我们要感谢索林集团（米兰，意大利）为我们提供了定制的肾脏灌注回路。此外，我们感谢XVIVO灌注公司（瑞典哥德堡）为我们提供了解决方案斯蒂恩，BBraun AG（梅尔松根，德国）与注射泵供给，里伯GMBH＆CO。KG（Reutlingen，德国）。

注意:研究方案的示意性概述示于图1。 <img alt="图1" src="/files/ftp_upload/52909/52909fig1.jpg" /> 图1.研究方案。本研究的常温体外肾脏灌注协议是基于一个猪的模型。的肾移植物的血管外科解剖并用500ml组氨酸 - 色氨酸酮戊二酸（HTK）冲洗后，将移植物可以检索。冷藏后（SCS）3小时，肾脏移植是常温灌注离体 （NEVKP）多个小时，直到指定的移植。 所有动物人文关怀符合&#39;"实验动物管理的原则&#39;&#39;&#39;实验动物的护理指南&#39;由美国国立卫生研究院公布制定的全国协会医学研究和"，加拿大安大略省。在多伦多综合研究所的动物护理委员会批准的所有研究。 1.动物<ol><li>在这个协议 - （33千克27）使用雄性约克夏猪。 </li></ol> 2.器官检索<ol><li>术前程序<ol><li>房子的雄性约克夏猪的研究设施为至少一周，以减少他们的压力水平。快猪诱导麻醉前至少6小时。 </li><li>通过注射氯胺酮（25毫克/千克），阿托品（0.04毫克/千克），和咪达唑仑（0.15毫克/千克）的肌内引发猪麻醉。接着，从壳体设施到手术室（OR）运输的动物。 </li><li>将猪上或桌子仰卧位。让它呼吸2升氧气与异氟醚5％油然而生。松弛后，暴露声带与喉镜，用2％利多卡因将它们喷雾到防止由插管痉挛。自动对焦之三插管6.5毫米的管道，阻止与3袖口 - 5毫升空气。 注:Capnometry揭示了气管插管的正确位置。 </li><li>降低了异氟醚气至2.5％。呼吸机设置为14 - 16次呼吸/分钟，潮气量到10 - 24毫升/公斤体重。密切监测猪。心脏速率和氧饱和度由脉搏血氧被记录下来。 </li><li>在无菌条件下，引入一个8.5大一×10cm的导管插入在Seldinger技术13的颈静脉。因此，用针穿刺静脉血管。引入线后，更换导管针。消除电线固定导管到皮肤上。管理将200ml每小时乳酸林格氏液在整个手术。 </li></ol></li><li>手术过程<ol><li>继消毒和覆盖手术区域，执行从xyphoid正中切口，以耻骨联合。为了改善曝光，延长的手术方法左外侧切口。涵盖了大，小肠道用毛巾并把它们放置到左侧向右侧肾脏最佳接入。 </li><li>分离从腹主动脉的下腔静脉（IVC）。结扎主动脉分支从主动脉的背面。 </li><li>完成后主动脉夹层到后面，绕过主动脉颅一个绑带，以肾分支机构。此外，把两个连写颅髂分叉。将左肾动脉周围领带。 </li><li>自由右肾从其附着的组织。解剖肾静脉，动脉，输尿管。 </li><li>打开隔膜和管理每公斤体重捐助1000 IU肝素到心脏。对于DCD模式，心内后3分钟全身肝素注射40 MVAL氯化钾诱发心脏骤停。心脏骤停的价值作为热缺血的起点。 </li><li>同时，收集血液，连接的CPDA袋（柠檬酸盐，磷酸盐，葡萄糖，一个线denosine），以引入到左上颈静脉导管。执行软旋（1500 XG无制动）。取出血浆和无菌条件下捉鬼外套（生物安全柜II级）和存储红细胞输血。 </li><li>导管插入与器官平齐线以上髂骨分叉的主动脉。扎连字在主动脉和左肾动脉。 </li><li>同花顺组氨酸，色氨酸酮戊二酸（HTK）解决方案的肾以100厘米水柱2 O的压力夹紧胸部静脉和经颈静脉导管采集血液。下面切开肾静脉腹腔静脉，以确保肾脏的最佳平齐。 </li><li>右肾的完整冲洗后，检索与主动脉的其中一部分的移植物。切开肾静脉和离开输尿管长。 </li></ol></li><li>移植肾的灌注表背准备<ol><li>无组织粘连肾脏。关闭主动脉颅部分用领带第二导管插入一个1/4"×3/8"减速器的下部。摘下领带小动脉分支从主动脉的到来。 </li><li>导管插入肾静脉与1/4"X 3/8"直接减速。插管具有8大一输尿管鼻饲管。 </li><li>置于冰上肾脏直到NEVKP的开始。 </li></ol></li></ol> 3.常温体外灌注肾（NEVKP） <ol><li>灌注电路的研制 灌注电路由新生儿体外循环设备（ 图2）。 <img alt="图2" src="/files/ftp_upload/52909/52909fig2.jpg" /> 所述灌注回路的图2的示意图。该电路由新生儿体外循环技术。该灌注溶液被收集于静脉储血。离心泵推动该溶液进氧合器，在那里它富含氧气并加热至37℃。 PA后通过肾动脉进入肾脏80毫米汞柱 - ssing动脉泡沫过滤器，灌注液被驱动用的60的压力。静脉流出（0 - 3毫米汞柱）导致灌注液放回静脉储血。注射器和输液泵确保与另外的化合物的供应量。尿液收集在整个灌注。灌注回路特性被连续记录。每小时静脉和动脉血液气体样品，和肾损伤标志物进行了分析。 <ol> <li>连接定制电路灌注到肾脏灌注设备。 </li><li>油管连接到静脉水库和氧合。动脉线管路连接到人工肺的动脉流出并定位在其支架气泡过滤器。连接吹扫管线。静脉线管连接到静脉水库的入口。 </li><li>对于灌注过程中的评估，插上温度探头插入动脉插座，连接流量计和BUbble传感器到动脉线路管道，和连接压力线。连接的水位传感器。 </li><li>连接静脉和动脉采样线的静脉和动脉取样口。 </li><li>定位器官腔上的立场，并通过预制孔介绍静脉和动脉管。修复管道表和室牢固。 </li><li>插入吸入管导入滚子泵和一个端部的位置进入腔室以收集液体。 </li><li>氧管路连接到含95％ 的 O 2/5％CO 2和氧合气体罐。加热单元管路连接到氧合器和器官腔。 </li><li>使用管夹关闭静脉和动脉流出线。应用另一种管夹的静脉储血的流出。 </li></ol></li><li>准备灌注的解决方案，额外的补充，并灌注电路<ol><li>用一个输液泵替换所产生的尿液乳酸林格氏液。 </li><li>用一个注射泵施用营养（葡萄糖0.5毫升/小时，氨基酸为0.5ml /小时）和胰岛素（5单位/小时）进入静脉水库。利用第二注射器泵输注血管扩张剂（维拉帕米，0.25毫克/小时）直接进入动脉管路。 </li><li>填写静脉水库灌注液。因此，倾林格氏乳酸（175毫升）中，STEEN溶液（200毫升），DRO（27毫升），肝素（1000 IU），碳酸氢钠以调节pH，和葡萄糖酸钙进入静脉水库。最后，加入洗净的红细胞（125毫升）中。 </li><li>切换心脏人工心肺机（HLM）上。激活的压力，温度，电平，和气泡传感器面板。激活的数据管理系统（DMS），以在整个灌注记录数据。激活所述加热单元的地暖灌注溶液和器官腔至37℃。打开氧气供应。 </li><li>打开管夹静脉水库后面并释放centrifug人泵头空气完全。启动离心泵以1000rpm和使溶液在整个电路推进。夹紧管道绕过动脉过滤器，并从动脉过滤器释放空气。 </li><li>零压力线。激活的注射器和输液泵。 </li></ol></li><li>移植肾灌注<ol><li>从冰中取出肾脏，并在器官腔定位在床上用品的肾脏。将尿导管进入尿液收集器。在确保静脉和动脉导管是自由的空气，堵塞连接到管道。 </li><li>关闭动脉和静脉管道线之间的快捷方式。通过调节离心泵的速度上设置的动脉压至75毫米汞柱。 </li><li>记录的压力，动脉流量，温度，和存在气泡的连续与DMS。整个灌注仔细观察值。在灌注，血液泄漏到室是山口经由吸入管放回静脉储血反恐执行。 </li><li>记录尿液生成量。静脉采集血液和尿液样本小时。监视灌注通过采取静脉和动脉血液气体样品和天冬氨酸转氨酶（AST），和乳酸分析。 </li><li>在灌注结束时，从肾动脉断开管和静脉，冲洗用冷HTK接枝，并在冰上储存在无菌风琴袋直到移植。 </li></ol></li></ol>

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The authors have nothing to disclose.

这项研究表明，N​​EVKP与红细胞系溶液可以以优异的成绩的时间在猪模型长时间来执行。在10小时的体外灌注肾脏表现出稳定的灌注参数，活跃肾脏代谢，平衡，和最小的肾损伤。 尿生产和肾损伤取决于灌注液的组合物。重要的是要保持一个生理范围内肿胀压力和灌注液的容积渗透摩尔浓度是重要的。特别是，一个低肿胀压力将导致非生理性高尿生产与显著肾水肿和肾损伤的增加标记。 STEEN溶液含白蛋白被选择在该模型来调节渗透压和模拟为肾生理条件。碳酸氢钠和葡萄糖酸钙被添加到系统，以实现pH值的生理值，HCO 3 </sub&gt;中，钠，钾，钙和氯离子。血管扩张剂的选择和用量是很重要的，以确保充分的血流量和氧供应。 常温的体外灌注肾的技术有一些限制。 体外灌注不与激素的支持肾脏，这可能会较长时间灌注产生负面影响有关。此外，新技术，在这个时间点上，与成本增加相关联。未来的改进可能简化工艺，降低成本。便携式肾灌注设备的发展可能允许完全避免冷肾存储的未来。 严重和持续的器官短缺导致边际器官（ECD或DCD移植肾）7的使用增加。目前，器官保存是基于静态冷库及低温灌注机。作为一个长期的冷缺血时间有显著小鬼在标准条件15和边际移植8,9肾功能的结果行为，新的保鲜技术，最大限度地减少冷库特别感兴趣16-19。 一个主要障碍利用边际移植更广泛的是无法评估之前移植器官的质量和活力。目前，唯一的临床参数，如供体年龄，供体相关的疾病，和移植物的热缺血时间被用于器官是否被接受或拒绝用于移植的决定。通过保持常温条件下的移植，移植物评估的基础上灌注的特点和数据是可能的。实时参数如肾血管流量，压力，肾内，耐尿生产，耗氧量，和肾损伤的参数（如AST和乳酸）应该是有用的参数来评估接枝的可行性。 在ADDIT离子，NEVKP在代谢活跃允许修复策略的应用，提高边际移植肾移植前。例如，抑制促炎途径，免疫调节，基因转移，以及干细胞给药可能是未来的技术中的保留时间来修改肾脏移植物和提高收件人的结果。

肾脏是最常见的移植实体器官。对于患有终末期肾脏疾病的患者，相比1-4透析肾移植提供了更好的寿命和生活质量的提高。持续存在的器官短缺表示在移植医学（表1）5的励磁一个严重的问题。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td>美国* </td><td>欧洲移植区** </td></tr><tr><td>对肾移植等待名单的患者</td><td> 101563（2月，2015年） </td><td> 10689（月，2014年） </td></tr><tr><td>已故捐赠者的肾脏移植在2014年</td><td> 10,650 </td><td> 3119 </td></tr><tr><td>平均等待时间为已故捐赠者的肾脏移植（年） </td><td>长达5年的* </td><td>长达4年** </td></tr></tbody></table> 表1.肾移植物短缺的美国和欧洲移植区。 肾移植的结果是负面的等待时间的影响，对患者进行长期透析6预后较差。这引发了边际肾移植作为附加供体来源，如从旧供体肾脏，与多个合并症给体（扩展标准供体（ECD），和心脏死亡（DCD）后捐赠肾脏的兴趣。边际施主肾脏，将已在过去下降了现在被认为是移植7。 对于使用边际肾移植的主要障碍是冷缺氧储存保鲜技术。目前，肾移植物静态地存储在冰上或在4℃下灌注没有氧气。寒冷缺氧保鲜技术与正在进行的移植过程中损伤肾保存相关联，并且不允许因为缺乏代谢的移植评估olism和尿液生产。特别是，边际肾移植容忍冷库不佳，导致显著肾损伤，以及移植肾功能延迟（DGF）8,9很高。 DGF是穷人长期移植物功能的预后因素。 体外肾灌注代表器官的保存，评估和修复的替代方法。在猪模型，有益的结果提交给肾脏常温条件10,11-下灌注体外 。在2013年进行了第一次临床试验证明了移植肾功能延迟率较低时，从扩展标准供体肾脏取出移植12前灌注于紧接1小时。 本文介绍了常温体外灌流肾（NEVKP）的模型。本研究的目标是降低所施加的冷缺血时间减至最少，并延伸NEVKP的时期。 NEVKP是一种替代的保存方法，其目的是降低能够由冷藏技术所造成的损害。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Neonatal cardiopulmonary bypass technology</td>
			<td>SORIN GROUP Canada Inc (Markhan, Canada)</td>
			<td>Custom made</td>
			<td>Neonatal venous reservoir D100 (500 mL, 1/16&#34; in- and outflow), neonatal oxygenator D100, centrifugal pump head (Revolution), arterial bubble filter (D130)</td>
		</tr>
		<tr>
			<td>Heart lung machine, St&#246;ckert S3</td>
			<td>SORIN GROUP Canada Inc (Markhan, Canada)</td>
			<td>Custom made</td>
			<td>Centrifugal pump, roller pump, control panel (sensors for pressure, flow, temperature, bubbles, and level), oxygen blender, heater unit</td>
		</tr>
		<tr>
			<td rowspan="3">Tubing</td>
			<td>SORIN GROUP Canada Inc (Markhan, Canada)</td>
			<td>01906BPC SG XS</td>
			<td>3/16&#34;x 1/16&#34;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>019071PC SG XS</td>
			<td>1/4&#34;x 1/16&#34;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>019060PC SG XH</td>
			<td>3/8&#34;x 1/16&#34;</td>
		</tr>
		<tr>
			<td>Tubing connectors</td>
			<td>SORIN GROUP Canada Inc (Markhan, Canada)</td>
			<td></td>
			<td>Various sizes</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>STEEN solution</td>
			<td>XVIVO (G&#246;teborg, SWE)</td>
			<td>19004</td>
			<td>200 mL</td>
		</tr>
		<tr>
			<td>Ringer&#39;s lactate</td>
			<td>Baxter (Mississauga, ON, CAN)</td>
			<td>JB2324</td>
			<td>175 mL</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Hospira (Montr&#233;al, QC, CAN)</td>
			<td>6625050</td>
			<td>pH dependent</td>
		</tr>
		<tr>
			<td>Calcium gluconate</td>
			<td>Pharmaceutical Partners of Canada (Richmond Hill, ON)</td>
			<td>C31110</td>
			<td>Calcium dependent</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sandoz Canada Inc (Toronto, ON, CAN)</td>
			<td>10750</td>
			<td>1000 IU</td>
		</tr>
		<tr>
			<td>Amino acid and glucose, Travasol 10%</td>
			<td>Baxter (Mississauga, ON, CAN)</td>
			<td>JB6760</td>
			<td>1 mL/h</td>
		</tr>
		<tr>
			<td>Fast acting insulin, Novorapid</td>
			<td>Novo Nordisk Canada Inc (Mississauga, ON, CAN)</td>
			<td>DS6H748</td>
			<td>5 IE/h</td>
		</tr>
		<tr>
			<td>Verapamil</td>
			<td>Sandoz Canada Inc (Quebec, QC, CAN)</td>
			<td>52216</td>
			<td>0,25 mg/h</td>
		</tr>
	</tbody>
</table>

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.

在以下使用的心脏跳动的肾脏检索模型6的实验结果。 原位冲洗和肾检索后，将移植物在冰上储存3小时（SCS），而红细胞制备。为临床上，这模拟所需的检索和后表准备的时间。 NEVKP下进行10小时。 为了保持生理条件和模拟体内周围为肾脏，器官腔应当被加热并密封。灌注和尿替代解决方案应该代表的生理值血气分析，胶体渗透压和渗透压摩尔浓度。由Yorkshire猪原位得到正常值（基线值），位于各图的说明中，分别（ 图3 - 13）。 NEVKP的目的是为了确保接枝用足够的氧和营养供应。由于缺血CA采用血管收缩，从而增加肾内阻力，实现了恒定的流动具有稳定压力对于充分氧合的良好指标。经由SCS后的器官的复温达到37℃的目标接枝温度后，流量值和肾内电阻保持稳定，大约60-80毫米汞柱的恒定生理压力在整个灌注（ 图3和4）。尿生产的量主要依赖于灌注溶液（ 图5）的组合物。 静脉和动脉PO 2小时测量表明肾脏的代谢活动。 （ 图6）14 -使用等式（PO 2 VEN）X流量/重量（PO 2技术）中的氧消耗量算出。在灌注的pH值，HCO 3，并且电解质是稳定，无需干预（ 图7 - 10）。实时AST和乳酸测量用于监测细胞损伤。在NEVKP期间（ 图11和12）没有检测到增长的细胞损伤的参数。灌注溶液的摩尔渗透压浓度是稳定的（ 图13）。组织学评估显示微小变化（ 图14 - 16）。 <img alt="图3" src="/files/ftp_upload/52909/52909fig3.jpg" /> 图与标准偏差（毫升/分钟）3.平均动脉流动。在整个灌注流量保持在生理范围内。猪的生理价值， 在现场测量:是指艺术。流量:170±57毫升/分钟（范围83 - 325毫升/分钟）。 <img alt="图4" src="/files/ftp_upload/52909/52909fig4.jpg" /> 图4.肾内阻力（IRR），平均和标准偏差（毫米汞柱/ </strong&gt; 毫升/分钟），平均动脉压（MAP）保持60和80毫米汞柱之间恒定。肾内电阻低于0.5毫米汞柱/毫升/分钟不断。 <img alt="图5" src="/files/ftp_upload/52909/52909fig5.jpg" /> 图5.总尿量，平均值和标准偏差（毫升），总尿体积主要依赖于灌注溶液的组合物。的肿胀压力和摩尔渗透压浓度越高，越低的尿产量。 <img alt="图6" src="/files/ftp_upload/52909/52909fig6.jpg" /> 图6.耗氧，平均值和标准偏差（毫升/分钟/克）。 <img alt="图7" src="/files/ftp_upload/52909/52909fig7.jpg" /> 图7. pH值静脉，平均值和标准偏差。将pH REMA插件不断在生理范围，而不碳酸氢盐的管理。猪的生理价值， 在现场测量:pH值7.46±0.06（范围7.34 - 7.63）。 <img alt="图8" src="/files/ftp_upload/52909/52909fig8.jpg" /> 图8. HCO 3静脉，平均值和标准偏差（毫摩尔/升）的 HCO 3保持在生理范围内，而不碳酸氢施用。猪生理值， 原位测定:HCO 3 30.3±2.4毫摩尔/升（范围21.6 - 35.8毫摩尔/升）。 <img alt="图9" src="/files/ftp_upload/52909/52909fig9.jpg" /> 图9.静脉钠浓度，平均值和标准偏差（毫摩尔/升）的钠保持在生理范围内。猪的生理价值，在现场测量:137.1＆＃177; 3.8毫摩尔/升（范围118.7 - 140.9毫摩尔/升）。 <img alt="图10" src="/files/ftp_upload/52909/52909fig10.jpg" /> 图10.静脉钾浓度，平均值和标准偏差（毫摩尔/升）的钾保持在生理范围内恒定。猪生理值，原位测定:3.85±0.46毫摩尔/升（范围3.5 - 5.36毫摩尔/升）。 <img alt="图11" src="/files/ftp_upload/52909/52909fig11.jpg" /> 图11.静脉天冬氨酸转氨酶，平均值和标准偏差。（AST; U / L）。在离体常温肾灌注，AST表明细胞损伤标记物。 AST值低灌注各地。 <img alt="图12" src="/files/ftp_upload/52909/52909fig12.jpg" /> 图12:乳酸<html> ，平均值和标准偏差（毫摩尔/升），在体外常温肾灌注，乳酸盐表示细胞损伤的标记。这些数值是在整个灌注稳定。 <img alt="图13" src="/files/ftp_upload/52909/52909fig13.jpg" /> 图13:血清渗透压，均 ​​值和标准差（毫渗量/ L）的灌流液恒定的渗透压低固定不变，但尿量。猪生理值，原位测定:282±1.7毫渗/ L（范围279 - 283毫渗透摩尔浓度/升）。 <img alt="图14" src="/files/ftp_upload/52909/52909fig14.jpg" /> 图14:组织学（H＆E）50X / 200X放大倍率皮髓质交界处呈现出温和的管状空泡化。坏死的迹象。ARGET ="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图15" src="/files/ftp_upload/52909/52909fig15.jpg" /> 图15:组织学（PAS）50X / 200X放大倍率皮髓质交界处呈现出温和的管状空泡化。坏死的迹象。 <a href="https://www.jove.com/files/ftp_upload/52909/52909fig15large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图16" src="/files/ftp_upload/52909/52909fig16.jpg" /> 图16:组织学（TUNEL染色）25X / 200X的放大倍率。很偶然的细胞核染色证实凋亡率很低。 <a href="https://www.jove.com/files/ftp_upload/52909/52909fig16large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation at JoVE.com

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

严重的器官短缺已导致增加了移植用的肾脏边际移植。这引发了另一种存储方式利息，因为边际特别是移植耐受不良的冷库。的常温体外肾灌注技术（NEVKP）代表了一种新的保存方法，肾脏移植在移植前。

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 ...

normothermic-ex-vivo-kidney-perfusion-for-preservation-kidney-grafts

Research

JoVE Journal

Medicine

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

19.0K 次观看.  Toronto General Hospital. 严重的器官短缺已导致增加了移植用的肾脏边际移植。这引发了另一种存储方式利息，因为边际特别是移植耐受不良的冷库。的常温体外肾灌注技术（NEVKP）代表了一种新的保存方法，肾脏移植在移植前。 

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

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

小鼠胰岛的分离和包膜下肾移植的方法

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

科研

医学

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Study of the animal organ physiology can be achieved by performing an experimental ex vivo perfusion system. The addition of a porcine kidney, as a homeostatic organ to our previously developed ex vivo liver perfusion model can be a principal step to achieve a better physiological environment.

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an ...

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 ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

鼠肾移植:移植排斥反应模型

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

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 ...

Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion

There is currently a severe shortage of liver grafts available for transplantation. Novel organ preservation techniques are needed to expand the pool of donor livers. Machine perfusion of donor liver grafts is an alternative to traditional cold storage of livers and holds much promise as a modality to expand the donor organ pool. We have recently described the potential benefit of subnormothermic machine perfusion of human livers. Machine perfused livers showed improving function and restoration of tissue ATP levels. Additionally, machine perfusion of liver grafts at subnormothermic temperatures allows for objective assessment of the functionality and suitability of a liver for transplantation. In these ways a great many livers that were previously discarded due to their suboptimal quality can be rescued via the restorative effects of machine perfusion and utilized for transplantation. Here we describe this technique of subnormothermic machine perfusion in detail. Human liver grafts allocated for research are perfused via the hepatic artery and portal vein with an acellular oxygenated perfusate at 21 &#176;C.

We describe a method of ex vivo machine perfusion of human liver grafts at subnormothermic temperature (21 &#176;C).

功能人类肝脏保存和恢复由Subnormothermic机灌注手段

There is currently a severe shortage of liver grafts available for transplantation.  Novel organ preservation techniques are needed to expand the pool of ...

Murine Kidney Transplant Technique

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique using the donor aorta to form the arterial anastomosis instead of the renal artery was developed and reported in 1993 by Kalina and Mottram 2 with a further advancement coming from the same laboratory in 1999 3. While one can become proficient in this model, a search of the literature reveals that many labs still experience a high proportion of graft loss due to arterial thrombosis. We describe here a technique that was devised in our laboratory that vastly reduces the arterial thrombus reported by others 4,5. This is achieved by forming a heel-and-toe cuff of the donor infra-renal aorta that facilitates a larger anastomosis and straighter blood flow into the kidney.

The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis.

小鼠肾脏移植技术

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

Intravascular Delivery of Biologics to the Rat Kidney

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

生物血管内送货到大鼠肾脏

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

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, ...

Orthotopic Kidney Auto-Transplantation in a Porcine Model Using 24 Hours Organ Preservation And Continuous Telemetry

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for kidney transplantation (KT). Even though, the use of allografts from extended criteria donors (ECD) could partially ease the shortage of organ donors, ECD organs carry a potentially higher risk for inferior outcomes and postoperative complications. Dynamic organ preservation techniques, modulation of ischemia-reperfusion and preservation injury, and allograft therapies are in the spotlight of scientific interest in an effort to improve allograft utilization and patient outcomes in KT.
Preclinical animal experiments are playing an essential role in translational research, especially in the medical device and drug development. The major advantage of the porcine orthotopic auto-transplantation model over ex vivo or small animal studies lies within the surgical-anatomical and physiological similarities to the clinical setting. This allows the investigation of new therapeutic methods and techniques and ensures a facilitated clinical translation of the findings. This protocol provides a comprehensive and problem-oriented description of the porcine orthotopic kidney auto-transplantation model, using a preservation time of 24 hours and telemetry monitoring. The combination of sophisticated surgical techniques with highly standardized and state-of-the-art methods of anesthesia, animal housing, perioperative follow up, and monitoring ensure the reproducibility and success of this model.

Large animal models play an essential role in preclinical transplantation research. Due to its similarities to the clinical setup, the porcine model of orthotopic kidney auto-transplantation described in this article provides an excellent in vivo setting for the testing of organ preservation techniques and therapeutic interventions.

使用24小时器官保存和连续遥测的猪模型中正交肾自动移植

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for ...

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此视频中的章节

小鼠胰岛的分离和包膜下肾移植的方法

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Subnormothermic的技术

鼠肾移植:移植排斥反应模型

供肝易地常温灌注机

功能人类肝脏保存和恢复由Subnormothermic机灌注手段

小鼠肾脏移植技术

生物血管内送货到大鼠肾脏

体外常温肝灌注的小动物模型

使用24小时器官保存和连续遥测的猪模型中正交肾自动移植