nutrient-rich perfusate to the heart. ESHP has been shown to be non-inferior to CS in the preservation of standard-criteria donor hearts and has also facilitated the clinical transplantation of the hearts donated after the circulatory determination of death. Currently, the only available clinical ESHP device perfuses the heart in an unloaded, non-working state, limiting assessments of myocardial performance. Conversely, ESHP in working mode provides the opportunity for comprehensive evaluation of cardiac performance by assessment of functional and metabolic parameters under physiologic conditions. Moreover, earlier experimental studies have suggested that ESHP in working mode may result in improved functional preservation. Here, we describe the protocol for ex situ perfusion of the heart in a large mammal (porcine) model, which is reproducible for different animal models and heart sizes. The software program in this ESHP apparatus allows for real-time and automated control of the pump speed to maintain desired aortic and left atrial pressure and evaluates a variety of functional and electrophysiological parameters with minimal need for supervision/manipulation.

Normothermic ex situ heart perfusion (ESHP), preserves the heart in a beating, semi-physiologic state. When performed in a working mode, ESHP provides the opportunity to perform sophisticated assessments of donor heart function and organ viability. Here, we describe our method for myocardial performance evaluation during ESHP.

工作模式下的心功能和代谢评估

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation ...

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 ...

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ quality exacerbated by excessive geographic transportation distance and stringent donor organ acceptance criteria pose limitations to current LTx efforts. Ex situ lung perfusion (ESLP) is an innovative technology that has shown promise in attenuating these limitations. The physiologic ventilation and perfusion of the lungs outside of the inflammatory milieu of the donor body affords ESLP several advantages over traditional cold static preservation (CSP). There is evidence that negative pressure ventilation (NPV) ESLP is superior to positive pressure ventilation (PPV) ESLP, with PPV inducing more significant ventilator-induced lung injury, pro-inflammatory cytokine production, pulmonary edema, and bullae formation. The NPV advantage is perhaps due to the homogenous distribution of intrathoracic pressure across the entire lung surface. The clinical safety and feasibility of a custom NPV-ESLP device have been demonstrated in a recent clinical trial involving extender criteria donor (ECD) human lungs. Herein, the use of this custom device is described in a juvenile porcine model of normothermic NPV-ESLP over a 12 h duration, paying particular attention to management techniques. Pre-surgical preparation, including ESLP software initialization, priming, and de-airing of the ESLP circuit, and the addition of anti-thrombotic, anti-microbial, and anti-inflammatory agents, is specified. The intraoperative techniques of central line insertion, lung biopsy, exsanguination, blood collection, cardiectomy, and pneumonectomy are described. Furthermore, particular focus is paid to anesthetic considerations, with anesthesia induction, maintenance, and dynamic modifications outlined. The protocol also specifies the custom device's initialization, maintenance, and termination of perfusion and ventilation. Dynamic organ management techniques, including alterations in ventilation and metabolic parameters to optimize organ function, are thoroughly described. Finally, the physiological and metabolic assessment of lung function is characterized and depicted in the representative results.

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

This paper describes a porcine model of negative pressure ventilation ex situ lung perfusion, including procurement, attachment, and management on the custom-made platform. Focus is made on anesthetic and surgical techniques, as well as troubleshooting.

非原位负压通气肺灌注:肺功能和代谢的评估

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ ...