Name: mouse model for pancreas transplantation using a modified cuff technique
Uploaded: 2017-12-16T13:00:00.000+00:00
Duration: 13 min 19 s
Description: Watch this Scientific Journal Video about Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique at JoVE.com

这项工作得到了赠款 #2008-1-596 和 #UNI-0404/1956 的 "交响 Wissenschaftsfonds (TWF)" (https://www.tirol.gv.at/en/), 并通过赠款 #2013-042018 的 "梅-开始 Förderungsprogramm" 的医学大学因斯布鲁克。

为了避免免疫反应, 并严格研究缺血再灌注损伤相关的移植损伤, 应采用体供者-受体对。在本协议中, 雄性 C57BL6 (H2b) 10-12 周大的小鼠体重26到28克, 被用作大小匹配的供体受体对。所有动物都被安置在一个屏障病原体免费设施, 并接受了人类的照顾, 符合 "实验室动物护理负责人", 由全国医学研究学会和 "实验室动物的关心和使用指南" 制定由国家科学院编写, 由国立卫生研究院出版 (NIH 出版物86-23 号, 修订 1985)。奥地利教育、科学和文化部批准了这份手稿中所描述的实验 (BMWF-66.011/0056-二/3 b/2011)。
1. 胰腺的采购
<ol>
	<li>麻醉用27口径 (G) 针进行腹腔 (ip) 注射嗪 (5 毫克/千克体重) 和氯胺酮 (100 毫克/千克体重) 的供体动物。</li>
	<li>用电动剃须刀将腹部的毛发剃光, 用胶带将鼠标固定在手术场上。</li>
	<li>用洗必泰浸泡的纱布擦洗手术场三次。</li>
	<li>用剪刀进行双侧下延长腹部中线切口。用不育的棉花棒轻轻地将内脏取出在左边, 用湿纱布裹住它们。用蚊子钳提起 xyphoid cranially, 为以下步骤提供最大的腹腔暴露。</li>
	<li>使用19克针, 注射400µL 1:4 肝素氯化钠溶液 heparinisation 入下腔静脉 (下静脉)。取出针后, 安乐失血 transecting 的主动脉。</li>
	<li>用弯曲尖端钳将纤维组织轻轻分离, 解剖肠系膜上动脉和右肾动脉之间的腹主动脉。破坏主动脉, 并与8/0 丝结扎。</li>
	<li>确定十二指肠韧带, 在 postpyloric 十二指肠和肝门之间运行。在远端结扎后, 将胆囊管入口下方的胆管分开, 并轻轻地解剖和横断面的门静脉尽可能地有足够的长度, 以执行吻合在接受者。</li>
	<li>使用弯曲尖端钳, 坦率地解剖腹主动脉从先前的结扎 (见步骤 1.6)。用弯曲的尖端钳破坏 periaortic 组织, 用8/0 的丝线结扎, 用剪刀将其横断面。<ol>
		<li>使用双极钳, 凝聚其所有腰椎分支和横断面的主动脉与剪刀尽可能密切的隔膜, 以提供足够的长度血管吻合。最后, 在左肾动脉上方横断面已系的主动脉。</li>
	</ol>
	</li>
	<li>灌注19克注射器与5毫升的4° c 组氨酸-色氨酸-二灌注溶液在顺的方式通过腹主动脉, 直到有一个明确的流出来自门静脉。应用低压, 避免水肿形成。 注: 执行步骤1.5 至1.9 的快速和标准化的方式, 以避免任何偏见的热缺血性恶化的恢复移植。</li>
	<li>用无菌棉棒将内脏换成腹膜腔。</li>
	<li>使用弯曲尖端钳, 分离胰腺逐步从 postpyloric 十二指肠开始, 并向前移动, 直到到达 Treitz 韧带。对于这些步骤, 确定胰腺与十二指肠壁之间的缺血性结缔组织区域。<ol>
		<li>坦率地解剖这些地区使用弯曲尖端钳, 以隔离桥之间的胰和十二指肠。在每一个孤立的血管结构周围传递一个8/0 的丝线结扎, 并将其绑住。</li>
		<li>最后, 用剪刀将血管结构横断面向十二指肠壁。以同样的方式, 用8/0 丝缝线将胰腺与肠系膜、横结肠和胃分开。 注: 在此过程中, 胆胰管结扎。</li>
	</ol>
	</li>
	<li>鉴别从脾到胃的 gastrosplenic 韧带和短胃分支, 通过提起胃 cranially 并用剪刀切开。把脾脏附着在回收的移植物上。 注: 为了最大限度地减少移植温, 灌溉胰腺持续使用10毫升注射器与19克针与冷氨酸氨酸-色胺-二灌注溶液储存在冰上。</li>
	<li>最后, 从捐献者的位置 (图 1B) 中取出胰腺, 用钳子抓住脾脏, 并将其转移到受体。<ol>
		<li>另外, 为了引发严重的缺血再灌注损伤, 将移植物存放在无菌、4° c 的灌注液中16小时, 然后植入受体动物体内。</li>
	</ol>
	</li>
</ol>
2. 收件人准备
<ol>
	<li>使用27克针, 麻醉的受体动物与一个 ip 注射嗪 (5 毫克/千克体重) 和氯胺酮 (100 毫克/千克体重)。</li>
	<li>用电动剃须刀剃掉右侧颈部区域, 并将鼠标放在手术场上仰卧位。使用磁带修复鼠标。避免过度前肢体, 以免损害呼吸。</li>
	<li>用洗必泰浸泡的纱布擦洗手术场三次。</li>
	<li>右旁, 轻微倾斜的皮肤切口从颈静脉切口到右下颌角。</li>
	<li>直接识别并调动右侧颈外静脉的侧支。用双极钳将它们凝固, 用剪刀将它们横断面。</li>
	<li>提起下颌下腺 cranially 的右叶, 识别血管蒂, 用双极钳烧灼。用剪刀 transecting 烧灼蒂, 取出耳垂。</li>
	<li>在类比2.5 步, 确定所有内侧分支的颈外静脉, 烧灼他们用双极钳和横断面他们用剪刀。</li></ol>破坏颈外静脉与弯曲尖端钳 cranially 尽可能, 并结扎它与两个8/0 丝连字, 留有足够的空间之间的绷带。
	<li>在两个先前放置的连字与直剪刀之间的颈外静脉横断面。</li>
	<li>通过聚乙烯袖口 (内径为0.75 毫米, 外径为0.94 毫米) 通过颈外静脉的近端, 并用静脉 microhemostat 钳固定袖口的手柄。</li>
	<li>在船残端上取下领带, 并埃弗特在袖口上。用圆形8/0 丝绷带 (图 1A) 将袖口上的外翻静脉固定。</li>
	<li>下部和远端烧灼右胸锁乳突肌的表面部分, 横断面与剪刀, 并删除它。</li>
	<li>轻轻地调动颈总动脉, 在分岔下破坏它, 并结扎两次, 确保不系颈动脉分岔。切断领带之间的船只。</li>
	<li>与步骤2.9 类似, 通过聚酰胺袖套 (内径为0.57 毫米, 外径为 0.6 mm) 通过颈总动脉的近端, 并用动脉微血管钳固定。</li>
	<li>取出血管端部的结扎, 用血管 dilatators 轻轻地加宽管腔。埃弗特的血管在动脉袖口, 并修复它与8/0 丝结扎 (图 1A)。</li>

3. 植入
<ol>
	<li>将移植物放入接受者颈部, 用脾作手柄, 头部侧向, 尾包括脾内侧, 腹尾。用棉签正确定位移植物。</li>
	<li>轻轻地将胰腺移植的门静脉移过受体动物的颈外静脉, 这是先前外翻的, 并固定在适当的袖口上 (见步骤 2.10)。用圆形的8/0 真丝绷带固定。</li>
	<li>将移植物腹主动脉的残端拉到受体动物外翻颈总动脉上。用圆周8/0 丝线结扎 (图 1C) 进行修复。</li>
	<li>确定脾血管靠近脾脏的脐部, 并用弯曲的尖端钳将其破坏。用8/0 条丝线结扎脾血管, 切除脾脏。最后, 缩短联系。</li>
	<li>使用钳子钳, 先取下静脉袖夹。然后取出动脉钳 注: 如果移植成功, 胰腺移植将立即灌注显示一个均匀的粉红色的颜色和可见的动脉搏动 (图 1D)。</li>
	<li>用常温盐水溶液润湿移植物。</li>
	<li>用直钳取出静脉袖的手柄。</li>
	<li>用6/0 缝合线闭合手术伤口。</li>
</ol>
4. 术后护理 (终点)
<ol>
	<li>按照该程序, 应用高达0.5 毫升的正常生理盐水皮下 (南卡罗来纳州), 以取代术中液体流失使用19克针。</li>
	<li>将接受者的动物放在加热垫上, 直到完全从麻醉中恢复。</li>
	<li>一旦醒来, 将接受的动物返回到住房设施, 在那里它可以有食物和水ad 随意。</li>
	<li>为防止术后疼痛, 在手术后施行 (1) Buprenorphin (0.1 毫克/千克 b.w.) 每12小时的前5天和 (2) 卡洛芬4毫克/千克 b.w. 每12小时的第一周。</li>
	<li>为了估计适当的营养摄入量, 每天监测每个接受动物的体重 (g)。在手术当天体重减轻超过 10-15%, 冷漠, 致残, 背部非常弯曲, 以及手术侧感染代表终点。<ol>
		<li>在这种情况下, 以及达到临床终点后, 牺牲的动物使用终端异氟醚吸入。</li>
	</ol>
	</li>
</ol>

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作者声明他们没有竞争的金融利益。

联合移植损伤是实体器官移植的固有功能, 其特点是微循环障碍。缺血期多代谢物的积累, 主要由活性氧和氮类介导的炎症叶栅的形成, 导致移植再灌注过程中的组织损伤4。这种级联不仅会危害短期, 而且会影响长期的成功, 从而对患者的生存有很大的作用14。到目前为止, 联合肾胰移植代表的选择治疗1型糖尿病与终末期肾脏疾病的病人15。一些研究表明, 成功的联合肾胰移植不仅能恢复和保护糖尿病患者的肾移植功能, 而且还能稳定甚至逆转继发性并发症, 包括神经病变以及微-和病变16,17,18。
尽管在动物研究中不断地减少、替换和提炼 (3 R), 但像 IRI 这样的复杂病理生理过程的复制在体外设置中是不可能的。因此, 动物模型仍然被认为是转化研究的理想工具19,20。与老鼠或其他动物模型相比, 这里描述的老鼠模型有几个优点。这些因素包括大量的基因定义的近交系小鼠菌株 (如转基因和淘汰赛菌株), 大量的分子分析工具, 以及一个简单和便宜的处理21。所述模型的主要优点在于 non-suture 袖套技术。通过使用本文提出的技术, 成功率和 #62; 90% 是可以实现的, 这比以前描述的模型22的效果要好得多。使用这种 non-suture 技术, 我们明显减少常见的并发症, 如休克休克, 血栓形成, 和狭窄的吻合12。这种方法的一个进一步的好处是包括额外的腹部位置的移植, 这是与快速术后恢复的接受者。此外, 宫颈位置使它完全适合体内分析, 如 exterioration 的活体成像, 无任何张力22。
该模型的主要缺点是阻断胰管, 这不像临床现实。在这个模型中, 外分泌引流是通过结扎胆胰管来管理的。从长期来看, 这导致了明显的纤维化和腺体萎缩, 而不导致移植性胰腺炎22。由于外分泌组织的这种恶化, 我们在移植后的30天早观察到, 我们认为这个模型不适合长期观察。与此相反, 未受损伤的内分泌功能使 gylcemic 控制接收器的一个简单的工具, 每天评估的功能移植13,23,24。
这些特点使这成为一个理想的模型, 分析早期移植损伤与长期保存期或不同的保存方案和技术。为了获得最佳的成功与这个模型, 必须考虑几个关键的步骤。胰腺本身很容易受到操控。因此, 在器官恢复和植入过程中使用棉花棒进行轻柔的处理能最大限度地减少机械损伤。应避免用钳直接抓取腺体, 因为这将不可避免地导致严重的移植损伤。由于同样的原因, 脾脏与胰腺一起被恢复, 并且被用作手柄。这也是建立在临床实践。进一步的陷阱包括冷灌注, 这是通过使用4° c 组氨酸-二灌注液灌注通过主动脉残端。因此, 通过轻轻灌注移植, 可以避免腺体过度肿胀。其余的灌注液应用于润湿移植物, 以使其在器官恢复过程中保持较低的温度。
对于接受者的准备, 仔细解剖外颈静脉和颈总动脉, 为成功的血管设置基础。特别是, 通过去除所有的支流, 而且周围的脂肪组织, 完全暴露的静脉, 是必要的, 以避免外部压迫和狭窄的残余脂肪组织。适当的袖口直径的选择是关键的。根据共同的经验, 对于体重在25到28克之间的小鼠来说, 动脉袖的内径为0.57 毫米, 在0.75 和0.8 毫米之间的静脉袖口是合适的。精确, 清洁的袖口边缘的切割是强制性的, 以避免撕裂的船只树桩。血管, 特别是动脉的扩张, 是利用血管 dilatators 的最佳方法。作为一个经验法则, 血管应该能够扩大到两倍的袖口的流明。在外翻的过程中, 把它固定在袖口上, 我们建议通过将血管夹放在皮瓣下使其稳定下来, 这样就能减轻这一关键步骤。
如上所述, non-suture 袖技术代表了一种简单的血管吻合方法, 可以在5分钟内完成。然而, 正确的定位移植在接受者的颈部区域是至关重要的正确的重建。因此, 必须对颈部区域的移植物的最终正确定位进行预测, 以保证静脉和动脉的安全、直接和无张力吻合。太长的船只必须避免, 因为这可能导致流出的障碍, 由于康祺。由于同样的原因, 在静脉吻合术中的袖柄也应在再灌注后去除。在胰腺移植局部 bleedings 的情况下, 用棉签轻轻按压出血侧5分钟即可取得成功的止血效果。这是唯一成功的方法来管理这种并发症。烧灼, 即使是高度选择性, 导致移植丢失几乎所有的情况下, 由于坏死性胰腺炎。
总之, 我们开发了一种方法, 在小鼠胰腺移植使用 non-suture 袖技术, 这是技术上和 microsurgically 可行的, 有很好的成功率。由于导管闭塞导致胰腺 progredient 纤维化, 因此该模型最适合于以早期移植损伤为重点的研究领域。这份手稿的目的是让研究人员在他们的实验室安全地建立这个模型。

同时肾-胰移植 (SPK) 代表了目前的护理标准, 糖尿病患者和终末期肾脏疾病。成功的移植导致长期胰岛素独立与稳定甚至回归糖尿病血管和更好的生活质量1。然而, 与其他常见的实体器官移植, 如肾脏和肝移植, 胰腺移植更容易发生缺血再灌注损伤 (IRI)。报告的发病率高达 35%, 不仅危害移植, 甚至病人, 生存2,3。
氧化应激, 微循环紊乱, 炎细胞因子的表达增加, 黏附分子最终导致内皮细胞的活化和完整性丧失, 都归因于这种 non-allogeneic 移植损伤4. 到目前为止, IRI 的确切分子机制在很大程度上是未知的, 并且可能因器官而异。
尽管在利用体外模型方面取得了重大进展, 但动物模型的发展对于加深对在胰腺移植后的 IRI 相关的移植改变的分子机制的认识至关重要。几个胰腺移植模型已开发的啮齿动物5,6, 但只有一个报告在老鼠7。这种高度苛刻的显微外科手术的致命弱点是低存活率为46%。然而, 老鼠模型代表最佳的模型为移植相关的研究, 因为最广泛的各种各样的分子分析工具可以适用于他们。基于对不同器官移植小鼠的广泛显微外科经验8,9,10, 我们开发了一种新的, 高重复性的异位, 宫颈胰腺的技术用 non-suture 袖技术移植小鼠和 #62; 90% 成功率。与此技术, 吻合相关的并发症减少到最低, 并取得了很高的成功率, 可以实现与缝合模型11。到目前为止, 只有一个类似成功率的鼠标模型被刘et al12描述。然而, 目前还没有使用该模型发表的研究报告。

<table><tbody><tr><td>Adventitia Scissors</td><td>S&amp;amp;T</td><td>S-00102</td><td>Straight</td></tr><tr><td>Dumont # 7 Forceps</td><td>FST</td><td>&amp;nbsp;11271- 30</td><td>Curved Tip 0.17 x 0.1 mm</td></tr><tr><td>Yasargil Clip Mini Permanent 7mm</td><td>Aesculap</td><td>FE720K</td><td></td></tr><tr><td>Micro vessel clip</td><td>S&amp;amp;T</td><td>B1 00396 V</td><td></td></tr><tr><td>Vessel dilatator</td><td>S&amp;amp;T</td><td>D-5a.2, 00125</td><td></td></tr><tr><td>Clip applier</td><td>S &amp;amp; T</td><td>CAF-4 00072</td><td>for venous cuff</td></tr><tr><td>Clip applier</td><td>Aesculap</td><td>FE572K&amp;nbsp;</td><td>for the arterial cuff</td></tr><tr><td>Polyethylene tube</td><td>Portex Ltd</td><td></td><td>Inner diameter 0.75 mm for venous cuff</td></tr><tr><td>Polymide tubing</td><td>Vention Medical&amp;nbsp;</td><td>141-0051</td><td>Inner diameter 0.8 mm (Alternative for polyethylene tube from Portex Ltd)</td></tr><tr><td>Polymide tubing</td><td>Vention Medical</td><td>141-0033</td><td>Inner diameter 0.57 mm for arteriail cuff</td></tr><tr><td>Bipolar forceps</td><td>Micromed</td><td>140-100-015</td><td></td></tr><tr><td>8/0 silk ligatures</td><td>Catgut GmbH, Merkuramed</td><td>17209008</td><td></td></tr><tr><td>Custodiol HTK solution</td><td>Dr. Franz K&amp;ouml;hler Chemie</td><td>59997</td><td></td></tr><tr><td>Ketamin Graeub</td><td>aniMedica GmbH</td><td>32554</td><td></td></tr><tr><td>Xylasol Graeub</td><td>aniMedica GmbH</td><td>50855</td><td></td></tr></tbody></table>

mouse model for pancreas transplantation using a modified cuff technique

小鼠模型在移植研究中有几个优点, 包括易于处理, 各种基因定义的菌株, 以及最广泛的分子探针和试剂的可用性来执行体内以及体外研究。根据我们在各种小鼠移植模型中的经验, 我们开发了一种模型, 目的是分析严重缺血再灌注损伤相关的早期移植损伤的机制。与以前描述的技术使用缝合技术对比, 这里我们描述一个新的过程使用 non-suture 袖口技术。
近年来, 我们在小鼠胰腺移植术中进行了超过300例, 总体成功率为 &gt; 90%, 在小老鼠胰腺中从未被描述成功。这种 non-suture 袖技术的骨干移植血管重建包括两个主要步骤: (I) 拉动接收器在聚乙烯/聚酰胺袖口和固定它与圆周结扎, 和 (II) 放置在捐助船只外翻接受者的船只, 并修复它与第二次圆周结扎。血管内皮层的连续性导致了血栓病变, 且通畅率高, 成功率高。
在这个模型中, 动脉吻合是通过拉动供体移植的腹主动脉在受体动物的外翻颈总动脉。静脉引流的移植是通过拉动门静脉移植的外翻外颈静脉的受体。这份手稿提供了器官恢复和器官植入程序的细节和关键步骤, 这将使研究人员有显微外科技术, 成功地在他们的实验室进行移植。

在过去的十年中, 我们在小鼠中进行了超过300胰腺移植。在建立该议定书之后, 总的生存率和 #62; 90%。术后出血是失败的主要原因, 其次是接枝血栓形成和随后的致命坏死性胰腺炎。在这两种情况下, 在24小时内达到了终点, 并牺牲了动物。本系列没有任何神经紊乱、吞咽困难和手术侧感染等症状。
为了研究移植移植物的内分泌功能, 从而验证模型的通畅性, 采用单剂量腹腔 (312.5 毫克/千克体重) 前处理对受体小鼠高血糖的诱导作用4手术前几天。如果血糖水平和 #62, 老鼠被认为是血糖; 300 毫克/dL。图 2A显示不同组的血糖水平。在移植后24小时内接受无长时间冷缺血的小鼠到达 normogylcemia, 并在整个观察期维持这种代谢状态。相比之下, non-transplanted 的动物仍然血糖。由于我们对缺血再灌注损伤相关的移植损伤对内分泌功能的影响有兴趣, 我们增加了第三组移植物暴露在16小时长的冷缺血时间 (CIT) 和45分钟的热缺血时间 (机智)。接受这些移植的小鼠没有到达血糖并且必须在 48 h 以后牺牲由于严重胰腺炎的发展, 被证明是致命的在这个模型13。
该模型是有用的各种项目, 旨在研究缺血再灌注损伤相关的早期移植损伤。进一步的调查包括, 除其他以外, 共聚焦活体荧光显微镜定量的微循环紊乱进行2小时后移植。将0.3 毫升的0.4% 荧光素异硫氰酸酯 (MW 150 000) 注入到生殖器中, 增强了移植物的微血管的对比。图 2B显示了一个天真的小鼠胰腺和移植的胰腺移植的正常毛细血管模式, 这是没有暴露在长时间的 CIT (图 2C)。相比之下,图 2D显示了由于将胰腺移植物暴露于长时间的 CIT 而导致微循环的破裂。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/54998/54998fig1.jpg"> 
图 1: 术中图片.(A) 为吻合而准备的接受血管的术中视图。外颈静脉 (1) 已外翻在静脉聚乙烯袖口和固定与循环8/0 真丝结扎。在类比中, 颈总动脉 (2) 已被外翻和固定在较小的动脉聚酰胺袖口。缩放条形图 1 mm (B)胰腺移植的原位。血管吻合术所需的门静脉 (1) 和腹主动脉 (2) 的残端。脾脏 (3) 与胰腺一起被检索并且使用作为把柄。脾脏在移植物再灌注前会被移除。缩放条形图1厘米(C)术中对吻合的看法。门静脉 (1) 被拉扯在外翻外颈静脉 (2) 的袖口和固定与一个圆环8/0 丝绸领带。同样, 腹主动脉 (3) 的主动脉残端被拉过外翻颈总动脉 (4) 的袖口。缩放条形图 1 mm. (D)术中对灌流性胰腺移植后5分钟再灌注后的看法: 静脉切除后, 动脉钳, 成功移植的胰腺移植显示一个均匀的粉红色的颜色。脾脏在再灌注前已被切除 (1: 结扎脾血管)。缩放栏1厘米.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54998/54998fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/54998/54998fig2.jpg"> 
图 2: 胰腺移植和共聚焦的内分泌功能在体内荧光显微镜.图 2A显示了无 CIT 移植小鼠血糖水平的折线图 (n = 10, PTX, 蓝线), non-transplanted 小鼠 (n = 11, 没有 PTX, 红线), 和小鼠接受移植暴露于长期 cit (PTX + 16 小时 cit, n = 10, 绿线)。所有接受者以前呈现血糖与312.5 毫克/千克 b.w. 链脲佐菌素 ip虽然没有 CIT 的所有移植接受者都能在整个观察期 (50 天) 内存活下来, 并具有完整的内分泌功能, 但 non-transplanted 小鼠在整个观察期内仍然血糖。接受16小时的移植物的小鼠没有从高血糖中恢复, 在移植手术后必须牺牲48小时, 因为体重下降超过10-15%。在最后一次血糖测定后50天, 在整个观察期内存活的小鼠被牺牲。采用共聚焦活体荧光显微镜观察移植后2小时的微循环。天真的胰腺充当了对照。图 2B显示了常规的毛细血管模式在幼稚的胰腺。在移植的移植物中也可以看到固定的毛细血管网 (图 2C)。相反, 在移植的移植物中, 暴露在长时间的 CIT (图 2D) 中观察到微循环的破裂。缩放条形图100µm. 图表中的数据被表示为±标准偏差。PTX: 胰腺移植;冷缺血时间;w/o: 没有<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54998/54998fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique at JoVE.com

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

在腹部实体器官移植中, 胰腺移植容易发生严重的缺血再灌注损伤-相关的移植损伤, 最终导致早期的移植丢失。该协议描述了一个模型的小鼠胰腺移植使用 non-suture 袖技术, 最适合于分析这些早期的, 有害的损害。

应用改良袖套技术的小鼠胰腺移植模型

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

mouse-model-for-pancreas-transplantation-using-modified-cuff

Research

JoVE Journal

Medicine

10.9K 次观看.  Medical University Innsbruck. 在腹部实体器官移植中, 胰腺移植容易发生严重的缺血再灌注损伤-相关的移植损伤, 最终导致早期的移植丢失。该协议描述了一个模型的小鼠胰腺移植使用 non-suture 袖技术, 最适合于分析这些早期的, 有害的损害。

视频: Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

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

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.

Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

补肾胶囊对糖尿病小鼠的胰岛移植

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

生物学

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer (PC). To address this deficiency, we describe a mouse model involving orthotopic implantation of PC followed by distal pancreatectomy and splenectomy. The model has been demonstrated to be safe and suitably flexible for the study of various therapeutic approaches in adjuvant and neo adjuvant settings.
In this model, a pancreatic tumor is first generated by implanting a mixture of human pancreatic cancer cells (luciferase-tagged AsPC-1) and human cancer associated pancreatic stellate cells into the distal pancreas of Balb/c athymic nude mice. After three weeks, the cancer is resected by re-laparotomy, distal pancreatectomy and splenectomy. In this model, bioluminescence imaging can be used to follow the progress of cancer development and effects of resection/treatments. Following resection, adjuvant therapy can be given. Alternatively, neoadjuvant treatment can be given prior to resection.
Representative data from 45 mice are presented. All mice underwent successful distal pancreatectomy/splenectomy with no issues of hemostasis. A macroscopic proximal pancreatic margin greater than 5 mm was achieved in 43 (96%) mice. The technical success rate of pancreatic resection was 100%, with 0% early mortality and morbidity. None of the animals died during the week after resection.
In summary, we describe a robust and reproducible technique for a surgical resection model of pancreatic cancer in mice which mimics the clinical scenario. The model may be useful for the testing of both adjuvant and neoadjuvant treatments.

In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.

胰腺癌矫形小鼠模型

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer ...

癌症研究

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

用改进的袖套法小鼠颈部心脏移植模型

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, immune cells, vessels, nerves, and extracellular matrix components. Over the years, different mouse models of PDAC have been developed to address the challenges posed by its progression, metastatic potential, and phenotypic heterogeneity. Immunocompetent mouse orthotopic allografts of PDAC have shown good promise owing to their fast and reproducible tumor progression in comparison to genetically engineered mouse models. Moreover, combined with their ability to mimic the biological features observed in autochthonous PDAC, cell line-based orthotopic allograft mouse models enable large-scale in vivo experiments. Thus, these models are widely used in preclinical studies for rapid genotype-phenotype and drug-response analyses. The aim of this protocol is to provide a reproducible and robust approach to successfully inject primary mouse PDAC cell cultures into the pancreas of syngeneic recipient mice. In addition to the technical details, important information is given that must be considered before performing these experiments.

Syngeneic mouse orthotopic allografts of pancreatic ductal adenocarcinoma (PDAC) recapitulate the biology, phenotypes, and therapeutic responses of disease subtypes. Owing to their fast, reproducible tumor progression, they are widely used in preclinical studies. Here, we show common practices to generate these models, injecting syngeneic murine PDAC cultures into the pancreas.

同系小鼠原位同种异体移植物模拟胰腺癌

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, ...

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

支架支持胰岛移植糖尿病小鼠附睾脂肪垫

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

生物工程

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

胰岛门静脉移植小鼠模型的研究

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

通过胆管 TNBS 输液治疗慢性胰腺炎的鼠标模型

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. ...

A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for investigating the mechanism of cardiac allograft rejection in preclinical studies because of their high homology with human genes. This understanding would help develop unique approaches to improving patients&#39; long-term survival treated with cardiac allografts. In a mouse model, abdominal donor heart implantation is commonly performed with an end-to-end&#160;anastomosis to the recipient&#39;s aorta and inferior vena cava using stitches. In this model, the donor&#39;s heart is implanted by end-to-end anastomosis to the recipient&#39;s carotid artery and jugular vein by the modified-Cuff technique. The transplantation surgery is performed without stitching and thus may increase the survival of the recipient since there is no interference with the blood supply and venous reflux of the lower body. This mouse model would help investigate the mechanisms underlying the immunological and pathological (acute/chronic) rejection of cardiac allografts.

In the present protocol, a mouse heart transplantation model is used for investigating the mechanism of cardiac allograft rejection. In this heterotopic heart transplantation model, operation efficiency is improved, and the survival of cardiac grafts is ensured by a cervical end-to-end anastomosis of heart implantation using a modified Cuff technique.

一种改进的袖带技术用于小鼠颈椎异位心脏移植模型

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for ...

Orthotopic Homograft Tumor Transplantation: A Technique to Generate Pancreatic Tumor Mouse Models

Source: An, X. et al.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/v/57460/" target="_blank">Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry</a>. J. Vis. Exp. (2018)
The video explains a stepwise surgical method for orthotopic implantation of a tumor fragment from a cancerous donor to the pancreas of a non-cancerous recipient mice for development of pancreatic cancer. Orthotopically implanted PDAC could recapture the tissue microenvironment of human pancreatic cancers than conventional subcutaneous (SC) models more accurately.

原位同种移植物肿瘤移植:一种生成胰腺肿瘤小鼠模型的技术

Source: An, X. et al.&#160;Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry. ...